LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı

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http://hdl.handle.net/11527/19400

Gözat

Protein engineering applications on industrially important enzymes

(Fen Bilimleri Enstitüsü, 2019) Özgün, Gülşah ; Karagüler, Nevin Gül ; Moleküler Biyoloji-Genetik ve Biyoteknoloji ; Molecular Biology-Genetics and Biotechnology

Büyüyen biyoteknoloji marketi, beraberinde biyokatalizörlerin geliştirilmesi ve yeni özellikler kazandırılmasına yönelik çalışmaların artmasına yol açmıştır. Endüstriyel ortamlar genellikle doğal enzimlerin kullanımı için uygun olmayan ekstrem şartlar barındırmaktadır, yüksek sıcaklık, basınç ve çok yüksek veya düşük pH gibi koşullarda, çoğunlukla doğal enzimler istenilen performansı sağlayamamaktadır. Başta protein mühendisliği olmak üzere birçok disiplin farklı endüstriyel alanlarda enzimlerin etkili bir şekilde kullanımına yönelik stratejiler geliştirmektedirler. Protein mühendisliği, endüstriyel sektörün özelliklerine ve enzimin kullanılacağı ortama bağlı olarak istenilen özelliklerin geliştirilmesinde, örneğin aktivite ve stabilitenin arttırılması, substrat veya koenzimin spesifisitesinin değiştirilmesi veya geliştirilmesi, optimum pH nın değiştirilmesi gibi stratejilere yönelik rasyonel tasarım, yönlendirilmiş evrim ve kombinasyonel uygulamaları kullanmaktadır. Protein mühendisliği stratejilerinin belirlenmesinde aynı zamanda enzime ait (yapı-fonksiyon) bilgilerin varlığı veya yokluğu etkili olmaktadır. Protein mühendisliği, moleküler biyoloji temeline dayanarak proteinin yapı fonksiyon ilişkisinin anlaşılmasına olanak sağlayarak, proteinin genetik düzeyde yeniden dizayn edilmesini ve istenilen özellikte biyokatalizörlerin oluşturulmasını mümkün kılar. Bu kapsamda tez, endüstriyel öneme sahip Bacillus subtilis lipazA (bsLipA) ve Candida methylica format dehidrogenaz (cmFDH) enzimlerinin üç farklı stratejinin kullanıldığı protein mühendisliği uygulamalarına odaklanmıştır. i) Optimum pH nın ii) ve koenzim spesifitesinin değiştirilmesi iii) termal stabilitenin arttırılması hedeflenen stratejiler olmuştur. Deneysel çalışmalar üç bölümde detaylandırılmıştır. Tezin birinci kısmında, bsLipA enziminin optimum pH'sının değiştirilmesi amacıyla rasyonel tasarım uygulamalarından bölgeye özel mutasyon yöntemi uygulanmıştır. Lipazlar (EC 3.1.1.3) trigliseridlerin serbest yağ asitleri ve gliserole hidrolizini gerçekleştirirlerken, aynı zamanda transesterifikasyon, aminoliziz ve asidoliziz reaksiyonlarını da katalizlerler. Mikrobiyal lipazların susuz ve az-sulu ortamlardaki potansiyeli, enzimin çok yönlü biyoteknolojik bir araç haline gelmesini sağlamıştır. Farklı endüstriyel alanlarda uygulama imkanı bulan lipazlar, son yıllarda sıvı veya süperkritik karbon dioksit (LCO2/SCCO2) gibi hijyen sistemlerinde hidrolitik enzim katkısı olarak kullanım potansiyeline sahiptir. LCO2/SCCO2 sistemlerinin temizleme etkisinin hidrolitik enzim ilavesi ile arttırılması mümkün olmasına karşın, çözgen olarak kullanılacak olan LCO2 / SCCO2' in polar olmayan bir çözgen olması ve düşük su içeriği sebebiyle, kullanılacak enzim sisteminin az sulu ve düşük pH' ya sahip çözgen sistemlerinde aktif olması beklenmektedir. Bacillus subtilis lipaz (bsLipA), geniş bir pH (4-11) aralığına sahip olmasına karşın optimum pH'sı 10'dur, bu sebeple LCO2 / SCCO2 çözgen sistemlerinde kullanımı için modifikasyonu gerekmektedir. Bacillus subtilis lipaz A (PDB ID: 1ISP) kristal yapısı ve Insight II programı kullanılarak enzimin optimum pH değişimini sağlayacak hedef mutantlar belirlenmiştir. Proteinin aktif bölgesinde katalitik özellikteki amino asitlerin pKa değerlerini etkileyebileceği düşünülen, yaklaşık 9 Å'luk mesafe içerisinde olan ve katalitik amino asitler ile doğrudan ilişkili olan amino asitlerin, bölgeye özel mutasyon tekniği ile tekli G11E, N18R, L102R, G103R, G104R, I157R mutantları oluşturulmuştur. N18R ve G103R mutantlarının template olarak kullanılması ile ikili (G11E-N18R, G103R- N18R, G103R- G11E, and G103R- G104R) mutantların da, bölgeye özel mutasyon tekniği ile oluşturulması planlanmıştır. Tezin ikinci kısmı cmFDH enziminin koenzim spesifitesinin değiştirilmesine yönelik yarı-rasyonel tasarım uygulamalarından bölge saturasyon mutagenez yöntemini kapsamaktadır. Tezin üçüncü kısmında ise cmFDH enziminin termal stabilitesinin arttırılmasına yönelik, , rasyonel tasarım uygulaması olan bölgeye özel mutasyon yöntemi uygulanmıştır. NAD+-bağımlı format dehidrogenaz enzimi (EC 1.2.1.2, FDH), format iyonunun karbondiokside dönüşümünü katalizlerken, NAD+ molekülünün NADH'e indirgenmesini sağlamaktadır. Gerçekleşen reaksiyonun basitliği, kullanılabilirliği, düşük maliyeti, termodinamik özellikleri ve son ürün olan CO2' in reaksiyondan kolaylıkla uzaklaştırılabilmesi gibi avantajlarından dolayı FDH, kimya endüstrisindeki kiral bileşiklerin sentezi için çok önemli olan NAD(P)H rejenerasyonunda potansiyel bir sistemdir. Fakat, doğada bulunan FDH'lerin çoğunluğunun NAD+ koenzimine spesifik olması ve düşük termal stabiliteye sahip olması, FDH'in kullanımını kısıtlamaktadır. Bu sınırlandırmaların aşılması amacıyla, tezin ikinci kısmında, cmFDH enziminin koenzim spesifitesinin değiştirilmesi için koenzim bağlama bölgesinde, koenzim spesifitesinden sorumlu amino asitler, Pseodomonas. sp.101 ve Candida boidinii FDH kristal yapıları baz alınarak, Insight II (Accelrys) programı ile oluşturulan cmFDH homoloji modeli yardımıyla belirlenmiştir. Belirlenen D195, Y196 ve Q197 bölgelerine ait dejenere primerler ile uygulanan bölge saturasyon mutagenez çalışması sonucunda her bölge için mutant kütüphaneleri oluşturulmuştur. NADP+ koenzimi ile aktivite gösteren aday mutantlar kolorimetrik tarama metoduyla belirlenmiştir. İki nesil oluşturulan adayların protein üretimi ve saflaştırılması neticesinde NADP+ koenzimine karşı olan ilgisi test edilmiştir. Yapılan çalışma sonucunda ikili mutantlardan D195S / Q197T ve D195S / Y196L mutant cmFDH enzimlerinin NADP+` ye karşı katalitik etkinlikleri yabanıl tip cmFDH enzimine kıyasla, sırasıyla 56000 ve 50000 kat artmıştır. Çalışmanın son kısmında, cmFDH enziminin termal stabilitesinin geliştirilmesi amacıyla, protein yüzeyindeki esnekliği yüksek olan oyuklar ve bu oyuklarda bulunan esnek amino asit kalıntıları hedef alınmıştır. Pseodomonas. sp.101 ve Candida boidinii FDH kristal yapıları baz alınarak, ExPASy programı ile cmFDH homoloji modeli elde edilmiştir. Oluşturulan model üzerinde FIRST algoritması kullanılarak, proteinin esnek oyukları ve hedef amino asit kalıntıları belirlenmiştir. Yapılan bilgisayar çalışmaları neticesinde belirlenen 12 aday; M131A, V133I, V139W, P140R, D158N, I162V, F186L, V219M, F247A, E272W, R277N ve K301R bölgeye özel mutasyon yöntemi ile oluşturulmuştur. Mutant adayların protein üretimi ve saflaştırılması neticesinde gerçekleştirilen kinetik ve sıcaklık çalışmaları sonucunda, birinci oyukta yer alan M131A mutant enziminin, rekombinant yabani tip cmFDH enzimine kıyasla yarı ömründe 4 °C'lik bir artışla diğer mutantlar arasında en iyi termal stabilite profili gösterdiği belirlenmiştir. Bu tez kapsamında yapmış olduğumuz tüm çalışmalar, endüstriyel kullanım için enzimlerin modifikasyonunda, doğru stratejiler kullanıldığında protein mühendisliği uygulamalarının, başarı sağladığını göstermektedir.
Investigation of the effect of ATP13A2 (PARK9) frameshift mutation on the protein function

(Lisansüstü Eğitim Enstitüsü, 2021) Kırımtay, Koray ; Karabay Korkmaz, Arzu ; 692806 ; Moleküler Biyoloji-Genetik ve Biyoteknoloji

Neurodegenerative diseases occur due to deformations in the nervous system as a result of structural and functional disorders in nerve cells. In practice, neurodegenerative diseases are classified according to clinical and pathological findings. Genetic and epigenetic factors play essential roles on the basis of the disease pathology. Mutations that occur in different genes or at various points in the same gene act at the onset of the disease by acting on different molecular mechanisms. In many neurodegenerative diseases, overlapping clinical findings at the beginning and progression of the disease are the most critical limiting factors in making the correct diagnosis in these diseases. Rare neurodegenerative disorders, such as complex Hereditary Spastic Paraplegia (HSP), Spino-cerebellar Ataxia (SCA), and Kufor-Rakeb syndrome (KRS), have many common symptoms. Spastic Paraplegia-78, a subtype of HSP, and KRS have been associated with mutations in the ATP13A2 gene. In these two diseases that are inherited in autosomal recessive manner, different mutations in the ATP13A2 gene cause many common symptoms, making clinical diagnosis difficult. Genetic and functional molecular studies are important for the accurate diagnosis of the disease. Within the scope of this thesis, two cases in a consanguineous Turkish family were examined genetically and cellularly. Clinically, patients were diagnosed with "autosomal recessive spastic paraparesis" and/or "autosomal recessive ataxia" due to the observation of spasticity, cerebral and cerebellar atrophy. In this thesis, gene mutations associated with the disease were screened by whole-exome sequencing in the family. Three variants of the ATP13A2 gene ("c.1422_1423del: p.P474fs", "c.G1426T: p.A476S" and "c.1429_1430insAAA: p.M477delinsKM") were detected as a result of whole-exome sequencing. The bioinformatics analysis determined that the "c.1422_1423del: p.P474fs" variant caused a frameshift and a premature termination codon. It was determined that the premature termination codon formed by the effect of frameshift causes the loss of N and P regions necessary for phosphorylation, which is important in the function of the ATP13A2 protein, resulting in the formation of a truncated protein of 519 amino acids. Following these data, ATP13A2 expression in primary fibroblast cells obtained from patients, their parents, and a healthy control was examined by qRT-PCR and Western blot method. According to the qRT-PCR result, it was determined that ATP13A2 mRNA was reduced by 60-70% in patients' fibroblasts compared to control fibroblasts. ATP13A2 mRNA was observed to decrease by 5-20% in the fibroblasts of the parents. When protein expression was examined by Western blot method, wild-type ATP13A2 protein with a molecular weight of 150 kDa was detected in the fibroblasts of control and parents. In comparison, the expected truncated protein around 50 kDa was not detected in the patient fibroblasts. The reason for not observing the truncated protein could possibly be to the fact that the produced truncated protein might have been degraded by proteasome machinery due to misfolding. In this context, truncated protein expression was investigated by inhibiting the proteasome mechanism with MG132. According to the Western blot result, mutant protein expression was not observed after MG132 application. Besides, BiP and PDI, which are endoplasmic chaperone proteins involved in protein folding, expressions were also examined, and no difference was observed in the expression of these two proteins. These results highlighted the possibility that the mutant ATP13A2 mRNA was targeted and degraded by NMD. To examine whether NMD degraded the mutant ATP13A2 mRNA, 5-azacytidine, which was shown to cause NMD inhibition by c-myc, was applied to fibroblasts. After 5-azacytidine administration, it was observed that the mutant ATP13A2 protein was expressed in patients' fibroblasts. However, the increase in both mRNA and protein levels in control fibroblast cells that do not carry mutations suggested that 5-azacytidine acts by a different mechanism independent of NMD. It is known in the literature that 5-azacytidine increases c-myc expression. Accordingly, after the 5-azacytidine treatment, c-myc expression was examined by Western blot, and an increase in c-myc expression was observed. In addition, the mutant ATP13A2 expression was detected in fibroblast cells differentiated from the patient's induced pluripotent stem cells in which c-myc had been expressing due to the presence of c-myc expressing Sendai virus. The reason for the increased ATP13A2 expression in both patient fibroblasts and control fibroblasts after 5-azacytidine suggested that the gene expression by c-myc may be transcriptionally regulated. When the ATP13A2 gene's promoter region was examined, the putative c-myc transcription factor binding sites were determined. These findings strengthen the possibility that the ATP13A2 gene can be regulated by c-myc. After 5-azacytidine treatment, NMD-independent increase in ATP13A2 expression was observed, fibroblasts were treated with NMDI-14, a specific NMD inhibitor, and a 2-fold increase in ATP13A2 mRNA level was observed in patient fibroblasts. In addition, mutant protein expression was detected in patients' fibroblasts. After determining that ATP13A2 protein was not expressed as a result of the mutation in the patients, based on the hypointense basal ganglion images on MRI, iron accumulation was examined by Prussian blue staining in the patient fibroblasts. Iron accumulation was found in the patient fibroblasts after staining. Later, the effect of iron deposition on cell viability was investigated by the MTT assay. It was found that patient fibroblasts were more sensitive to iron overload. It is known that increased intracellular iron increases reactive oxygen species and triggers oxidative stress. In the presence of oxidative stress, the Nrf2 transcription factor is localized to the nucleus and provides the expression of several genes involved in oxidative stress defense. For this reason, the intracellular localization of Nrf2 was examined by immunocytochemistry. It was observed that Nrf2 was localized in the nucleus in the patient's fibroblasts, while it was mostly cytoplasmic in the parents' fibroblasts. These data show that patients have iron overload and oxidative stress due to accumulation.
Discovery of novel enzymes using proteomic approaches

(Graduate School, 2021-01-26) Kılınç Öztuğ, Merve ; Karagüler, Nevin Gül ; Akgöz, Müslüm ; 521142107 ; Molecular Biology-Genetics and Biotechnology

Thermophilic microorganisms that survive and grow in extreme environments, above temperatures of 50 °C, have been well studied over the last decade allowing us to increase our knowledge of the compositional and functional potential of these microbial communities. These microorganisms are of great importance for industrial processes since they express heat-resistive enzymes with the potential to serve as a biocatalyst in the future. Developing proteomic and metaproteomic approaches to discover novel enzymes from environmental samples is growing research of interest owing to the advanced mass spectrometry (MS) based techniques. In this study, proteomics and metaproteomics approaches were applied to discover novel enzymes from harsh environmental conditions. Geothermal sources are among the habitats of thermophilic bacteria. In Turkey, there are many spas that have the potential habitat for numerous thermophilic bacteria, and this offers a good opportunity for the discovery of new thermophilic microorganisms. In this study, a thermophilic bacterial consortium of the Armutlu Hot Spring in the Yalova region of Turkey was investigated in a culture-dependent manner using proteomic approaches.
Alteration of titanium surfaces using hyaluronic acid coated mesoporous silica nanoparticles for local drug release

(Graduate School, 2021-12-17) Erşan, Yeliz ; Karataş Yazgan, Ayten ; Önder, Sakip ; 521181123 ; Biology-Genetics and Biotechnology

Nosocomial infection is still an important problem for developed and developing countries as it decreases the effectiveness of the treatment as well as increases the healthcare expenses due to the prolonged stays in units. Most of these infections are biomaterial-based and caused by biofilm forming bacteria on biomaterial surfaces. Therefore, systemic drug administration is used to prevent biomaterials associated infections and to increase success of the implantation. Otherwise, revision surgery is generally required. Revision surgery means more pain for the patient, and it does not guarantee that osteointegration between the implant and surrounding tissue will be as strong as the first implementation. Local drug release using drug eluting implant materials suggests a great opportunity to prevent implant associated infections. Functional coatings containing therapeutic agents can be deposited on these surfaces by using different surface coating techniques such as layer by layer deposition (LBL), electrophoretic deposition (EPD), physical/chemical vapor deposition (CVD/PVD) etc. Local drug release from these surfaces does not only prevent implant associated infections, it also prevents the systemic toxicity. Moreover, sustained local drug release is possible with drug eluting materials. In the present study, a functional coating based on hyaluronic acid (HA) and mesoporous silica nanoparticles (MSNs) was proposed to prevent implant associated infections. For this purpose, drug loaded HA coated MSNs were prepared and deposited onto the Ti implants using EPD technique. Mesoporous silica nanoparticles (MSNs) were synthesized by the sol-gel/emulsion method and analysed using different characterization techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS). After that, MSNs was silanized by using APTES (3- Triethoxysilylpropylamine) to form amino groups (-NH2) on the MSNs surfaces. In order to form HA coatings on MSN surfaces, HA was dissolved in MES buffer, NH2 -MSNs were added into the solution, EDC (N-(3-Dimethylaminopropyl)-N'-ethyl- carbodiimid-hydrochloride) and NHS (N-hydroxysuccinimide). Characterization of silanized (-NH2-MSNs) and HA-coated MSNs (HA-MSNs) were done by using SEM, Zeta Sizer and Fourier Transform Infrared Spectroscopy (FT-IR). SEM micrographs showed that almost homogeneous spherical MSNs ranging from 185 nm to 240 nm were synthesized. DLS analysis was showed that their sizes are ca. 8.7 μm. The cause of the difference was due to the environment MSNs exposed during characterization. SEM analysis is performed on dry nanoparticles while watery environment is used for DLS analysis. TEM analysis gave similar results with SEM for the sizes of the MSNs. Porous structure of synthesized MSNs were shown in this analysis. Moreover, characteristic peaks at 696 cm-1 and 1540 cm-1 that are attributed to –NH2 groups after silanization and peaks at 1639 cm-1 that are attributed –CH groups following the HA coating were determined in FTIR analysis. According to these results MSNs, -NH2-MSNs and HA-MSNs are synthesized and modified successfully. Ti plates were treated mechanically and chemically prior to deposit nanoparticles. Deposition onto the Ti surfaces were carried out using two different procedures. In the first procedure, HA-MSNs were dissolved in ethanol (70%) and HA coated MSNs deposited onto the Ti substrates for 1, 3 and 5 min. at 30V. In the second procedure, different ratios of MSNs and HA solution (MSNs:HA (w/w; 1:0.5, 0.5:1, 1:1)) were prepared and electrodes were then placed in this solution. Same coating parameters were applied for both coating procedures. The surfaces were analysed using SEM, FTIR and X-ray spectroscopy (EDS) to examine the surface morphology and chemical composition of the surfaces following the coating process. Characterization studies showed that both procedure 1 and procedure 2 can be used to obtain homogenous coatings on the surfaces. Neverthless, MSNs:HA should be 1:0.5 because coatings that were deposited using higher HA concentrations detached from the surfaces due to the thick film formation. In addition, SEM and EDS analysis showed that coating thickness can be increased with prolonged deposition time. The coating thicknesses were determined using mechanical profilometer and different thicknesses from 0.48 to 1.9 micrometer. were measured on the surfaces for different coating times. According to results of analysis, thickness was increased while increasing deposition time. Drug loading and release studies were carried out in PBS for free (synthesized MSNs, -NH2-MSNs, HA-MSNs) particles and coated particles (HA-MSNs) that were prepared using two different procedures. Ciprofloxacin as a model drug was used in this study, and it was both loaded into free MSNs and coated surfaces by diffusion. In accordance with the drug loading studies, drug loading efficiency was higher for HA-MSNs (ca. 80 %) when compared to HA free MSNs (ca. %40). Moreover, it was shown that drug release was possible using free MSNs for ca. 15 hours at 37 oC. Moreover, synthesized and silanized MSNs did not show sufficient drug loading and release rate like HA- MSNs. Finally, drug release from the coated Ti surfaces were examined. According to the drug release profiles, it was possible to have 10 hours drug release was determined from the Ti surfaces. In sum, HA-coated MSNs can be used as a functional coating to design a drug eluting Ti implant material and to prevent implant associated infections.
Investigating protein variations of Bag-1 in wild type and Bag-1 knockout MCF-7 breast cancer cells

(Lisansüstü Eğitim Enstitüsü, 2022) Kılbaş, Pelin Özfiliz ; Doğanay, Gizem Dinler ; Arısan, Elif Damla ; 723988 ; Moleküler Biyoloji-Genetik ve Biyoteknoloji

The multifunctional Bag-1 protein which is known for its anti-apoptotic role has many critical direct or indirect interaction partners in the cell. According to these interactions, the Bag-1 protein plays an important role in the decision mechanism between survival or death of cancer cells. In previous studies determining the chromosomal location of the Bag-1 gene, it has been stated that this gene is associated with neurodegenerative diseases such as Alzheimer's and Parkinson's, and cancer. It has been shown that the change in the expression level of Bag-1 protein in the cell is associated with many different cancer types. In addition, it has been demonstrated that Bag-1 may provide clinical benefit as a prognostic marker in the determination of breast cancer, which is the first among women with its incidence. Elevated levels of Bag-1 are generally associated with breast cancer growth, development, and aggressiveness. In particular, interactions with the Hsc70/Hsp70 chaperone family are effective in the long-term survival of breast cancer cells under stress conditions. Our previous studies showed that silencing the Bag-1 protein increases drug-induced apoptosis in breast cancer. Although there are several studies on the association of changes in the expression level of the Bag-1 protein with cancer, the biological aspect of the complete deletion of the Bag-1 gene in breast cancer cells is not detailed explained. Therefore, this thesis study consists of two parts covering the molecular function of the deletion of the Bag-1 gene in MCF-7 breast cancer cells. The first part of the thesis consists of the generation of Bag-1 knockout cells with the CRISPR/Cas9 system, the validation and characterization, and the investigation of the effect of Bag-1 deficiency on the viability and cellular response of MCF-7 cells. The second part of the thesis includes the profiling of differentially expressed microRNAs and the determination of the molecular targets of these significantly differentially expressed miRNAs in wild-type and Bag-1 knockout MCF-7 breast cancer cells.
Improving bone tissue integration of hard tissue implamants using bioactive materials

(Graduate School, 2022) Kerem, Gizem ; Kılıç, Abdulhalim ; 867049 ; Department of Molecular Biology-Genetics and Biotechnology

Hard tissue implants which made of different materials are used widely in medical applications in bone and tooth deficiencies. Metallic and ceramic materials are preferred as hard tissue implants because of their strength and toughness. Due to low density, high corrosion resistance and biocompatibility properties of titanium and titanium alloys, their usage as an implant has gradually increased. Implant materials should not cause an immunogenic reaction in the body and also in recent years thanks to new technologies in biomaterial field there are some applications to improve interaction in a positive way between implant and implantation tissue. Some morphological changing applications like grooving and acid etching, some physicochemical activation treatments like hydroxyapatite (HA) coating and titanium oxide (TiO2) coating, some biochemical activation treatments like coating with biopolymers and immobilization of bone morphogenic proteins and inducer chemicals are studied on titanium surface to improve bone formation, bone bonding and antibacterial property. At this study, titanium (grade 2) surfaces are chosen to increase its bone tissue integration by designing bioactive surface by using chitosan microspheres as a natural polymer and water-soluble dexamethasone (DEX) as an inducer for SAOS-2 cell differentiation. Chitosan microspheres were produced by using single emulsion-crosslinking method. Different amount of glutaraldehyde with different percentages was used as a crosslinker agent to obtain optimum DEX loading into chitosan microspheres. Crosslinking time parameter was also changed for optimization. Dexamethasone loading was tried in two ways first one is adding drug into dissolved chitosan solution then produce chitosan microspheres, second one is adding produced chitosan microspheres into the drug solution. DEX was loaded into chitosan microspheres via diffusion. After optimization process, chitosan microspheres were produced by using glutaraldehyde as a crosslinker. Chitosan microspheres were placed into DEX solution for loading process. Drug release studies were performed by using DEX-loaded chitosan microspheres, and released amount of DEX was determined. Drug loading efficiency was found as 50.16% and release of DEX was observed for 12 hours and the released amount of the loaded DEX was calculated as ~32.6%. Before chitosan microsphere coating onto titanium surfaces, they were treated chemically (polished, oxidized and silanized to produce amino groups on titanium surfaces). Then the samples were activated by glutaraldehyde and coated with chitosan microspheres.
The computational study of the interaction of POT1 with SSDNA and TPP1

(Graduate School, 2022) Gürbüz Önder ; Balta, Bülent ; 740109 ; Molecular Biology-Genetics and Biotechnology Programme

Linear DNA sequences in eukaryotes have telomere sequences at the chromosome ends. In humans, telomeres consists of repeating TTAGGG sequences. Shelterin complex mainly interacts with telomere sequences. It interacts with both telomeric dsDNA and ssDNA. It protects it from DNA repair mechanism. POT1 is one of the members of shelterin complex. It interacts with telomeric ssDNA. It controls the length of the telomere sequence by forming complex with TPP1. The N-terminal domain of POT1, POT1N, binds to ssDNA while C terminal domain of POT1, POT1C, is known to interact with TPP1. The interaction between POT1 and ssDNA depends on many factors. In the shorter sequences, POT1 prefers to bind 3' end of the sequence by 10 fold. The preference changes as TPP1 binds to POT1. POT1 slides on ssDNA after forming complex with TPP1. There is no information about the mechanism of action in the literature. TPP1 interacts with POT1 and increases its affinity to ssDNA. The affinity of POT1 on ssDNA increases after interacting with POT1 and POT1-TPP1 interaction is regulated by cell cycle as well. It is likely that the POT1-TPP1 complex has at least 2 different conformations and these conformations have different interactions, conformations and geometries. There is no information about these structures in the literature. The preference of POT1 on ssDNA, potential initial binding and sliding mechanisms, POT1-TPP1 complex and the allosteric effect of TPP1 were investigated by standard Molecular Dynamics (MD) simulations and Replica Exchange Molecular Dynamics (REMD) simulations. Free energy of the system was calculated by Molecular Mechanic/Poisson-Boltzmann Surface Area (MM/PBSA) method. Another candidate for the mechanism was investigated by Potemtial of Mean Force (PMF) method. According to our results, the binding preference of POT1 depends on the length of ssDNA. In a longer DNA sequence (22 nucleotides), POT1 has the highest abundance in the middle of sequence, ~27%, followed by 5'end, ~ 17% and 3' end ~ 16%. In a shorter DNA sequence (16 nucleotides), POT1 prefers binding to the 3' end over 5' end (~32% and ~22%). This results shows that the preference of POT1 is influenced by ssDNA length. Our results indicate that extra nucleotide in the longer ssDNA simulations form secondary structure with each other and this might explain 5' end preference over 3' end. In the shorter ssDNA simulations, 3' end is loosely bound to POT1 ~23% of the time and ~8% in the solvent, while 5' end has ~8% protein bound and ~14% in the solvent and this explains 3' preference in the shorter ssDNA simulations. Our results show that there are multiple potential initial binding and sliding mechanisms. Most of the time, POT1 slides only 1 OB fold at a time while other OB fold conserves its position. The sliding happens per nucleotide in this mechanism. The relative abundance of these OB shifted structures were ~ 21%. Here, OB2 fold shifted structures had higher abundance than OB1 fold shifted ones which confirms the literature. Another potential mechanism involves the repetetive nature of telomere sequence. POT1 might prefer to skip a telomere repeat and OB fold can bind to the next telomere sequence. When OB1 fold binds to telomere sequence, OB2 fold moves 6 nucleotides and then OB1 folds shall slide on ssDNA. Furthermore, when ssDNA was interacted with only OB1 fold or OB2 fold, it clearly showed that OB1 fold prefers to bind ssDNA. The relative abundance of these structures were ~4.5% and ~1.5%, respectively. When whole sequence was shifted by 1 nucleotide, which is considered a potential mechanism, the structure has the lowest abundance. In order to test this for 2, 3, 4 and 5 nucleotides, 5 new initial structure was created. In these structures, ssDNA was 11 nucleotide long. The crystal structure and 6 new structures were simulated. G results show that all of these structures are not a candidate for a potential intermediate structures during the sliding. There are 2 different complexes for POT1-TPP1. In the first complex, it should slide easily on ssDNA and it should have less specific interactions. In second complex, it should bind it tightly and have specific interactions. Both structures were constructed based on O. nova. In the simulations, they survived more than 1 µs.
In vitro and in silico investigation of NFIB-SUMO interactions

(Graduate School, 2022) Özkan, Ayberk ; Kumbasar, Aslı ; 718174 ; Molecular Biology-Genetics and Biotechnology Programme

Nuclear Factor I family of transcription factors are involved in regulation in diverse physiological processes, including neuronal terminal differentiation, gliogenesis, stem cell quiescence as well as in pathologies such as tumorigenesis and cancer progression. NFI family is encoded by four genes: NFIA, NFIB, NFIC and NFIX. NFI proteins contain a highly conserved N-terminal DNA binding and dimerization domain, while their C-terminal domains diverge. Because of their highly similar DNA binding and dimerization domain, NFIs bind to a palindromic consensus site with similar affinity in vitro, potentially regulating the same set of target genes in vivo. Any functional differences between the family members may arise from the more diverse C-terminal domain provides which can promote either transcriptional activation or repression. NFIs may regulate target gene expression via different mechanisms of action. NFI can bind directly to DNA and regulate the expression of the target gene or interact with another protein to affect gene expression indirectly. Moreover, NFIs can interact with histone proteins and cause alterations in the nucleosome structure, thereby being involved in the formation of the transcription complex. NFIs can directly interact with and facilitate recruitment of basal transcription factors. NFIs can also bind co-activator or co-repressors to control transcriptional activation. In addition, NFIs can, along with other transcription factors, co-regulate target gene expression. Finally, NFIs can promote dissociation of DNA methyltransferase from target gene promoters and activate transcription. Any alterations in the production or action mechanisms of NFI proteins lead to important developmental defects as well as cancer. One member of the NFI family, NFIB, is an essential gene as demonstrated by studies on knockout on mice: silencing of NFIB leads to perinatal death due to lung defects. NFIB controls stem cell differentiation in different cell types such as, adipocytes, megakaryocytes, melanocytes and hippocampal neural progenitors. Interestingly, in humans, mutation of one copy of the NFIB gene can result in intellectual disability and brain malformations. These findings underscore the importance of NFIB as a transcriptional regulator, however, the mechanism by which NFIB acts or regulatory events upstream of NFIB have not been fully elucidated. Indeed, scarce data exists regarding NFI post-translational modifications. Phosphorylation, glycosylation, acetylation, sumoylation may regulate the activity of NFI. Among these modifications, sumoylation is conserved by eukaryotic organisms. Sumoylation regulates many cellular mechanisms such as nuclear transport, chromosome segregation, and transcription activation/repression. SUMO (small ubiquitin like modifiers), which is generally observed as a suppressor in transcriptional regulation, can be conjugated to many transcription factors and affects the activity of these factors. The SUMO gene family has five mammalian isoforms: SUMO1, SUMO2, SUMO3, SUMO4 and SUMO5. SUMO peptides are activated by a series of enzymatic processes. These processes are required to form mature SUMO, which is active and may able to conjugate specifically to the target protein. The sumoylation consensus sites and SIM (SUMO interacting motif) on target proteins enable SUMO to specifically recognize and bind to these proteins and regulate their activities. Sumoylation can affect transcription factors in several ways. SUMO can compete with other modifications, may interact with co-activators, and can control the binding of the transcription factor to its target site on chromatin. In addition, sumoylation can control intracellular localization of transcription factors. Sumoylation of NFI has been shown in vitro. Interestingly, a study on neuroblastoma cells exposed to oxidative stress, identified NFIB among sumoylated proteins modified on sumoylation consensus sites. However, sumoylation of NFIs have not been further explored, in silico and in cell culture. In this study, we set out to investigate the functionality of sumoylation consensus site and SUMO interacting motifs of NFIB, using in silico methods and forced expression in cell culture. Currently, there is no experimental or modeled 3D structure of NFI proteins. Information about NFI protein structure is quite limited. As mentioned above, NFI proteins contain an N-terminal DNA binding and dimerization domain and C-terminal transactivation domain. NFI proteins carry four conserved cysteine residues in their DNA binding and dimerization domains, three of which are required for DNA binding activity. Another piece of evidence regarding NFI structure comes from the homology with the MH1 domain of SMAD3. Both proteins have highly similar Cys-His box motifs consisting of three cysteine residues and one histidine residue. Nevertheless, this homology is below 30%. Here, due to the lack of high homology and also fold similarity, we used ab initio modeling method to predict structure of NFIB DNA binding and dimerization domain. Subsequently, these predictions were compared to each other. For this comparison, we focused on the cysteine residues which are required for DNA binding activity as well as the conserved MH1 Cys-His box motif. Then, selected structure prediction models were assessed by molecular dynamic simulations. Finally, with REMD (Replica Exchange Molecular Dynamics) the model that showed higher stability and quality was verified. We performed molecular docking simulations to investigate NFIB SIM-SUMO1 interactions. We found that SUMO1 would preferentially bind to a specific NFIB SIM. Meanwhile, to investigate NFIB-SUMO1 conjugation in vitro; site-directed mutagenesis was performed for generation of a sumoylation consensus site mutant and a SIM mutant. HEK293T cells were co-transfected with SUMO1 and wild-type NFIB or NFIB mutants and NFIB was immunoprecipitated for analysis of NFIB-SUMO1 conjugation. Future experiments are required to validate the putative NFIB sumoylation consensus site and SIMs in cell culture.
Investigation of the mitochondrial metabolism of Helicobacter-activated B cells

(Graduate School, 2022) Şentürk, Zeynep Nur ; Yazgan Sayı, Ayça ; Molecular Biology-Genetics and Biotechnology Programme

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, and spiral-shaped bacterium and a member of the Helicobacteraceae family. H. pylori was discovered in 1982 by Warren and Marshall. Helicobacter infection can lead to multiple gastro pathologies such as chronic gastritis, gastric cancer, peptic ulcer disease, and mucosa-associated lymphoid tissue lymphoma. Whereas more than 50% of the world population has been infected with H. pylori, 80% of them are asymptomatic. Similar to H. pylori; H. felis is a gram-negative, urease-positive, spiral-shaped, and microaerophilic bacteria. Studies have shown that H. felis can induce gastric atrophy, metaplasia, dysplasia, chronic and persistent inflammatory response, and gastric cancer in mice models. Because H. pylori have less capacity to activate an efficient immune response in mice compared to Helicobacter felis (H. felis); H. felis is used to generate mice models for studying this pathogen. B cells play critical roles in adaptive immunity with antigen presentation to T cells and antibody production. Recently, new B cell subsets have been shown to exert anti-inflammatory and immune suppressive properties were discovered. These B cells are termed regulatory B cells (Bregs) by Bhan and colleagues. In mice; mainly CD19+CD21hiCD23hiCD24hi transitional 2 marginal-zone precursor cells (T2-MZP), IL-10 producing CD1dhiCD5+ B10 cells, CD19+CD21hi CD23- marginal-zone (MZ) B cells, Tim-1+ B cells, CD19+CD5+ B1a cells, CD9+ B cells, CD138+ plasma B cells, and CD138+CD44hi plasma blasts are identified as Breg subsets. For maintaining host immune tolerance and balance effector immune responses, these Breg cells secrete IL-10, IL-35, and TGF-ꞵ cytokines. In addition to cytokines, Breg cells also use cell membrane-bound molecules such as CD39, CD73, programmed death-ligand 1(PD-L1), or aryl hydrocarbon receptors for their functions. Stimulation of B cells with H. felis; signals via TLR2 and MyD88 and results with IL-10 producing Bregs. Bregs can induce differentiation of naive CD4+ T cells to IL-10-producing regulatory Tr1 cells by direct B and T cell interactions for suppressing Helicobacter-associated pathologies. Metabolism is the collection of all anabolic and catabolic reactions which are the generation and breakdown of cellular substances respectively. Oxidative phosphorylation (OXPHOS) is one of the major metabolic pathways inside the cell. It occurs in the mitochondria and consists of the tricarboxylic acid cycle (TCA) and electron transport chain reactions for generating ATP. Shortly, in OXPHOS electrons formed from the tricarboxylic acid cycle (TCA); are combined with molecular oxygen (final acceptor of electron transport chain) and this results in many oxidation/reduction reactions where energy is released for the production of ATP from ADP. Mitochondria are double membrane organelles that have both outer and inner mitochondrial membranes (OMM and IMM) and are found in eukaryotic cells. Membrane transporters and electron transport chain (ETC) complexes of mitochondria localize on the inner mitochondrial membrane. IMM encloses a viscous structure called a mitochondrial matrix. Enzymes, mitochondrial DNA (mtDNA), ribosomes, and nucleotides are placed in the mitochondrial matrix. Mitochondrial DNA encodes 37 mitochondrial genes including 22 transfer RNAs, 2 ribosomal RNAs, and 13 important oxidative phosphorylation polypeptides: ND1, ND2, ND3, ND4L, ND4, ND5, ND6 (parts of Complex I); Cytochrome b (parts of Complex III), COI, COII, COIII (parts of Complex IV) and ATP6, ATP8 (parts of Complex V). To provide expression of a mitochondrial gene, mitochondrial DNA needs to be transcribed by mitochondrial transcription factor A (Tfam), mitochondrial RNA polymerase (POLRMT), and mitochondrial transcription factor B1 and B2 (Tfb1m and Tfb2m). Two of the most critical features of multicellular life are metabolism and immunity. These can be explained as the need to distribute nutrients across cells, tissues & organs and protect from injury and inflammation. In recent years, studies have focused on elucidating the metabolism of immune cells in the context of their survival, activation, differentiation, and functions. For the activation of immune cells, signals which are triggered by metabolic intermediates and ATP molecules are required. In order to maintain proper immune cell activation, differentiation, and function, mitochondrial metabolism which generates energy plays a critical role. Studies have demonstrated that B cell activation with B cell receptor (BCR) or different Toll-like receptor (TLR) ligands changes mitochondrial dynamics. LPS-stimulated B cells enhance mitochondrial mass, and co-stimulation of B cells with BCR ligand IgM and TLR9 ligand CpG increases mitochondrial biogenesis. In addition to that, anti-CD-40 and IL-4 stimulated B cells to undergo OXPHOS. However, there is no information in the literature about the mitochondrial metabolism of Helicobacter-activated B cells. The main aim of this study is to elucidate the mitochondrial metabolism of Helicobacter-infected B cells. For this purpose, B cells were magnetically isolated from spleens of C57BL6 mice and treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Afterwards, cells were collected at respective time points for mitochondrial mass and membrane potential staining by using Mitoview Green and Mitoview 633 or TMRE dyes respectively in the flow cytometry. The supernatant of these cells is used for the IL-10 ELISA experiments for checking their IL-10 secretion. H. felis, PAM3CSK4, and LPS-stimulated B cells increased their IL-10 production most noticeably at 24h and 48h indicating the suppressive capacity of that cells. Also, compared to the unstimulated control group, all of the stimulant groups increased both the mass and membrane potential of mitochondria at 24h and 48h time points. The second aim of our study was to investigate whether B cells with high mitochondrial membrane potential (Mitoview 633+ B cells) produce IL-10 or not. For that, after B cells were isolated from IL-10 GFP reporter (VertX IL10 egfp) mice, they were treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Mitochondrial membrane potential were analyzed by flow cytometry. Afterwards, at 6h, 24h and 48h we evaluated mitochondrial membrane potential of the IL-10 producing B cells with quadrant analysis using flow cytometry. At 6h time there were no significant changes on IL-10+ Mitoview 633+ B cells. But at 24h and 48h time points; H.felis, PAM3CKS4, and LPS-stimulated B cells increased IL-10+ Mitoview 633+ B cells. These data show that in all stimulated B cell groups; high portion of the IL-10 producing B cells also have high mitochondrial potential. Our data shows, H. felis, PAM3CSK4, and LPS-stimulated Mitoview 633 + B cells increased their IL-10 GFP signal both at 24h and 48h time points compared to the unstimulated control group. The third aim of our study was to investigate mitochondrial biogenesis markers: mtDNA:nDNA ratio and mitochondrial transcription factor A (Tfam) gene expression. To perform Q-PCR experiments isolated and treated B cells were collected for DNA and RNA isolation at 6h, 24h, and 48h. For mtDNA:nDNA ratio analysis in Q-PCR; respectively cytochrome c oxidase subunit I (COX1) and 18S ribosomal subunit gene (RPS18) were targeted by using gene-specific primers. While H. felis, PAM3CSK4, and LPS-stimulated B cells decreased their mtDNA:nDNA ratio, they increased Tfam expression level compared to the unstimulated control group. This study showed that H. felis-activated B cells have active/functional mitochondria, and increased oxidative phosphorylation for energy production and their IL-10 production can be related to their mitochondrial metabolism.
Comparative whole genome sequencing and bioinformatic analysis of afreeze-thaw stress-resistant, industrial Saccharomyces cerevisiae strain

(Graduate School, 2022) Şimşek, Burcu Tuğba ; Çakar, Zeynep Petek ; 737560 ; Molecular Biology - Genetics and Biotechnology Programme

Yeasts have been around for thousands of years; they have benefited people in many fields such as science, medicine, food and agriculture. In particular, Saccharomyces cerevisiae is used in multi-enzyme pathways for the expression of protein biocatalysts and to synthesize chemicals and small molecular weight compounds important for medicine and nutrition. Due to these advances, S. cerevisiae is currently the primary model organism for the study of eukaryotic biology and human diseases. S. cerevisiae is a unicellular eukaryote. It has 16 chromosomes with subcellular organelles containing and these organelles commonly found in eukaryotes. S. cerevisiae has a classical eukaryotic cell cycle (including G1, S, G2, and M). Different strains of S. cerevisiae have been established to fill the gaps and requirements in genetic, biochemistry and physiology research. The CEN.PK family is frequently used in industrial biotechnology research, while the BY strain family derived from the S288c strain is mainly used in genetic studies. Yeast contains a large number of orthologous genes in the human genome. By examining the expression of some genes in yeast, the mechanism in more complex eukaryotes can be understood. S. cerevisiae has highly developed homologous recombination and contributes to the basic knockout operation of genes. Furthermore, S. cerevisiae is an important model for understanding the role of stress response genes in living organisms. S. cerevisiae cells can experience different environmental stress conditions such as metal toxicity, heat or cold shock during growth, essential nutrient limitations, hyperosmotic or hypoosmotic pressure, and ethanol toxicity. To overcome these stress conditions, S. cerevisiae cells have been developed to detect stress signals and respond to these signals through general or specific stress response and protection programs. Cryopreservation is a long-term storage method of various living cells, and the freeze-thaw tensile strength is important in cryopreservation. However, this method includes freezing and thawing processes that cause fatal damage to cells. Under freeze-thaw stress conditions, cells are exposed to more than one type of stress. These are; cold during freezing, dehydration, osmotic, ice crystal formation and oxidative stress during thawing. Therefore, it is important to obtain freeze-thaw tolerant organisms and to examine all freeze-thaw tolerance mechanisms. Yeasts are organisms that have a high survival rate when rapidly frozen at -80 °C. However, it is usually applied to commercial products at -20 °C and is highly damaging to cells, predominantly lethal to cells. Applications of freeze-thaw stress in S. cerevisiae are concerned with inducing this cross-resistance to overcome the effects of freeze-thaw stress. Additional mechanisms at gene expression levels are thought to be triggered and maintained during freeze-thaw exposure to achieve multiple stress tolerances and freeze-thaw stress tolerances. Metabolic engineering; it is defined as enhanced production of metabolites and cellular activities. It is done with through manipulation of the enzymatic, transport and regulatory functions of the cell by modifications of cellular networks including metabolic, gene regulatory and signaling networks using recombinant DNA technology. Metabolic engineering strategies can be divided into two groups as rational engineering and inverse metabolic engineering. Evolutionary engineering is a common strategy used in biological research to achieve the desired phenotype by improving its properties such as high environmental tolerance and improvement of product yield. Evolutionary engineering differs from metabolic engineering in that it is based on random methods; genetic modifications are not directed. Ploidy is the number of complete sets of chromosomes in a cell, which means the number of possible alleles for autosomal and pseudoautosomal genes. Many eukaryotic creatures have two sets of chromosomes (diploid) or more than two sets of chromosomes (polyploid). During the evolution of plants, animals, and fungi, ancient whole-genome duplication (WGD) or hybridization events frequently result in diploid and polyploid conditions. Increased chromosomal sets, development, cellular stress, disease, and evolution all cause polyploidy. Yeasts, which belong to the kingdom of fungi, can exist in both haploid and diploid forms. Polyploid yeasts, on the other hand, are widespread. Allopolyploid cells are formed when two or more cells from closely related but not identical species fuse together. Euploidy refers to the stance in which cells have a chromosomal number that is an integral multiple of the characteristic circum haploid number. Due to the common occurrence of polyploidy and aneuploidy in yeast, variable chromosome numbers elicit characteristics that may be beneficial in specific circumstances. As a result, the physiology and fitness of cells with different ploidy levels may differ. Bioinformatics is a highly interdisciplinary field that drives knowledge discovery from biological data using computational analysis. Today, bioinformatics is becoming an important part of most life science research. The process by which the DNA sequence of gene expression is copied into a gene product or RNA is explained by the central dogma of molecular biology. Microarray and more recently RNA sequencing; it has been widely used to measure gene expression levels. In this thesis, ploidy and genomic differences between the industrial Saccharomyces cerevisiae strain R625 and the freeze-thaw resistant evolved strain P8 obtained from R625 by evolutionary engineering were analyzed to gain insight into the complex molecular mechanisms of ploidy and freeze-thaw stress resistance.
Expression, purification and characterization of high-fidelity DNA polymerase

(Graduate School, 2022) Türk, Kübra ; Doğanay Dinler, Gizem ; 783309 ; Molecular Biology-Genetics and Biotechnology Programme

DNA polymerases found in all living cells discovered to date are the enzymes that synthesizes a new DNA strand complementary to template single stranded DNA. These enzymes do not only play a major role in the transmission of genetic information across generations during cell division, they also form the basis of Polymerase Chain Reaction (PCR), which is one of the most important in-vitro diagnostic techniques today. In addition to synthesis ability, DNA Polymerases may also have other properties including processivity, which is known as the ability of continuous polymerization, fidelity, which is known as the synthesis accuracy, and nucleotide selectivity. Thermostable DNA polymerase enzymes are mostly preferred in PCR-based studies because it is high importance that the stability of the enzymes used do not decrease depending on temperature. Taq DNA polymerase is the first discovered polymerase, which is a well-known enzyme used in a wide range of applications. Following the discovery of Taq DNA polymerase, the high-fidelity DNA polymerase was discovered in 1991 as a highly thermophilic DNA polymerase. Due to its high thermostability and proofreading properties, high-fidelity DNA polymerase is widely used in the applications that require high accuracy such as molecular cloning. High-fidelity DNA polymerase is an enzyme with a length of 775 amino acids and a molecular weight of about 90 kDa. This enzyme can perform 3'-5' exonuclease (proofreading) activity, which allows the addition of the correct nucleotides by removing the wrong nucleotides added to the structure during DNA synthesis. Due to this feature, it reduces the error rate during synthesis (1.3×10-6 mutations/base pairs/duplications), resulting in about 8 times less errors compared to Taq DNA polymerase. Many researchers have produced this protein by cloning it from Pyrococcus furiosus, a hyperthemophilic archaea, into different strains of Escherichia coli. The purification step is simplified by adding an affinity tag to the N- or C-terminus during the cloning. Based on these tags and various biophysical properties of the protein, purification protocols were created by affinity chromatography or ion exchange chromatography. In this study, we aimed to purify and characterize the high-fidelity DNA polymerase enzyme by taking the advantage of its thermal stability and 10X Polyhistidine-tag after bacterial production with high efficiency and low cost. For this purpose, commercially purchased pET16B. High-fidelity polymerase's plasmid DNA with a 10X Polyhistidine-tag at the N-terminus was used. The plasmid pET16B. High-fidelity polymerase was transformed into competent E. coli BL21(DE3) cells containing GroEL/GroES chaperonins to ensure soluble expression of the protein. In the first step of purification of High-fidelity DNA polymerase, which is a thermostable protein, all the folded proteins obtained from bacterial cells were heated and centrifuged to separate impurities with less thermal stability. High-fidelity DNA polymerase in soluble form was purified using IMAC affinity chromatography. The pure product was taken into a storage buffer containing 50% glycerol by filtration. The GroEL/GroES chaperonin system is a system that enables unfolded proteins with a molecular weight of 2-100 kDa to be folded in vitro and in vivo. Given that GroEL/GroES system can increase the folding of co-expressed recombinant proteins of different sizes by up to 70%, this system was employed in the production of High-fidelity DNA polymerase. However, while this system increases the amount of target protein, it can also increase the amount of impurities. Therefore, the purification of High-fidelity DNA polymerase was highly challenging. So that, various buffer compositions were used in order to optimize one step IMAC purification. Co-expression system was induced using IPTG for expression of pET16B. Expression of the polymerase regulated by the Lac operon. Since the growth temperature was chosen in the range of 12-20°C, where the metabolic rate of the cell and thus the growth rate was selected, the amount of protein folded by the chaperones was increased. Most of the impurities that increased with the target protein were eliminated with the 90°C heat treatment step. While heat sensitive proteins are eliminated from the environment, thermostable high-fidelity DNA polymerase enzyme, GroEL, and GroES proteins are still present. The separation of GroEL and GroES was achieved by applying IMAC affinity chromatography to increase the purity of the high-fidelity DNA polymerase enzyme. Purification results were analyzed by SDS-PAGE and immunoblotting methods. At the end of 500 ml bacterial production and purification process with three biological repetitions, high-fidelity DNA polymerase enzyme of similar quality with its commercial counterparts was produced, which can be used for a total of 60 000 PCR reactions with ~90% purity. The protein band on the SDS-PAGE gel was excised and analysed by peptide mapping using Liquid Chromatography-Mass Spectrometry (LC-MS) system to confirm that the produced protein is the target protein. According to the analysis, it was concluded that the purified protein was the target DNA polymerase. In order to determine if the purified protein is correctly folded or not, the secondary structure analysis of the protein with a purity over 90% was performed using Circular Dichroism (CD) in the far-UV (<260 nm) range. As a result of the study, the protein showed an apparent α-helix secondary structure with two minima at 208 and 222 nm wavelengths and a maximum at 190 nm wavelengths. Functional analysis of the protein on its folded state was completed by performing the Polymerase Chain Reaction (PCR). The 825 bp DNA region with 49.6% G-C content, and 1947 bp DNA region with 61% G-C content was amplified. These regions were selected considering the processivity of the enzyme. No-template control was used as negative control. The amplified regions were analyzed comparatively with commercial enzymes and it was observed that the target regions were successfully amplified. Commercially available high-fidelity DNA polymerase enzymes do not have endonuclease contamination and exonuclease contamination. Within the scope of quality control experiments, both endonuclease and exonuclease contamination of three biological replicates of our high-fidelity DNA polymerases were compared with commertial ones. λ DNA and λ DNA digested with HindIII were used as positive control. As a result, it has been shown that commercial enzymes and our high-fidelity DNA polymerase enzyme do not have endonuclease and exonuclease contamination. Another important test for demonstrating the quality of commercial enzymes is testing whether the protein remains stable under different conditions. For testing the stability of the polymerase, the effect of freeze-thaw stress repeated 20 times and also the effect of incubation of the enzyme for 5 days at room temperature were evaluated. As a result of these experiments, it has been shown that the freeze-thaw process during the general use of purified enzyme does not cause a negative effect on the activity of the protein, and that if the purified enzyme is forgotten at room temperature for up to 5 days during use, they can polymerize without reducing their activity. In conclusion, with this study, we have produced high-fidelity DNA polymerase with the same stability and processivity as commercial polymerases produced by large biotechnology companies with high efficiency and purity.
Investigation of protonation state dependent conformational dynamics of the nucleotide binding domain of Hsp70 protein homolog DnaK via computational methods

(Graduate School, 2022) Uçar, Umut Çağan ; Balta, Bülent ; 771654 ; Moleküler Biyoloji-Genetik ve Biyoteknoloji Bilim Dalı

70 kDa heat shock proteins (Hsp70) are a ubiquitious and well-conserved protein family with chaperone functions. They supervise protein folding process and assist in proper protein folding and renaturation by preventing partially folded or misfolded proteins from forming amorphous aggregates and amyloid fibrils. In terms of structure, Hsp70s are composed of approximately 45 kDa N-terminal nucleotide binding domain (NBD) with ATPase activity, 25 kDa C-terminal Substrate Binding Domain (SBD) with substrate peptide binding pocket. Also, there is a conserved hydrophobic linker segment connecting the 2 domains. Hsp70 proteins do not work alone. Rather, they need aid of some other proteins called "cochaperones" to work optimally. The two auxillary cochaperones required by the Hsp70s are the 40 kDa Hsp40s(J domain proteins) and nucleotide exchange factors (NEFs). Together with the cochaperones, Hsp70 proteins form 3-membered Hsp70 chaperone machinery. For Hsp70 chaperone system to carry out all of its functions, the NBD and SBD domains must communicate with each other during the functional cycle. This mutual signal transfer and crosstalk between the domains is an intricate example of "allosteric regulation". Basically, nucleotide status of the NBD exerts an effect on substrate affinity. Reciprocally, binding of a substrate protein to the SBD stimulates ATPase activity of the NBD. In ATP bound state of the NBD, the two domains are in docked conformation. On the other hand, in ADP bound or nucleotide free state of the NBD, these two domains are undocked and become independent of each other. In this undocked conformation, the two domains are connected to each other with the hydrophobic linker. The ATP-bound state of the NBD with docked NBD-SBD conformation binds and releases substrates at much higher rates than nucleotide free or ADP-bound state; thus, ATP-bound state of the Hsp70 is known as "low-affinity state" state with substrate binding pocket open. In contrast, when the NBD lacks any nucleotide or is bound by ADP, rate of substrate binding and release occur at way slower rates. This state of the Hsp70 proteins is called as "high-affinity state" with closed substrate binding pocket. Hence, Hsp70s shuttle between open and closed states. The hydrophobic linker is an undispensable component of the Hsp70 chaperone system. By functioning as a dynamic signal switch, the linker conveys messages in either direction, from NBD to SBD and from SBD to NBD, keeping both domains in touch. In literature, the linker, particularly the hydrophobic 388 VLLL 392 sequence, have been demostrated to be vital for both interdomain communication and dynamics of the ATPase domain. Likewise, there are many residues of the NBD playing key roles in the Hsp70 cycle, particularly in the mechanism of ATP hydrolysis. Especially, D8, K70, E171, D194, T199, and D201 residues of E.coli Hsp70 homolog DnaK and correspondants of these residues in other Hsp70 homologs have been given attention. In this study it was aimed to elucidate the conformational dynamics of the nucleotide binding ATPase domain (NBD) of E.coli DnaK in nucleotide free state by means of computational simulation techniques. First of all, so as to understand if protonation states of critical residues of interest have any effect on the opening-closure dynamics of the NBD in nucleotide free state, initially open and closed structures with different protonation states were prepared. By changing the protonation states of residues D194, D201, and H226 in distinct combinations, charge states +12, +13, and +14 were attained. Four protonation states, namely D194 protonated (194pr), D201 protonated (201pr), both D194 and D201 protonated (2pr), and both D194 and D201 unprotonated (2dep) were tested for both initially open and initially closed conformations. In each case, another residue of interest H226 was taken in protonated form. To broaden conformational space scanned and explore contribution of linker position to the dynamics of the NBD, positions of the linker were changed manually multiple times for each protonation state. According to our standard molecular dynamics (MD) simulation results (each 500 ns) of DnaK 1-392 construct of initially open conformations with these 4 protonation states, it was seen that protonation state can dramatically influence the tendency of initially open conformation towards closure. The cases in which D194 were unprotonated (2dep and 201pr) exhibited a tendency to close. Especially, 2dep became totally closed after 200 ns simulation period. On the other hand, 194pr had no tendency towards closure at all. On the other hand, all of the initially closed conformations for each of the 4 protonation states of the DnaK 1-392 retained their closed forms. Based on these simulation results, it can be deduced that the energy barrier between open and closed conformations was highest for 194pr and lowest for 2dep. In order to both decide on relative abundance of open - closed structures and circumvent sampling problems encountered during classical MD simulations, we carried out temperature replica exchange molecular dynamics (T-REMD) simulations for each protonation state. In addition to the 4 protonation states with protonated H226 residue investigated during MD simulations, 2 additional states, namely D194 protonated-H226 deprotonated (194prHID226) and D201 protonated-H226 deprotonated (201prHID226), were prepared by only removing the epsilon H atom of H226 from 194pr and 201pr cases, respectively. To elucidate whether the linker favors closed conformations, apart from these 6 different protonation cases with residues 1-392 of NBD, 6 further constructs were prepared by stripping the last 4 residues 389VLLL392 of the linker away from each of 1-392 construct. In total, 12 T-REMD simulations, 6 with 1-392 and 6 with 1-388 residues, and each with 300 ns-long were performed. Based on our T-REMD results, the most essential point to be understood is that closed conformations are much more favorable compared to open ones. Considering the ratios of open conformations, even the highest fractions of open conformations obtained in the case of 194pr388 did not exceed 25%. Looking at other cases, the percentage of open conformations can be as low as 2-3% for 201pr cases. If we look at 194pr cases, irrespective of the presence of last 4 residues of linker and protonation state of H226, open structures are most abundant in the cases of 194pr. Among 194pr and thereby all other protonation states-structures, 194pr388 (226 protonated) promotes, by far, the open structures most. Additionally, the role of the length of linker in favoring closed structures over open ones becomes more prominent when H226 is protonated with nearly 7% higher closed frames in 194pr392 than 194pr388. As opposed to 194pr, regardless of linker or protonation state of H226, 201pr cases are out and away the protonation states that exhibit lowest tendency towards opening with fraction of open structures no more than 5%. Unlike 194pr, there is no evident impact of linker on opening-closure behavior. Protonation state of H226 seemed not that important in either protonation state. In agreement with the standard MD simulations, 2dep was monitored to tend to have high number of closed frames. Indeed, 2dep comes after 201pr in terms of closed structure fractions. The contribution of the linker to the closure of the NBD was minor and seen only the last 200ns. As to 2pr states, the 2nd highest fraction of open frames, both in the presence and absence of 4 terminal residues of the linker, after 194pr were obtained. Another point of interest in this study was to scrutinize the pH dependence of DnaK ATPase domain. The active site of the ATPase domain comprises multiple charged amino acid residues; therefore, it can be expected from Hsp70 proteins to be susceptible to changes in pH conditions. To the best of our knowledge, only two experimental studies in early 2000's underlining pH dependent behavior of isolated DnaK NBD have been conducted thus far. On the other hand, no computational study has paid attention to change in the dynamics and activity of either full-length protein or NBD alone with respect to pH. Hence, which of the candidate residues in the active site are in protonated or deprotonated form around physiological pH remained elusive. To solve this mystery, thermodynamic integration (TI) simulations were performed, again for both DnaK 1-392 and DnaK 1-388. For TI simulations, for each of 12 protonation states used in the T-REMD, 2 initially closed structures alongside one open structure were picked from cluster analysis performed on the T-REMD trajectories. Additionally, pKa values of E171 and epsilon position of H226 were calculated in various protonation/deprotonation scenarios of D194 and D201. Regarding pKa values of epsilon H226, we inferred that pKa values were more or less the same around 9, indicating the fact that neither protonation states of active site aspartate and glutamate residues nor the lenght of the linker altered pKa values of H226. Protonation state of E171 is likely to depend on the protonation states of D194 and D201. When these aspartates were both deprotonated, E171 was protonated. D194, D201, and H226, on the other hand, must be in protonated states around physiological pH conditions. Consequently, according to our pKa values, we got +12 charge state, not +13 or +14. All in all, combining all atom MD simulations with REMD simulations and free energy TI simulations, the following questions were tried to be clarified in this thesis study: 1) Effect of protonation state on opening-closure abundance and dynamics of NBD 2)Whether the linker induces a change in the ratio of open to closed conformations. 3) pH dependence of the NBD through demonstration of the possible protonation/deprotonation status of critical residues 4) Whether linker promotes change in pKa of any of critical active site residues. At the end, we came to these conclusions. Dynamics of the DnaK NBD opening-closure were protonation state dependent. Intriguingly, protonation of 2 active site aspartate residues have opposite effect in such a way that protonation of D194 increases the abundance of open conformations, whereas D201 promotes more closed form of the NBD. In addition, at different charge states, there must be multiple protonation states in equilibrium. Regarding the linker, there was no direct indication of the linker being involved in opening/closure or pH dependence of the isolated DnaK NBD. Likewise, no apparent role of the linker in shifting the pKa of any critical residue in the active site.
Recombinant production and characterization of aquaporin protein isolated from geobacillus thermoleovorans ARTR1 and virgibacillus sp. agtr strains

(Graduate School, 2022) Uysalcan, Şevval ; Karagüler, Nevin Gül ; Genceli Ateş, Esra ; 521191138 ; Molecular Biology - Genetics and Biotechnology Programme

Cells have a fluid mosaic model that provides the passage of organic and inorganic molecules, ions and water, which are survival for the cell, that form the structure of cell membranes and contain protein, carbohydrates and cholesterol. Proteins located in the membrane of the cell are called biological membrane proteins and they constitute approximately 25-30 % of all proteins. However, the partially hydrophobic surfaces of membrane proteins are still a largely unconquered area due to their lack of flexibility and stability. Biological membrane proteins have three subgroups depending on their location are analyzed in category. Integral membrane proteins, one of the biological membrane proteins which are permanently attached to the cell membrane. Detergents, non-polar solvents, or sometimes denaturing agents are used to separate this protein from the biological membrane. Integral membrane proteins are a permanent part of the membrane and can penetrate the membrane to form transmembrane proteins. Peripheral membrane proteins, another biological membrane protein, temporarily bind to the membrane or integral membrane proteins by hydrophobic, electrostatic, and other non-covalent interactions. These proteins can dissociate from the membrane after treatment with a solution containing a high pH or high salt concentration. Lipid-anchored proteins, on the other hand, are proteins on the surface of the cell membrane that covalently bind to lipids embedded in the cell membrane. Today, the structural and functional studies of the above-mentioned biological membrane proteins have been largely hampered by the difficulties in their production and overexpression, which are necessary for the structural studies of membrane proteins. Compared to other protein classes, the determination of the structure, expression, and purification of membrane proteins is greatly hampered by difficulties. Aquaporin, a biological membrane protein of great medical and industrial importance, are integral proteins that form pores in cell membranes and facilitate the transport of water between cells. This protein is the membrane protein that allows the passage of water molecules and rejects all other solutes. In the literature, there is a number of researches on the discovery of aquaporin from different cell membranes.While aquaporins, which are at the forefront of biomimetic membrane production in prokaryotic cells in the industrial area, are known for the passage of molecules that provide the vital conditions of the cell in plant cells and their necessity during some stress conditions (drought), in mammalians aquaporins are associated with many diseases, especially leukemia. The biggest difficulty encountered during the studies carried out in this direction is the obstacles in ensuring the overexpression of the integral biological membrane protein aquaporins and the optimization of their purification afterwards. Aquaporins have a similar basic structure: aquaporin monomers consist of six transmembrane helical segments and two short helical segments surrounding cytoplasmic and extracellular vestibules connected by narrow aqueous pore. Between the helices are five regions (A-E) with an asparagine-proline-alanine ("NPA motif") pattern, two of which are hydrophobic (B, E) that fold in or out of the cell membrane. Another part of the channel is the "ar/R selectivity filter", a set of amino acids that allows aquaporin to selectively allow or block the passage of different molecules. Aquaporin monomers can assemble as tetramers in membranes, with each monomer functioning independently. The primary function of most aquaporins is to transport water across cell membranes in response to osmotic gradients created by active solute transport. Aquaporins provide a 10-100-fold increase in water transfer across the cell membrane. Because of their unique properties, they need to be produced in large quantities for different purposes, including structure and activity analysis or new biomimicry materials. Different species of aquaporins may have different permeability and rejection properties for the same solute. While aquaporin may vary depending on solute concentrations, the selectivity of aquaporin also varies depending on load and size. It creates a positively charged energy barrier due to the amino acid Arginine in its structure. In this way, aquaporin prevents the passage of loaded or neutral solvents. Over the past 5 years, there has been great interest in the biology of aquaporins (AQPs) in over 500 studies published on aquaporin cloning, genetics, tissue localization, developmental and regulated expression, transgenic mouse models, and structure/function analyzes. When aquaporins, which are found in all cells from bacteria to mammals, are investigated in the literature, it is seen that mostly AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8 species are water specific. AQP3, AQP7 and AQP9 are called aquaglyceroporins and also carry glycerol and/or other small uncharged molecules. Most studies on recombinant aquaporins have so far been functional, regulatory or structural studies of aquaporins. In addition to these studies, the application of aquaporins with high water permeability and high solute rejection with biomimetic membranes for water desalination and reuse has attracted great interest in recent years. Prokaryotic aquaporins were first identified in Escherichia coli, and microorganisms such as Rhodobacter sphaeroides, Lactococcus lactis, Saccharomyces cerevisiae, Mathanothermobacter marburgensis, Photobactetrium profundum SS9 and Halomonas elongate have been used in studies to purify aquaporin protein. Also the recombinant production of aquaporin is being studied by using cell-free expression systems. Studies with prokaryotic AQPs show that bacteria help cope with osmotic, oxidative stress and nutritional fluctuations. However, there are limited studies in the literature for aquaporins produced from prokaryotic organisms and especially prokaryotes under extreme conditions. Virgibacillus sp. AGTR strain isolated from Acıgöl (Burdur, Turkey), which is a lake with high salinity has taken its place in the literature as a new halophilic bacterium strain. The genome information of this organism was obtained, and it was determined that it contains two aquaporin-encoding genes, one of which is 831 bp and the other 795 bp. Also, a new thermophilic bacterium Geobacillus thermoleovorans ARTR1 was isolated from another extreme environment, Armutlu (Yalova). The gene sequence encoding aquaporin with a length of 819 bp was determined in this organism. To analyze the stability of extremophilic aquaporins in industrial processes requiring harsh conditions (high temperature, high pH), in total, three extremophilic aquaporin genes were isolated from both halophilic Virgibacillus sp. AGTR and thermophilic Geobacillus thermoleovorans ARTR1 strains. These isolated genes were transferred to the pET28a (+) vector and produced recombinantly in the Escherichia coli C43 host cell. In this process, genes related to the PCR method with appropriate primers were amplified and ligated with a pET28a (+) vector. IPTG induction was subjected to time, temperature, and molarity trials, respectively. Then the optimal condition of 1 mM IPTG, 37 °C and 4 hours induction was selected. Under this optimal condition, gene expression was visibly achieved. Since it was observed that the protein remained in the inclusion body formed in the centrifuge after sonication, it was continued with the pellet after centrifugation and examined by SDS-PAGE in the control supernatant. After this process, the protein was obtained purely by various His-tag purification methods. Proteins expected to appear at an average size of 29 kDa were confirmed by SDS-PAGE and Western Blot Analysis. Liposomes are small artificial vesicles of spherical shape that can be formed from cholesterol and non-toxic natural phospholipids. Because of their size and hydrophobic and hydrophilic character (as well as biocompatibility), liposomes are promising systems for drug delivery. Liposomes can mimic the cell membrane. Different lipid compositions can be used to obtain the desired liposome type. DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), an example of Phosphatidylcholines (PC), was used in this study. The proteoliposome consists of liposomes containing aquaporin. For 2.5 mg/ml DOPC lipid, aquaporin with a concentration of 0.5 mg/ml was dissolved in 10 ml of PBS and 0.05M OG detergent was added to obtain a proteoliposome, and aquaporin-free liposome was obtained. Control liposome and proteoliposome were treated with 0.85M NaCl and the water transfer rate of aquaporins was measured with a steady flow light scattering spectrometer device. Here, it was determined that the water passage of liposomes with aquaporin was faster. Aquaporin-bearing proteoliposomes lose water at a much higher rate than control liposomes. This was explained as an indication that aquaporin obtained from the Geobacillus Thermoleovorans ARTR1 strain was working. The identification and characterization of novel aquaporin proteins from both halophilic and thermophilic bacteria within the scope of this project are of great importance in terms of their contribution to the literature.
Investigation of the effect of valproic acid, an hdac inhibitor, on the relationship between oxidative stress and autophagy in human eosinophil

(Graduate School, 2022) Turan Üzel, Göksu ; Çıracı, Ceren ; 521181126 ; Molecular Biology-Genetics and Biotechnology Programme

Epigenetics bridges the gaps between phenotype and genotype. Any process that modulates the gene activity without causing any change in DNA sequence is called epigenetic modification. Epigenetic modifications are divided into three main groups: histone modifications, DNA methylation, and non-coding RNAs. These changes are reversible because they do not alter DNA sequence. However, dysregulation in modifications can lead to several diseases such as cancer, metabolic, and neurological disorders. Inhibitors that suppress modifications such as DNA methylation, histone deacetylase, histone acetyl transferase, and protein methyltransferase have been developed to reverse the epigenetic processes that drive the pathogenesis of diseases and are used as epigenetic drugs for treatments. One of the most widely used inhibitors in therapy is histone deacetylation inhibitors (HDACi). Valproic acid (VPA), a branched short-chain fatty acid obtained from valeric acid, is one of the drugs used as HDACi. VPA is generally used as an anticonvulsant drug for the effective treatment of different types of diseases such as epileptic seizures, migraine headaches, bipolar disorder. In addition, VPA has the promising potential to be effective in cancer therapy and controlling allergic responses because it can mediate the expression of important genes involved in anti-tumor immunity and activation of cells. Nevertheless, even when VPA is used therapeutically, studies have revealed that VPA induces oxidative stress, and can activates autophagy pathway, inflammasome response in different cells as a side effect. In this thesis, our aim is primarily to investigate the effects of valproic acid-induced stress on the activation of the antioxidant pathway, inflammasome complexes, and autophagy pathways that maintain cell balance in Eol-1 cells. Eol-1 human eosinophilic cells were used in this study since eosinophils have a key effector role in the response to allergic reactions. When the balance of reactive oxygen species (ROS) production in the cell is disrupted, oxidative stress arises in cells. ROS are highly reactive molecules and, elevated levels of ROS can damage cell compounds. Therefore, regulation of reactive oxygen species (ROS) generation is crucial for the proper functioning of cells. To maintain homeostasis, cells develop a series of antioxidant responses to reduce the toxicity caused by oxidative stress. In this case, the main regulator in the cell is the antioxidant transcription factor nuclear factor erythroid 2-associated factor 2 (Nrf2). Under normal conditions, Nrf2 interacts with Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and leads to degradation of Nrf2 by the 26s proteasome, thus it is a negative regulator of Nrf2. Oxidative stress causes a conformational change in Keap1, leading to the dissociation of Nrf2 and Keap1. This enhances the acetylation and nuclear translocation of Nrf2. Proteins which transcribed by Nrf2 are involved in the activation of both anti-inflammatory and pro-inflammatory pathways in the cell to inhibit excessive immune responses caused by oxidative stress. Our results showed that VPA stimulation increased the cellular ROS formation in a dose-dependent manner at 24 h post-stimulation without any change in viability of Eol-1 cells. Moreover, the activation marker CD69 was upregulated via VPA treatment in dose-dependent manner. At lower concentrations, VPA augmented the protein level of Nrf2 and acetylated Nrf2, while attenuating the protein level of Keap1 in a dose-dependent manner. The immune system represents cells, tissues, organs, and components that come together to form a defense network to fight various pathogens or diseases by the excessive reaction and invasion of the microorganisms and to maintain the homeostasis of the host. The immune system consists of two arms: innate immunity and adaptive immunity. The first defense against pathogens is provided by innate immunity and white blood cells such as granulocytes (basophils, mast cells, eosinophils, neutrophils), monocytes, and Langerhans cells which are the main effector cells in innate immunity. The innate immune response is controlled by membrane/cytoplasmic receptors, inflammatory proteins, secreted cytokines, and chemokines. Pattern recognition receptors (PRRs) are mainly categorized as Toll-like receptors (TLR), NOD-like receptors (NLR), C-type Lectin (CLR) and RIG-I-like receptors (RLR). They can recognize different pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). Ligand-receptor interaction initiates inflammatory responses. The major canonical inflammatory responses are driven by NLRP3, NLRC4 inflammasome complexes against microbial structures and danger singals such as bacteria, mitochondrial ROS, and ATP. The NLRP3 and NLRC4 proteins combine with ASC and caspase 1 separately to form complexes. Activated caspase-1 cleaves pro forms of IL-1B, IL-18, and gastermin D (GSDMD) into mature forms. Firstly, GSDMD oligomerizes, then migrates to the membrane to form pores, so mature cytokines can be released from the pores. Cell membrane integrity is disrupted, and cells eventually die; this process is called pyroptosis. The release of cytokines and chemokines induces the migration and activation of many inflammatory cells, which is important for the proper immune response in microbial defense. According to our findings VPA treatment reduced the protein level of NLRC4 in a dose dependent fashion, but interestingly NLRP3 and caspase-1 cleavage, cleaved IL1β protein levels were elevated at lower doses in Eol-1 cells. IL-1B cleavage increased in parallel to the activity of caspase-1 and the inflammasome complex. Although cleaved IL1B was increased intracellularly, IL-1B and IL-10 secretion via VPA stimulation did not significantly change as compared to non- treated group. Autophagy is a mechanism responsible for maintaining homeostasis in the cell under various stress conditions. Cells remove damaged and unnecessary cell components through lysosomal degradation in response to cellular stresses such as nutrient deficiency or high levels of reactive oxygen species (ROS). As the most studied autophagy type, in macroautophagy, cytoplasmic components merge with the lysosome with the help of vesicles called autophagosome, forming autolysosome structures and the components inside the autophagosome are degraded. Various genes/proteins and pathways are involved in the maintenance of autophagy. The MAPK (mitogen-activated protein kinase), Akt (alpha serine/threonine-protein kinase) and mTOR (mammalian target of rapamycin) pathways are in the upstream of the autophagy pathway and are responsible for the regulation of cell growth, cellular metabolism, cell survival, and proliferation of cells. ERK1/2 is phosphorylated in the presence of autophagosome and thus it can be used as a positive marker in the autophagy pathway. Conversely, Akt and mTOR pathways negatively regulate the autophagy pathway. Phosphorylation of the Akt signaling pathway affects activation of the mTOR pathway as mTOR is downstream of Akt. In addition to these, microtubule‑associated protein light chain 3 (LC3) is the main marker in the autophagy flux. The cytosolic form LC3-I is converted to the LC3-II form through autophagy activation. LC3-II binds to the inner and outer membrane of the autophagosomes and preautophagosomal structure (PAS). Also, Beclin 1 is used as a marker for autophagy activation because it is crucial for the autophagosome structure and suppresses mTOR activity while inducing ERK activity. Our findings indicated that following VPA treatment, p44/42 MAPK (ERK1/2) protein increased at ascending doses, and phosphorylation of p44 MAPK was upregulated at lower doses. On the other hand, while the protein level of Akt didn't change after VPA, phosphorylation of Akt on both serine 473 and threonine 308 was downregulated via VPA stimulation. Moreover, the mTOR protein levels and the phosphorylation of mTOR decreased at high VPA doses, while protein levels of LC3B II was increased by VPA stimulation. However, increasing concentrations of VPA decreased the Beclin-1 protein level. Our results suggest that VPA stimulation induced the autophagy pathway independently of Beclin1in Eol-1 cells. Based on our current data, VPA led to oxidative stress by increasing ROS production levels and activated the antioxidant transcription factor NRF2 in Eol-1 cells. Also, VPA activated Eol-1 cells. VPA induced inflammasome complex formation without any change in the secretion of proinflammatory cytokines. Finally, VPA activated the autophagy pathway independently of Beclin1 in Eol-1 cells. Our results demonstrated the side effects that should be considered in the therapeutic use of VPA. In addition, the fact that VPA increases the activation of Eol-1 cells raises the question of whether VPA can not be used as a drug in allergic responses since it is not desired to increase the severity of the reaction in allergic responses.
Expression, purification, and characterization of recombinant human IL-2

(Graduate School, 2022-01-18) Akgün, Buse ; Doğanay Dinler, Gizem ; 521181103 ; Molecular Biology – Genetics and Biotechnology

Cytokines, which are small proteins secreted by the immune system, are in charge of directing the immune system. Through their formation, differentiation, and activation functions, cytokines govern the maintenance of innate and adaptive immune responses. They are primarily formed by mononuclear phagocytes, dendritic cells, and antigen-presenting cells. Interleukin (IL) is a kind of cytokine that acts as an immunomodulatory protein. It induces a variety of cell and tissue responses. Interleukins mediate the interaction of leukocytes (white blood cells) and initiate a response by attaching to high-affinity receptors on the surface of the cells. They play a critical role in the regulation of cellular formation, differentiation, and activation that occurs over the course of inflammatory and immunological responses. Each family is assigned an IL based on sequence homology, receptor chain similarity, and functional qualities. Interleukin-2 (IL-2) was the first cytokine discovered to stimulate the growth of T lymphocytes. T cells, B cells, natural killer (NK) cells, lymphokine-activated killer cells, and macrophages all require IL-2 to regulate their proliferation and differentiation. Mier et al. discovered the molecule and named it "IL-2" since it was produced by and acted on leukocytes. Its discovery is regarded as a milestone in immunology. However, there is one issue that is common to all lymphokines when it comes to the molecular and functional characterization of IL-2, and it is due to their production in small quantities. The cloning of cDNA for IL-2 was a significant turning point in 1983, precipitated by the discovery of IL-2. The Jurkat T cell leukemia cell line was employed for the IL-2 cDNA clone development. IL-2 is a 15.5 kDa glycoprotein that belongs to the cytokine family four α-helical bundles. There are 153 amino acid residues in a single polypeptide chain of IL-2. IL-2 binds to and communicates with a receptor complex composed of three different subunits known as IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2R (CD132). Different combinations of these three components bind to IL-2 with varying degrees of affinity. The αβγ heterotrimer, βγ dimer, and α chain monomer all bind to IL-2 with "high," "intermediate," and "low" affinity, respectively. Binding of IL-2 to the IL-2R heterodimer complex activates several pathways. In response to an interaction between interleukin-2 and its receptor, kinases connect to cytoplasmic areas of the receptor subunits, resulting in the tyrosine phosphorylation of many proteins and the activation of a number of signaling pathways, including JAK/STAT, PI-3K/AKT, and Ras/MAPK. IL-2 activity promotes cell survival, proliferation, cell cycle progression, and targeted gene transcription. Due to its ability to activate both T and NK cells, IL-2 was the first cytokine to be successfully used in cancer treatment. The US Food and Drug Administration authorized high-dose IL2 for the treatment of melanoma and renal cell carcinoma in xxii 1992 and 1998, respectively. Moreover, the use of recombinant IL-2 therapy may help researchers understand better the coronavirus disease 2019 (COVID-19), which is caused by a virus that leads to severe acute respiratory illnesses and has rapidly spread throughout the world. As a prospective treatment for this condition, the use of rIL2 may be beneficial for patients since it has the potential to accelerate disease recovery by increasing the number of lymphocytes in the body. A major difficulty is figuring out how to direct IL-2 activity toward Teffs and away from Tregs, which inhibit the immune system. IL-2 is available in two recombinant forms derived from E. coli, but only aldesleukin is FDA-approved. Recombinant IL-2 differs structurally from its natural version. IL-2 recombinant is not glycosylated and lacks N-terminal alanine. To avoid the formation of an incorrect disulfide bond, serine has been substituted with cysteine at amino acid position 125. The pharmacological actions of endogenous and recombinant human IL-2 are similar. In this study, E. coli Rosetta (DE3) was used as the host cell. Induction of protein expression was accomplished by the use of IPTG. Following that, inclusion bodies, which develop in the cell as a result of excessive protein expression, were separated and solubilized from cell lysates and refolded by step-wise dialysis. Anion exchange chromatography was used to separate the target protein from the rest of the protein mixture. After purification, the yield was determined to be 0.114 mg per liter of cell culture. SDS-PAGE and immunoblotting methods were used to validate the effectiveness of the purification. The molecular weight is estimated using intact mass analysis through LC/MS. The CE-SDS analysis revealed that rIL-2 has a purity of around 80%. In addition, the pI value of the protein was determined as 7.31 using the capillary isoelectric focusing method. The peptide mapping on LC-MS/MS is used to figure out the main structure of the protein that has been purified. The secondary structure of pure human interleukin-2 (hIL-2) was investigated using circular dichroism (CD), and the results revealed that it included a high concentration of alpha helices. The biological action of our IL-2 is determined by phosphorylation of one of the MAPK pathway proteins, extracellular signal-regulated kinase 1/2 (ERK), on human monocytic cells, THP-1. An active protein has been produced as a result of this work. The experimental results indicate that the procedures established for generating and purifying the rIL-2 protein may be employed to create a pure product that maintains its bioactivity.
DADA2 hastalarının periferik kan mononükleer hücrelerinde total ada aktivitesinin analizi

(Graduate School, 2022-02-09) Demirci, Turna ; Turanlı Tahir, Eda ; 521171122 ; Molecular Biology, Genetics, and Biotechnology

Deficiency of Adenosine Deaminase Type 2 is an autosomal recessive disease caused by biallelic mutations in the ADA2 gene. It was first defined as monogenic vasculitis syndrome in 2014 as a result of studies conducted by two different groups independently. Although it has been shown that the prevalence of ADA2 Deficiency maybe 4 in 100,000, the prevalence of the disease may differ between ethnic groups, depending on the degree of consanguinity and the presence of founding variants. Adenosine deaminase is an enzyme involved in the regulation of adenosine homeostasis and purine metabolism by converting adenosine to inosine and 2'-deoxyadenosine to 2'-deoxyinosine. There are two isoforms of adenosine deaminase in humans, and one of them, the 57-kDa homodimer ADA2 protein, is produced by the Adenosine Deaminase 2 (ADA2) gene. The N-terminal portion of the ADA2 protein is responsible for growth factor activity, while the C-terminal portion is responsible for adenosine deaminase activity. In addition to the catalytic domain, the ADA2 protein also has a protein dimerization domain and a cell surface binding domain. ADA2 proteins bind to different cell surfaces via glycosaminoglycan chains and to T cells via adenosine receptors. In this way, it shows both cytokine-like and autocrine-type growth factor properties. Although the ADA2 protein is involved in macrophage polarization, it also has an important regulatory function for neutrophil activation. In addition, it significantly reduces the formation of neutrophil extracellular traps, which are caused by extracellular adenosine and can lead to the activation of proinflammatory cytokines. Despite the clinical manifestations of DADA2 being very diverse, episodic clinical findings are usually observed in patients with fever and systemic inflammation. The most common type is vasculitis findings. In addition to dermatological and neurological symptoms, it is also rarely defined by renal involvement and gastrointestinal system findings. More than half of patients have attacks of non-infectious fever. Symptoms include recurrent oral and genital ulcers, musculoskeletal symptoms, recurrent abdominal pain, inflammatory bowel disease, and immunodeficiency. Hematologic findings include cytopenia, anemia, and rare bone marrow failure. The diagnosis of the disease is made based on the detection of pathogenic variants on the ADA2 gene or the measurement of ADA2 activity in serum/plasma. Treatment methods are selected depending on the symptoms and the severity of the disease. Currently, anti-TNF-α is the most common treatment modality, especially for patients with signs of vasculitis. Hematopoietic stem cell transplantation can be used in the treatment of hematological diseases. In addition, although it is not a suitable choice for long-term treatments, fresh frozen plasma infusions are also among the treatments applied. Enzyme-linked immunosorbent assay (ELISA) is a method used to detect and quantify protein in soluble substances, based on antigen-antibody interaction and measuring enzyme activity by colorimetric analysis. The purpose of this study was to compare the total adenosine deaminase (ADA) activity in peripheral blood mononuclear cells of patients diagnosed with DADA2 with the control group. 8 patients diagnosed with ADA2 deficiency and 5 healthy individuals were studied. Two of the patients are Syrian and have a G47R/G321E heterozygous mutation. 3 of the patients have G47R homozygous. Total ADA activity was measured in lysates prepared from subjects' peripheral blood mononuclear cells using a colorimetric ADA Activity Assay kit that is a commercial kit. ADA activity was calculated by following the protocol written in the kit, and then the statistical comparison of the results was analyzed by performing the t-test. The disease-causing variant p.G47R, which occurs in the dimerization domain, affects the stability of the homodimer required for enzyme activity of the ADA2 protein. Therefore, due to the decrease in ADA2 catalytic activity in patients with p.G47R mutation, it is expected that the total ADA activity will be lower than in the healthy group. As a result of statistical analysis, a significant difference was observed in ADA activity (p=0.0008). As expected, ADA activity was lower in the patient group compared to the healthy group. In addition, when patients with heterozygous mutations were compared with patients with homozygous mutations, lower ADA activity was observed in patients with heterozygous mutations. In this case, it can be said that the G321E mutation plays an important role in catalytic activity.
Preparation and characterization of PCL/lignin sponges for bone tissue engineering applications

(Graduate School, 2022-05-09) Elmalı, Gizem ; Kök, Fatma Neşe ; 521181108 ; Molecular Biology-Genetics and Biotechnology

Organs and tissues may be damaged by accident, congenital anomalies, diseases and similar reasons. Although living tissues have the capacity to heal these damages, large defects or severe tissue losses can only be repaired to a certain extent. Tissue engineering techniques have been developed to trigger, assist and accelerate the healing process, or directly replace the targeted tissue. In tissue engineering, the most suitable three-dimensional scaffold structure is produced to match the characteristics of the targeted tissue and provide the initial structural integrity and organizational backbone for cells to replace the damaged tissues and organs. Especially bone and its related diseases can cause a significant impact on a person's health and quality of life if they are not treated and healed properly. Millions of bone injuries have been occurring each year and they are not only affecting people's life but also cause to a serious burden on country's economies. Bone tissue engineering was presented as a solution to address these problems. In this thesis, a synthetic and natural polymer blend was used to create a scaffold that is suitable for bone tissue engineering. Polycaprolactone (PCL) was used as synthetic polymer, while alkaline lignin was used as natural polymer. Solvent casting, particulate leaching and freeze-drying methods were used to create porous sponge-like scaffolds. PCL and lignin samples were prepared at different lignin concentrations (0, 10, 15, and 20 mg/ml) while keeping the PCL concentration constant at 200 mg/ml, PCL200, PCL200/Lignin10, PCL200/Lignin15 and PCL200/Lignin20, respectively. A sample with PCL concentration of 100 mg/ml and lignin concentration of 20 mg/ml was also prepared to see the effect of PCL concentration (PCL100/Lignin20). For all scaffolds, except for the control sample, NaCl crystals were used as porogen. Lignin containing scaffolds were appeared more brownish than pure PCL scaffolds. Since the structural integrity of PCL100/Lignin20 sample was very poor, and PCL200/Lignin20 without the salt addition cannot be removed from the mold surface, they were not used in further experiments. Even though, mechanical properties of the scaffolds have not been studied in this thesis, based on preliminary visual and manual analysis rest of the PCL/Lignin type scaffolds were observed to be more flexible than PCL constructs. Water uptake analysis showed that there is no significant difference between the samples with different lignin concentrations. The water uptake behavior of the scaffold, however, was dramatically increased with the lignin addition. While the water uptake for PCL200 was 44%, it was 380-400% for the samples with lignin after 24 h. In Fourier-Transform Infrared Spectroscopy (FTIR) analysis, typical peaks for PCL are identified for all scaffold types. Even though special peaks for lignin could not be observed, a reduction in the intensity of a peak, which represent –OH groups, was determined in all PCL/Lignin scaffolds compare to the PCL200. This can be evidence of reactions and new bonds between PCL and lignin polymers. Since water uptake and FTIR analysis showed that there is no significant difference between different lignin concentrations, it was decided to continue with only one sample type, PCL200/Lignin10, for further experiments. The porosities of pure PCL200 and PCL200/Lignin10 determined as 73% and 76%, respectively using liquid displacement technique. Pore sizes were investigated with ImageJ software on SEM images. For PCL200 scaffold, 31% and 58% of the pores on the surface were in the range of 100-300 µm and 300-600 µm, respectively. For PCL200/Lignin10, 30% and 43% of the pores were in the range of 100-300 µm and 300-600 µm, respectively. In addition, many pores that are smaller than 20 µm were observed for both sponges. The melting temperature of PCL200/Lignin10 scaffold was %5.4 higher than PCL200 scaffold as determined by Differential Scanning Calorimetry. The increased melting point might be the result of a new bond between PCL and lignin. Hydrolytic degradation was not seen within 7 days for both samples, but enzymatic degradation in lipase presence was observed for both sponges. After 7 days, only 43% weight loss was recorded for PCL200/Lignin10, while PCL200 sponge was completely degraded after the fifth day. Lignin's natural resistance to enzymatic degradation or the modifications to form an alkaline lignin might be the reason of this unexpected decrease in degradation. Biomineralization process was triggered by soaking scaffolds to modified-simulated body fluid solution. The mineralization rate was analyzed by SEM. SEM micrographs revealed significant mineral accumulations for both scaffold types at the end of the 7th day. When the 7th day samples were compared, it was seen that the hydroxyapatite-like structures were mostly deposited on top of each other in the PCL200 sample, while the aggregations were more evenly spread over the surface on the PCL200/Lignin10 sample. Furthermore, even though an intense accumulation was observed on the scaffold surface of the PCL200, the inside of the pores as appeared to be smooth. Lignin containing samples, on the other hand, had more mineral deposition within the pores. The elemental analysis of the formed hydroxyapatite-like structures was analyzed by EDX. It was observed that the Ca/P ratio of the accumulations on the PCL200/Lignin10 samples at all determined times was closer to the ideal hydroxyapatite Ca/P ratio (1.67). Finally, cell proliferation assay was performed with hFOB cells to examine the effects of lignin addition on cell adherence and proliferation on the surface. The proliferation of hFOB was significantly higher on PCL200/Lignin10 scaffolds compared to PCL200. The proliferation on PCL200/Lignin10 sponge was found to be 212% and 50% higher after day 3 and 7, respectively. Further studies are necessary in order to evaluate the usability of PCL200/Lignin10 sponge scaffolds in bone tissue engineering. However, the findings obtained within this thesis suggest that this blend is promising candidate for bone tissue engineering.
Investigation of familial multiple sclerosis genetics

(Graduate School, 2022-05-16) Everest, Elif ; Turanlı Tahir, Eda ; 521152104 ; Molecular Biology-Genetics and Biotechnology

Multiple sclerosis (MS) is a chronic, neuroinflammatory, neurodegenerative disease of the central nervous system. Several lines of evidence have shown that the primary pathophysiological mechanism of MS is the infiltration of autoreactive lymphocytes through the blood-brain barrier, attacking central nervous system components such as myelin and resulting in oligodendrocyte death. This process has been thought to be responsible for axonal pathology and neuronal loss, which result in progressive neuronal dysfunction in some patients. Over the recent years, the roles of astrocytes, microglia, and pericytes have also been increasingly shown in MS pathology. To date, several studies have revealed disease-related cellular pathways that emphasize the different pathological components of the disease; however, underlying mechanisms in MS development and progression are yet to be elucidated. Consistently with its heterogeneous clinical presentation and complex pathophysiology, MS also has a complex inheritance pattern and develops in genetically susceptible individuals under environmental influences. Many studies have been carried out using different approaches and methods to identify genomic regions and variants that cause genetic predisposition to MS, identifying hundreds of common variants as well as candidate rare variants that increase the risk of MS. Today, MS associations of 233 common variants, as well as hundreds of suggestive associations, have been identified. However, all significant common variants, together with the suggestive effects, can cumulatively explain approximately half of MS heritability. Meta-analyses have shown that rare variants can further explain up to 5% MS heritability, still leaving a large proportion of MS genetics unknown. In this thesis study, it was aimed to reveal novel information on MS genetics and pathogenesis. Multiplex MS families with more than two affected family members were collected to identify possible novel genes that contribute to the high MS aggregation in these families. Seven multiplex MS families with the highest number of affected individuals and parental consanguinities were selected, and SNP genotyping (710K or 2.5M, Illumina) was performed (N=41). Candidate MS-associated genomic regions were identified through linkage analysis and homozygosity mapping. Exome sequencing (N=56) revealed that there were no fully penetrant, homozygous, rare, exonic variants segregating within the families. However, two variants were found to be segregated with the disease with an autosomal dominant inheritance pattern in the LRRC6 gene (rs139131485) in family FMS01 and RNF217 (rs73580047) gene in family FMS05, which may increase the risk of MS in corresponding families. Additionally, many incompletely penetrant, rare and low-frequency variants were identified. Subsequently, a weighted sum score analysis including previously identified common MS-associated risk variants and polygenic risk score (PRS) analysis were conducted in MS families (24 affected, 17 unaffected), 23 sporadic MS cases, 63 individuals in 19 non-MS control families, and 1272 independent, ancestry-matched controls to determine whether an increased burden of known MS-associated common variants explain the increased MS risk in these families. Logistic regression analyses showed that familial MS cases had higher sum scores (OR=2.16, P=0.002; OR=2.4, P=0.014) and PRS (OR=1.84, P=0.0077; OR=2.27, P=0.049) compared with the population controls and control families, respectively. Moreover, affected individuals in the MS families had higher weighted sum score and PRS values compared with the unaffected family members; however, the differences were not significant after Bonferroni correction. When individual families were observed, it was seen that the higher sum score and PRS trends in MS cases were evident in only three of the families, and in others, there were no apparent differences in the sum score and PRS values between the affected and unaffected family members or the unaffected individuals had higher sum score and PRS values compared with their relatives with MS, further supporting the polygenic inheritance of MS. Sporadic MS cases had significantly higher PRS compared with both affected and unaffected individuals in MS families, control families, and population controls (P=0.02, P=0.0055, P=0.003, and P=0.0008, respectively), supporting the presence of higher rare risk variation loading in the familial cases. There was no significant difference in the sum scores of familial and sporadic MS cases, possibly due to the high degree of convergence between common and rare risk variation in significant loci for MS. As part of this thesis study, we also performed an integrated bioinformatic analysis using genomic and proteomic data of an unrelated MS group. For this, first, SNP genotyping (300K, Illumina) was performed for 11 unrelated MS cases selected from our MS family cohort whose cerebrospinal fluid samples had been previously included in our proteomic study, in which 2D-gel electrophoresis, mass spectrophotometry, and pathway analyses had been conducted, revealing 151 differentially expressed proteins between MS cases with different clinical MS phenotypes and non-MS controls. To integrate the genomic and proteomic datasets of this patient group to reveal the most relevant disease pathways, pathway enrichment analyses of MS-associated SNPs and differentially-expressed proteins were conducted using the functional enrichment tool, PANOGA. Nine shared pathways were detected between the genomic and proteomic datasets after merging and clustering the enriched pathways. Among those, complement and coagulation cascade was the most significantly associated pathway (hsa04610, P=6.96×10−30). Other pathways involved in neurological or immunological mechanisms included adherens junctions (hsa04520, P=6.64 × 10−25), pathogenic Escherichia coli infection (hsa05130, P=9.03×10−14), and prion diseases (hsa05020, P=5.13×10−13). We conclude that despite the overall increased genetic burden in familial MS cases, weighted sum score and PRS distributions among affected and unaffected family members within individual families revealed that known susceptibility alleles can explain disease development in some high-risk multiplex families, while in others, additional genetic factors remain to be identified through more detailed genomic analyses such as genome sequencing. Additionally, integrating multiple omics datasets of the same patients helps reduce false negative and positive results of genome-wide SNP associations and highlights the most prominent cellular players among the complex pathophysiological mechanisms in MS.
Development of novel BCL-2 inhibitors for glial tumors by using in vitro and in vivo systems

(Graduate School, 2022-05-31) Çalış, Şeyma ; Turanlı Tahir, Eda ; Avşar, Timuçin ; 521162117 ; Molecular Biology – Genetics and Biotechnology

Glioblastoma Multiforme (GBM) is the most malign form of glial tumors, which accounts for the majority of brain tumor cases worldwide. There have been different approaches to treat GBM effectively, and with the advancements made for the last decade molecular pathology, target driven therapy, and personalized medicine gained attraction. One of such promising targets for GBM is Bcl-2 induced intrinsic apoptosis pathway. Anti-apoptotic members of Bcl-2 induced intrinsic apoptosis pathway have an important role in the regulation of GBM cell death. In this thesis study, we screened seven potential Bcl-2 inhibitor compounds and evaluated their effects on proliferation of GBM cells as well as their inhibitory capacity of Bcl-2 protein. Of those, I further analyzed three of them namely 58, 243, and ind-199. 58 and ind-199 compounds did not show any significant anti-proliferation effect on GBM cells. Eventually, we decided to elucidate the mechanism of action of 243 compound, a thiazolidine derivative BH3 mimetic, which was the most promising one according to the in vitro proliferation experiments. I performed colony formation assay to assess proliferation of YKG1 GBM cells, additionally to the proliferation assay with A172 GBM cells. While 243 inihibited cell growth significantly compared to control group, Bcl-2 inhibitor ABT-199 did not inhibit cell proliferation. Moreover, I tested 243 on YKG1 tumorspheres to determine its effectivity on tumor initiating cancer stem cells (CSC). Both ABT-199 and 243 had inhibitory effect on CSC proliferation, however 243 was significantly more effective than ABT-199 when compared to control group. Since 243 is a Bcl-2 inhibitor, I analyzed key players of Bcl-2 family and intrinsic apoptosis pathway. I have analyzed gene expression levels of BCL2, BCLXL, BAX, CASP3, CASP7, and CASP9. Furthermore, I also analyzed genes related with cell death which are CASP8 and TP53. Time dependent quantitative RT-PCR results suggested that, GBM cells that are treated with Bcl-2 inhibitors ABT-263 and 243 acts differently in case of gene expressions related to apoptosis. Next, we wanted to show apoptotic cell death with Annexin V-PI assay. Interestingly, we did not detect significantly elevated apoptosis in A172 cells when they are treated with either ABT-199 or 243. Similarly, cell cycle analysis showed that 243 did not have any effect on cell cycle, altough ABT-199 induced G1 phase arrest. Moreover, I determined expression levels of apoptosis related proteins PARP, Caspase-3, and Caspase-9. I used staurosporine treatment as a positive control to induce apoptosis. None of the treatment groups apart from staurosporine increased cleaved-PARP expression. Similarly, I checked if there is a difference in expression of Pro-caspase-3 and Pro-caspase-9, and observed that only stauroporine treated group expressed lower levels of Pro-caspases, indicating that cleaved forms of both Caspase-3 and 9 were produced upon staurosporine treatment only. At this point, we hypothesized that both ABT-199 and 243 could only induce limited apoptotic cell death because BCL2 expression was relatively low in A172 cell line. Expectedly, when I compared gene expression levels among different cell lines, I observed that BCL2 expression was very low in A172 cells, and it was abundant in SH-SY5Y neuroblastoma cells. Therefore, I decided to analyze apoptosis of SH-SY5Y cells after a treatment with ABT-199 and 243. Within only 48 hours of treatment with both inhibitors, I observed apoptotic cell death of SH-SY5Y cells. Hence, we had a new hypothesis that when BCL2 expression is low, upon Bcl-2 inhibitor treatment, cells may die through autophagy since Bcl-2 forms a complex with autophagy related protein Beclin 1. I showed that 243 treatment significantly upregulated autophagy related genes such as BECN1, ATG5, and MAP1LC3B, whereas ABT-199 induced autophagy on limited level. Moreover, autophagy indicative LC3B-II expression was significantly upregulated on a protein level with the 243 treatment, when compared to control as well as ABT-199 treatment. Additionally, I determined protein expression level of p53, which has a role in the interplay between apoptosis, cell cycle, and autophagy. I observed that p53 protein expression was increased upon both ABT-199 and 243 treatment, when compared to control group. Expectedly, when we performed in silico computational analysis, Beclin 1:Bcl-2 interaction and binding of 243 to their BH3 binding domains, we observed that 243 binds to Bcl-2 through important interactions. Since 243 and Beclin 1 binds to Bcl-2 from the same domain, when cells are treated with 243, Beclin 1 cannot bind to Bcl-2 and therefore it is released to initiate autophagy. In addition, we demonstrated that 243 significantly reduced in vivo tumor growth and prolonged survival in orthotropic brain tumor models, compared to vehicle group as well as ABT-263 treated animals. Furthermore, I assessed the anti-proliferative effects of 243 on primary glial cell lines as well. 243 exerted anti-proliferative effect on all patient derived glioma cell lines that have different grades and histopathology, except OLG3 cell line which is a grade 2 oligodendroglioma. According to quantitative RT- PCR results of OLG3, OLG7, and GBM9 cell lines I observed that OLG3 has a lower expression level of BCL2. These results suggest that patients with high BCL2 expression might benefit from 243 treatment. Taken together, our results indicate that 243 disrupts Beclin 1:Bcl-2 complex, hence activates autophagic cell death, and may serve as a potential therapeutic for the treatment of GBM.
Targeting bag-1S/C-raf interaction for therapeutic intervention in cancer

(Graduate School, 2022-06-05) Tatlı, Özge ; Doğanay Dinler, Gizem ; 521152114 ; Molecular Biology-Genetics and Biotechnology

In this context, this study aims to map the interaction surface of the complex formed by Bag-1 and C-Raf, which was accomplished through the use of both molecular and structural techniques. For this, the three dimensional structure and domain architecture of the small isoform of Bag-1 were first examined by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS), and the regions on Bag-1S that can accommodate small molecule binding were probed to assess its "druggability". To this end, Bag-1S was first purified from cell lysate using Ni-NTA affinity purification through the incorporated hexahistidine tag, and subsequently, the tag was cleaved with TEV protease. A subsequent Ni-NTA purification was carried out in a flow-through mode to collect Bag-1S separate from His-tagged TEV enzyme and impurities that contained neighboring histidines. The purified Bag-1S showed an apparent 33-kDa band in gel electrophoresis. Sample purity was estimated at over 90% using ImageJ analysis of SDS-PAGE gel. To monitor the deuteration level of the Bag-1S isoform, HDX-MS experiments with five time-points that ranged from 12 s to 24 h were carried out and revealed the identification of ~150 peptides of the Bag-1S with a sequence coverage of 98%. Using HDX-MS data, peptide-specific deuterium incorporation rates were projected onto the modeled structure of Bag-1S and deuterium uptake was analyzed on the Bag-1S full-length structure. BAG domain exhibited a more solvent-protected and stabilized structure compared to the UBL (ubiquitin-like) domain. While turn regions are more labile, the regions where the helical conditions exist remained unexchanged during the entire monitored time. Multiple interaction partners of the adapter protein Bag-1 engage specifically with the BAG domain. Interestingly, the interaction sites of these partners coincide with the regions that are most solvent-protected. The interaction site is supposed to be located in the solvent-protected region of the BAG domain, which is surrounded by charged and hydrophilic regions. This solvent-protected region in the BAG domain likely possesses an interaction region, revealing a potential "druggable" binding site. To further evaluate the binding stoichiometry of the Bag-1S with C-Raf, cross-linking assays were performed in the subsequent experiments. To accomplish this, C-Raf and Bag-1S proteins were affinity-purified, which was followed by the combination of purified proteins to form an in vitro complex. Covalent coupling of the formed complexes was then performed with a cross-linking agent, DSS (disuccinimidyl suberate). According to the results obtained after immunoblotting of cross-linked samples, Bag-1S and C-Raf formed a 2:2 stoichiometric complex, suggesting that Bag-1S might contribute to C-Raf activation by triggering its dimerization. After the Bag-1S/C-Raf interaction was affirmed and stoichiometrically tested, on-membrane in vitro binding experiments were conducted to selectively identify the interface of the complex. The purified C-Raf was immobilized on a PVDF membrane and incubated with purified Bag-1S in vitro. Bag-1S-bound peptides were recovered and analyzed by LC-MS/MS after the formed complex was subjected to limited tryptic digestion on the membrane. A 20-amino acid length peptide was identified as a plausible C-Raf interacting peptide in the BAG domain of Bag-1S. Further, an in silico docking study was also conducted using the protein structure of the kinase domain of C-Raf (PDB ID 5OMV) and the modeled full length protein structure of Bag-1. In some of the poses with the lowest docking energy score, K137, T140, Q144, K149, and L156 residues of Bag-1S were found to occupy the Bag-1/C-Raf binding site. This region coincides with the plausible "druggable" interaction site identified in HDX-MS and on-membrane in vitro binding experiments. Site-directed mutagenesis experiments were then carried out to confirm the identified binding interface and to evaluate if mutations in the determined peptide sequence affect the binding of Bag-1S/C-Raf or not. Upon mutagenesis, K149A and L156R substitutions significantly decreased the endogenous levels of p-C-Raf (S338) and p-MEK1/2 (217/221) in MCF-7 cells. Consistently, TAP-pull down experiments demonstrated that these substitutions impaired the interaction of Bag-1S with C-Raf, without affecting its HSP70 contact. They also led to a significant decrease in the survival of MCF-7 cells compared to wild-type Bag-1S. In addition, while these mutations did not affect the interaction of Bag-1S with its known direct interaction partners, Bcl-2 and HSP70, they resulted in the disruption of its interaction with the complexes involved in other regulatory cell survival pathways, including B-Raf, Beclin 1, and Akt. Subsequent in vitro binding experiments did not reveal a binary interaction of Bag-1S with either Beclin 1 or B-Raf, at least under our experimental conditions. Therefore, it has been hypothesized that the formation of a Bag-1/Beclin 1 or Bag-1/B-Raf complex might require the presence of C-Raf as a mediator. Further, Bag-1S interacting C-Raf region was identified by on-membrane in vitro binding experiment coupled with LC-MS/MS. Four different peptides derived from native Bag-1 and C-Raf sequences corresponding to the plausible interaction segments of the complex were designed and then synthesized by using solid-phase synthesis. The ability of the peptides to hamper the formation of a Bag-1S/C-Raf complex was tested in vitro. Of these peptides, Pep 3 that targets C-Raf binder region of Bag-1S significantly altered Bag-1S/C-Raf interaction. Pep 3 not only impeded the binary interaction of C-Raf with Bag-1S but also disrupted BAG-associated complexes of Bag-1 in TAP pull-down experiments. Inhibition of multiple Bag-1S interactions afforded by Pep 3 bolsters its potential to impair the prolonged survival of cancer cells. We therefore not only affirmed that this region on C-Raf is responsible for Bag-1 binding, but also discovered a novel peptide inhibitor targeting Bag-1S, which has the potential to be improved for cancer therapy.

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