LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora
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ÖgeInvestigation of the spastin's role in the invasion capacity of glioblastomas(Graduate School, 2024-01-05)Glioblastoma Multiforme (GBM) is the most lethal form of glioma, which are the most frequent brain tumors. Even though multimodal therapy is employed to treat GBM, tumor recurrence makes treatment almost impossible due to its robust migration/invasion potential. For the improvement of GBM therapy, it is critical to identify the proteins involved in the disease's migration/invasion process. Tumor cells require special cellular extensions controlled by cytoskeletal components to achieve migration/invasion capabilities. Spastin, a microtubule-severing protein, is mainly expressed in neurons and controls dendrites and axonal extensions of neurons. Given that the formation mechanism of these extensions in post-mitotic cells is comparable to that of specialized cell protrusions in mitotic cells, Spastin might have roles in tumor cell migration/invasion. Interestingly, Spastin has been discovered to be co-localized with actin filaments in GBM cells, suggesting that it may play a role in GBM migration ability. However, this topic has not been investigated in the literature until this study. This thesis aims to clarify the molecular mechanism underlying the shift in the intracellular localization of Spastin in GBM cells, and the potential significance of this mechanism in GBM migration/invasion ability. This study discovered for the first time that Spastin takes an active role in GBM migration. Furthermore, Spastin was discovered to interact with Pin1 via phosphorylation of Pin1 recognition motifs located in its microtubule-binding domain. Moreover, this interaction was found to direct of Spastin towards actin filaments, which promotes migration/invasion ability of GBM cells. These findings suggest that Spastin might be a therapeutic target for several tumors with a high migration/invasion capacity, like GBM.
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ÖgeMolecular dynamics studies on proteins involved in genetic variation and metabolism: DMC1 and lipase(Graduate School, 2024-12-23)Both homologous recombination and enzymatic processes, such as those catalyzed by lipases, play essential roles in biological systems, with wide-reaching applications in science and industry. Scientists have studied homologous recombination for a long time to understand its evolution, mechanics, and biological significance. The involvement of the Dmc1 protein in the context of homologous recombination is the focus of this study's thorough investigation of these features. Recombinases, such as Rad51 and Dmc1, which are found in eukaryotes, are essential for homologous recombination and DNA repair. Despite significant sequence similarities, Rad51 and Dmc1 serve different purposes; although Rad51 is essential for DNA repair, Dmc1 participates in homologous recombination during meiosis. More details about its structure and activities are revealed, including the involvement of ATP binding sites and the precise amino acids required for ssDNA binding. Loop areas have been found to be essential for DNA binding. Dmc1 has been characterized by several crystal structures showing an octameric ring configuration in the absence of ATP and DNA. Additionally, it has crystal structures in filament form. These different structures provide valuable insights into the structural flexibility of Dmc1 and its mechanism of interaction with DNA. Molecular dynamics simulations were performed on human Dmc1 protein in various oligomeric states to investigate its structural and dynamic behavior. This study aimed to explore the effects of nucleotide binding, protonation states, and peptide bond isomerization on the stability and conformational dynamics of Dmc1's N- and C-terminal domains. Key simulations included standard molecular dynamics, thermodynamic integration for pKa calculations, and umbrella sampling for free energy profiling. Protonation states of residues E162 and H295, cis-trans isomerization of the D223-S224 peptide bond, and nucleotide-binding states (ATP, ADP, or nucleotide-free) were systematically examined. Root mean square deviation (RMSD) analyses showed distinct equilibration dynamics for the C-terminal domain, while the N-terminal domain displayed significant mobility. Structural analysis revealed the connection between the protonation of E162 and its influence on DNA-binding residue R230. Besides, when E162 is protonated, the ring structure of the protein remains stable. when the D223-S224 peptide in a cis configuration, ATP binds to the Walker A and Walker B motifs in a canonical manner, similar to how ATP binding occurs in other ATPases. However, when the peptide bond is in the trans configuration, the interactions between ATP, Mg²⁺, and the Walker A and Walker B motifs are disrupted, weakening the binding. In the nucleotide-free state, the trans configuration with E162 appears more stable. Upon ATP binding, the structural behavior changes, and multiple configurations become possible. Simulations indicate that trans isomer with protonated E162, trans isomer with unprotonated E162, and cis isomer with unprotonated E162 likely exist in comparable amounts, suggesting a dynamic equilibrium driven by ATP binding and associated conformational flexibility of the protein. Additionally, molecular dynamics simulations on lipase enzymes offered comparative insights into structural flexibility and catalytic efficiency. Lipase dynamics highlighted the role of active site flexibility in substrate binding and enzymatic activity, providing a broader perspective on protein behavior. Overall, the simulations enhanced the understanding of Dmc1's dynamic behavior, interdomain interactions, and potential DNA-binding mechanisms, contributing to deeper molecular-level insights into homologous recombination processes.
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ÖgeProteomic approaches for the identification and quantification of clinically relevant biomarkers(Graduate School, 2024-10-16)Cardiovascular diseases are a significant health issue affecting people worldwide. Early diagnosis of cardiovascular diseases is crucial for the successful treatment of conditions such as heart attacks and for preventing death. Cardiac troponin I (cTnI) is a vital biomarker for the diagnosis and risk assessment of heart attacks. In healthy individuals, cTnI levels are found below 45 nanograms per liter. During and in the hours following a heart attack, it is released into the blood stream due to heart tissuedamage, leading to an increase in its levels. cTnI can be detected in the blood in various forms, including a ternary complex (cTnC-cTnT-cTnI), a binary complex (cTnI-cTnC), and free forms. It is highly susceptible to proteolysis and enzymatic changes. Consequently, various forms of cTnI, including proteolyzed, phosphorylated, oxidized, and reduced forms, can be found in the blood. All these variables lead to differences in cTnI measurements. There are many cTnI tests available on the market. The different variations circulating in the blood can be recognized by different monoclonal antibodies specific to different epitopes of cTnI. The various versions of cTnI, along with the different antibodies used, have increased the correlations between commercial tests more than tenfold, yet standardization remains challenging. Laboratories use different clinical decision thresholds depending on the test used. Different assay cutoffs have the potential to confuse physicians, leading to the misinterpretation of cTnI results; hence, there is urgency for cTnI standardization. The standardization and/or harmonization of cTnI assays is considered a high priority by the International Consortium for Harmonization of Clinical Laboratory results (ICHCLR). According to ISO 17511, the standardization of the measurement of a biomarker requires a metrological traceability chain. This chain begins with a primary reference measurement procedure (RMP), which assigns quantity values to a primary reference material (RM). Primary RMs are used to assign values to a secondary RM. With this secondary RM, values are assigned to working and product calibrators for routine quantification of the biomarker in patient samples. This traceability chain allows the values reported for patient care to be traced back to the International System of Units (SI). In this way, metrological traceability supports the long-term stability and comparability of routine laboratory measurement results. The standardization or harmonization of cTnI measurement requires the development of RMs and RMPs. The traceability chain proposed by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of Troponin I (IFCC WG-TNI) incorporates all these standardization steps. One of the tasks that the IFCC working group is focused on to establish the proposed traceability chain is the development of a higher-order RMP. In this thesis, the focus has been on developing two different analytical methods to support the development of a RMP. Both analytical procedures involve targeted and bottom-up proteomic approaches. In both methods, isotope dilution mass spectrometry (IDMS) has been used to determine the absolute amount of cTnI. For quantifying proteins using the IDMS method, two different strategies have been employed: the protein-based calibration strategy and the peptide-based calibration strategy. Each method has its own advantages and disadvantages. The first developed analytical method allows for the determination of cTnI from human serum using a protein-based calibration strategy. In this context, human cardiac troponin complex material (NIST SRM 2921) has been selected for use as a calibrant. The troponin complex was purified from human heart tissue and consists of three subunits: troponin T (cTnT), troponin I (cTnI), and troponin C (cTnC). As an internal standard, isotopically labeled cTnI protein with the same sequence as cTnI has been used. To extract cTnI from a complex matrix like serum, an immunoaffinity enrichment strategy has been employed. As the first step of immunoaffinity enrichment, two different diameters of magnetic particles were selected: micro (Dynabeads® MyOne™, 1 μm) and nano (Nanomag®-D, 130 nm). A monoclonal antibody capable of binding to cTnI was immobilized on both types of magnetic nanoparticles, and their cTnI enrichment efficiencies were compared. Magnetic nanoparticles (Nanomag®-D, 130 nm) were chosen for further experiments because the peak areas of two selected tryptic peptides of cTnI were relatively higher. Next, the maximum loading capacity of the magnetic nanoparticles was determined. It was found that when 100 μg of antibody was added to 1 mg of particles, 59.2 ± 5.7 μg/mg of antibody could be bound. Using the synthesized nanoparticle-antibody conjugate, the required amount for cTnI enrichment from 1 ml of serum was calculated, and it was determined that 10 μl of conjugate was sufficient to capture all cTnI in 1 ml of serum for analysis. As a result of these optimizations, the isotope dilution liquid chromotograpy tandem mass spectrometry (ID-LC-MS/MS) method using the developed protein-based calibration strategy allows the measurement of cTnI in the range of 0.6 to 24 μg/L (R > 0.996). The limit of quantification (LOQ) was determined to be 1.8 μg/L, and the limit of detection (LOD) was 0.6 μg/L. Intermediate precision was found to be below 9.6%, and repeatability ranged from 2.0% to 8.7% for all quality control materials. The accuracy of the analyzed quality control materials was between 90% and 110%. Total measurement uncertainties (n=6) were found to be below 12.5% for all levels. The second developed ID-LC-MS/MS method allows the determination of cTnI in human serum using a peptide-based calibration strategy. In this method, two tryptic peptides (TLLLQIAK and NITEIADLTQK) of cTnI were selected and synthesized as calibrants. Isotopically labeled versions of the selected peptides were used as internal standards. Peptide impurity correction amino acid (PICAA) analysis was performed to assign values to the synthetic peptides, thereby producing SI-traceable primary peptide standards. Peptide-based calibration approach also employed two surrogate matrices to construct the calibration curve. The surrogate matrices were evaluated based on parameters such as linearity, accuracy, repeatability, intermediate precision, and trueness. It was observed that both matrices yielded similar results, indicating consistency in their performance. To ensure complete cleavage of the cTnI protein and enhance proteolysis yield, optimizations such as trypsin digestion methods, enzyme-to-protein ratio, and digestion time were performed. The best trypsin cleavage yield was obtained using the Filter-Aided Sample Preparation (FASP) method with a 1:10 enzyme-to-protein ratio and overnight digestion. The developed analytical method using the peptide-based calibration strategy enables the quantitative determination of cTnI in the range of 0.6–21.6 μg/L. Intermediate precision RSD was less than 28.9%, and repeatability RSD was less than 10% across all concentration levels. The recovery rate ranged between 72% and 151%. Four patient serum samples with suspected heart attack were measured using the developed method, and the results showed discrepancies of more than 50% compared to those obtained with immunoassay. Finally, the performance of the peptide-based calibration strategy was compared with the protein-based measurement strategy. In conclusion, this thesis has developed two different ID-LC-MS/MS methods using a targeted and bottom-up proteomic approaches. Both methods were compared with each other in terms of effectiveness. This efforts aim to support the metrology community in adopting new approaches and developing SI-traceable peptide and protein primary standards and/or reference procedures tailored to specific needs. Standardization and harmonization of cTnI across laboratories are undeniably complex tasks. However, the IFCC WG-TNI believes that cTnI measurement is standardizable. Given the critical role of cTnI in patient management, the significant effort invested is worthwhile. The proposed measurement methods will play a role in supporting the activities of the IFCC WG-TNI. These studies are necessary and logical steps towards the harmonization of results obtained from different test kits.
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ÖgeInvestigating molecular interaction networks of nuclear factor one transcription factors(Graduate School, 2024-12-19)Gene expression is a tightly regulated process involving multiple steps, with transcription factors (TFs) serving as key regulators. TFs bind to specific DNA sequences, interact with other proteins, and modulate the expression of target genes. Among these, the Nuclear Factor I (NFI) family plays a significant role in development and cancer. NFI transcription factors are key regulators of gene expression, influencing cellular proliferation, differentiation, and specialization across various tissues. In vertebrates, the NFI family includes four members: NFIA, NFIB, NFIC, and NFIX. Alternative splicing of NFI genes gives rise to multiple isoforms. These proteins contain a highly conserved N-terminal DNA-binding and dimerization domain, enabling them to recognize and bind a consensus sequence (TTGGC(N5)GCCAA) as either homo- or heterodimers. While performing their regulatory functions, NFI family members interact with other proteins and the chromatin. However, aside from a few specific interactions detected in certain contexts and the interactions of NFIs with other transcription factors, the interactomes of NFIs, their dynamics, and their functions remain largely unexplored. In this thesis, we employed complementary omics-level techniques, i.e., interactomics (affinity purification mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID)), and chromatin immunoprecipitation sequencing (ChIP-Seq), to obtain a comprehensive view of the NFI proteins and their interactions. The MAC-tag approach was utilized, enabling combined AP-MS and BioID analyses. The HA epitope within the MAC3-tag, along with the BioID (UltraID) sequence, allows detection of tagged protein expression by immunoblotting and immunofluorescence, and the use of the same Flp-In™ T-REx™ 293 cell lines in ChIP-Seq for studying protein-DNA interactions. In addition to the MAC-tag approach in isogenic inducible Flp-In™ T-REx™ 293 cell lines, we utilized lentivirus transduction and electroporation methods to achieve forced expression in neuroblastoma and glioblastoma cell lines, which are generally difficult to transfect using standard methods. Our experimental approach encompassed all four predominant isoforms of NFI family members and the atypical isoform NFIB4, which lacks the DNA-binding domain and was initially linked to megakaryocyte differentiation. Interaction partners were identified through proximity labeling with varying biotinylation times, while stable interactors were captured using AP-MS analysis. In parallel, ChIP-seq experiments were performed to uncover potential NFI target genes. We also investigated NFIA's regulatory impact on one of its interacting partners, the transcription factor SOX2. Additionally, we used AlphaFold 3 to predict DNA binding, modeling interactions between NFI proteins and their ChIP-seq target DNA sequences, enabling us to identify key regions of the NFI proteins involved in DNA binding. We observed that, despite exhibiting some redundancy, each family member had unique high-confidence interactors and target genes, highlighting distinct roles within the transcriptional regulatory networks. Time-dependent interactome mapping revealed the dynamic nature of NFI interactions with chromatin remodelling and transcriptional regulator complexes. UltraID-based proximity labeling identified extensive, evolving interactions with chromatin remodeling and transcriptional complexes, particularly SWI/SNF and Mediator. AP-MS confirmed stable associations with SWI/SNF complex proteins, and most of the interactors are validated by co-expression and pull down experiments, underscoring the collaborative roles of NFIs and SWI/SNF in chromatin remodeling. While previous studies associated NFIA and NFIB with the Mediator complex in neural stem cells, our findings reveal that NFIC also interacts with Mediator complex; however, these associations appear transient or context-dependent and are only detected by proximity labeling. The interactome of the atypical short isoform, NFIB4, is enriched with proteins involved in mRNA regulation, suggesting that NFIs may have roles beyond traditional DNA binding and transcriptional modulation. Meanwhile, structural analysis using AlphaFold3 revealed four conserved binding regions—loop, helix1, helix2, and segment—that are critical for DNA interaction. Additionally, predictions with ChIP-seq results showed that NFIs can bind to motifs with variable spacer lengths, indicating flexibility in motif recognition. By integrating ChIP-seq with RNA interference (RNAi) experiments, our study identified SOX2 as an important target of NFIA. NFIA knockdown led to an altered genomic binding profile of SOX2, accompanied by notable shifts in pathway enrichment among SOX2 target genes. Specifically, pathway analysis of potential SOX2 targets post-NFIA silencing revealed enrichment in the cAMP signaling pathway. Our findings reveal that NFIs interact with a diverse array of chromatin remodelers, transcriptional regulators, and other proteins, highlighting their extensive roles in regulating gene expression. Time-resolved interactomics demonstrated dynamic and context-dependent interactions with key complexes, such as SWI/SNF and Mediator, suggesting that NFIs actively participate in coordinating transcriptional activity. Additionally, integration of ChIP-seq data with RNA interference experiments showed that NFIA modulates the activity of other transcription factors, including SOX2, altering its genomic binding profile and influencing pathway enrichment among its target genes. These observations, coupled with the isoform-specific interaction profiles and their distinct sets of target genes, position NFIs as central regulators capable of modulating complex transcriptional networks and influencing cellular processes across diverse contexts. Therefore, we propose that NFIs function as master regulators, modulating other transcription factors and interacting with various proteins across multiple cellular processes. While NFIs may exhibit pioneering capabilities, this requires validation through direct evidence of their ability to bind closed chromatin. Future studies could determine whether NFIs access nucleosome-occupied or otherwise inaccessible chromatin, helping to confirm their potential role as pioneering transcription factors. In conclusion, this thesis advances our understanding of the NFI family's roles in gene regulation, revealing both established and novel interactions that shape cellular function across various contexts. By employing complementary omics-level approaches, we identified distinct interaction profiles and target genes for each NFI member, underscoring their individual roles within transcriptional regulatory networks. The unique interactome of the NFIB4 isoform, particularly its association with mRNA regulatory proteins, suggests a broader functional scope for NFIs that may extend beyond traditional DNA binding to influence post-transcriptional processes. Additionally, our findings on NFIA's regulatory effects over SOX2 binding and pathway enrichment point to NFIs as master regulators with the potential to guide cellular fate decisions and differentiation. These insights lay the groundwork for future research aimed at exploring the mechanisms of NFI function in chromatin remodeling and gene expression, as well as their therapeutic implications in diseases such as cancer and neurodevelopmental disorders.
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ÖgeExploration of novel serine protease do-like HtrA from acigöl(Graduate School, 2023-12-06)Enzymes involved in industrial biotechnological processes take place in conditions of extremely high temperature, high pH, and high salinity or when there are organic solvents that have made it necessary to discover enzymes resistant to these conditions. Microorganisms in extreme environments adapt to varying levels of stress such as very high pH, temperature, salt, and pressure. For the last 20 years, researchers have focused especially on extreme environments for the discovery of enzymes that are resistant to extreme conditions, with the hypothesis that the enzymes of microorganisms adapted to these conditions can also work under extreme conditions. In this context, microorganisms can be isolated from their environment and their enzymes can be characterized by traditional microbiological methods. Besides this, new enzymes have been discovered by the method called 'metagenomics', which is not based on culture. Environments with high salt concentration are divided into two in terms of their ionic compositions. Many high salt concentration environments were formed by the evaporation of seawater, also called 'thalassohaline'. Their salt content is similar to seawater and the pH varies from basic to slightly acidic. Environments with high salt concentrations, called 'Athalassohaline', are completely different from seawater in terms of ionic composition. Acıgöl, which is our study area, is a lake with high salt concentration, which is included in the 'Athalassohaline' state group. In this study, samples from Acıgöl were employed. Acıgöl is located between the provincial borders of Denizli and Burdur in the Aegean Region of our country. Looking at the chemical composition of the lake, it is seen that Na+, K+, Cl−, and SO4 2− ions are dominant. The salinity of the Acıgöl changes between 5.8%-13%, pH between 7.8-8.2, and temperature varies seasonally between 8 °C and 32 °C. These changing extreme conditions force the microorganisms in the lake to cellular and enzymatic adaptation. These organisms adapted to high salt concentration are called 'Halophilic' microorganisms, meaning salt-loving. Based on this information, the main subject of the study is the discovery of enzymes of halophilic microorganisms that can be used in difficult industrial processes. The primary objective of this study is to obtain new proteases, which are of industrial importance, by function-based screening of culturable microorganisms. In line with this goal, firstly, soil samples taken from Acıgöl were diluted in Nutrient Broth and spread on nutrient agar petri dishes containing 10% NaCl and 1% skim milk, and the species containing protease activity were determined. It was determined by the transparent region around the colonies that the isolate had protease activity, resulting from the breakdown of skim milk. With this screening method, halophilic species in Acıgöl, which actively produce protease, were determined. Sixmorphologically different species were determined. Twoshowed protease activity, and the species with t huge zones were chosen for further studies. In the second part of the study, the whole genome of the determined species was sequenced with the New Generation Sequencing method (Illumina HiSeq 2500 platform), and its serine proteases and other biotechnologically potential enzymes were determined. According to the sequencing results, it was determined that the entire genome of the isolated species was 4,708.499 bp (base pair) in length, had a G+C ratio of 36.66%, and had 4536 gene-coding sequences. In addition, it was revealed that 99.81% ratio similarity to Virgibacillus marismortui species according to 16S rDNA sequence similarity. The whole-genome average nucleotide identity (ANI) value was obtained as 99.44% and digital DNA-DNA hybridization was computed as 88.8%. The average amino acid identity ratio (AAI: Average Amino acid Identity) was calculated as 98.69%. In addition to genomic analyses, the isolated species was also examined phenotypically and biochemically. It was determined that the species was gram positive (Gr+), both alkaliphilic and moderately halophilic, motile, endospore-forming, and protease-producing bacterium. The isolated strain shows optimum growth at 37 °C with salinity and pH ranging from 5-10% and 6 and 9, respectively. As a result of this polyphasic analysis, it was conclueded that the isolate was a subspecies of Virgibacillus species, and it has been brought to the literature with the name Virgibacillus sp. AGTR. All genome information is stored in the NCBI database. Accession number JAJERH000000000. The last step of the study aimed to produce by recombinantly and characterize the serine protease from a new isolate. Among the four serine proteases determined by whole genome analysis, the Serine protease Do-like HtrA with the lowest sequence similarity rate and fewer studies in the literature was selected for recombinant production. The Serine protease Do-like HtrA is a member of the Trypsin-like serine protease superfamily (Tryp_SPc Superfamily) and S1-C subfamily. HtrA (high-temperature requirement A), a periplasmic heat-shock protein, it has two different functions. While it shows molecular chaperone properties at low temperatures, it shows proteolytic activity at high temperatures. The structure of this kind of protease differs slightly from other commercial and well-studied proteases. Due to these properties, it could be used specifically in the pharmaceutical industry. For the recombinant production of Serine protease Do-like HtrA, primers that contain EcoR I and SacI restriction sites were designed to be specific to the start and end sequences of the gene of interest (targeting the 5' and 3' ends). By using the genome of the isolated Virgibacillus sp. AGTR strain as a template, the target protease gene was amplified and ligated into the pET-28-a(+)expression vector. The cloned vector was inserted into E. coli BL21, E. coli C43 (DE3), and RosettaTM 2 expression cells to determine the best expression host cell. As a result of the purification study, the RosettaTM 2 cell was selected for expression. Expression studies were performed with 0.1 mM, 0.5 mM, and, 1 mM IPTG concentrations at 30 ºC and 37 ºC for up to 6 hours. The highest level of expression was achieved with 0.1 mM IPTG in 4 hours at 30 °C. Successfully expressed protease gene was purified by the His-tag method. The estimated molecular weight of the protein was 42100 Da and the isoelectric point was 4.53 which is calculated using the ExPASy program. As a result of purification, the molecular weight of the enzyme (42.1 kDa) was compatible with the predicted value, according to SDS-PAGE and Western blot tests.
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