Biology subjects

Sahin, C.

Publications and source records attributed to Sahin, C..

8 recordsLinked to original sources

bioRxiv2026

Long noncoding RNA NRAV can regulate interferon-stimulated gene expression in melanoma

The long non-coding RNA Negative Regulator of Antiviral Response (NRAV) is known to suppress antiviral immunity by regulating interferon response, but its functional role in tumor immunology remains poorly understood. We examined the relevance of NRAV in melanoma and found that high NRAV expression was associated with poor survival, reduced inflammatory pathway activation, and resistance to immune checkpoint blockade. Bulk and single-cell transcriptomic profiling indicates that NRAV expression is selectively enriched in malignant cells suggesting a potential cancer cell-intrinsic function. To examine whether NRAV can regulate inflammatory responses in melanoma cells, we manipulated the levels of NRAV in the BRAF-mutant A375 melanoma model and characterized the expression of key interferon-stimulated genes (ISGs) following type-I and type-II interferon stimulation. Our findings reveal that the stable NRAV overexpression blunts the induction of key ISGs, whereas NRAV knockdown reciprocally amplifies their transcription. Subcellular fractionation revealed that NRAV is predominantly localized to the nuclear compartment of melanoma cells and the overexpression of NRAV altered regulatory histone marks on the target ISG promoters including MX1 and IFITM3. Collectively, these findings establish NRAV as a tumor-intrinsic epigenetic regulator of interferon signaling, highlighting its potential contribution to melanoma immune evasion.

cancer biology↗

bioRxiv2024

The SH Protein of Mumps Virus is a Druggable Pentameric Viroporin

Viral infections are on the rise and drugs targeting viral proteins are needed. Viroporins constitute a growing group of virus-encoded transmembrane oligomeric proteins that allow passage of small molecules across the membrane. Despite sparsity in viroporin structures, recent work has revealed diversity in both the number of transmembrane helices and oligomeric states. Here we provide evidence that the small hydrophobic protein (SH) from mumps virus is a pentameric viroporin. From extensive biophysical data, a HADDOCK model of full-length SH shows its intracellular C-terminal region to form an extended structure crucial to stabilization of the pentamer. Heterologous expression of wild type SH and variants in Xenopus laevis oocytes reveals the viroporin as a chloride channel, facilitated by conserved hydroxyl-carrying residues lining the pore. The channel function of SH is inhibited by the small-molecule BIT225, highlighting the potential for antiviral targeting through SH.

biophysics↗

bioRxiv2023

A suicidal and extensively disordered luciferase with a bright luminescence

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 [A] and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase. Significance statementEnzymes are typically characterized by an overall globular structure with a hydrophobic core and a defined cavity for binding of substrate, containing the active site amino acid residues. Gaussia Luciferase is a widely used luminescent reporter with a very strong, albeit short-lived, flash of light due to rapid auto-inactivation. We show, using solution NMR, that while this luciferase shows some secondary structure elements held together by disulfide bonds this highly unusual enzyme is extensively disordered with essentially no hydrophobic core. Although the enzymatic mechanism remains unknown, we have identified two essential arginine residues but, in the structure, these do not point into a common active site. In spite of this, the enzyme has high substrate specificity suggesting that it undergoes major structural rearrangements upon binding of substrate.

biochemistry↗

bioRxiv2023

TCGEx: A visual interface for multifaceted analyses of The Cancer Genome Atlas gene expression data

The Cancer Genome Atlas (TCGA) initiative has been essential for revealing key mechanisms in human cancer leading to the development of novel therapeutics. Analysis of the cancer transcriptomics data in the TCGA and other public repositories require coding skills that are associated with a steep learning curve for most non-specialists. To enable a wider utilization of these data, we introduce The Cancer Genome Explorer (TCGEx), a web-based visual data analysis interface that can perform a number of sophisticated analyses ranging from survival modeling and gene set enrichment analysis to unsupervised clustering and linear regression-based machine learning. In addition to providing access to preprocessed data from TCGA and immune checkpoint inhibition studies on cBioportal and CRI-iAtlas, the TCGEx platform allows users to upload and investigate their own data. Using this tool, we investigated the molecular subsets of human melanoma and identified microRNAs associated with intratumoral interferon signaling. We validated these findings using independent data from clinical trials involving immune check-point inhibitors for melanoma and other cancers. Moreover, our analyses unveiled a subset of cytokines predictive of positive responses to diverse immune checkpoint inhibitors prior to treatment initiation. Built on the R/Shiny framework, TCGEx modules offer customizable features to tailor the analysis to different study contexts and help generate publication-ready plots. TCGEx is freely available at https://tcgex.iyte.edu.tr, and it provides an interactive solution to extract meaningful insights from cancer transcriptomics data and guide scientific inquiry.

bioinformatics↗

bioRxiv2023

Genomic adaptation underlies nutrient-driven reprogramming in the malaria-causing parasite

Parasites often depend on metabolic adaptation to the host environment. An adaptive feature of malaria-causing parasites is digestion of hemoglobin (HB) to acquire amino acids (AAs). Here we describe a link between nutrient availability and translation elongation-dependent regulation of gene expression as an adaptive strategy. We show that, unexpectedly, tRNA expression in P. falciparum does not match the decoding need as tRNAs decoding AAs that are rare in HB are lowly expressed. This discrepancy renders codons of HB-rare AAs inefficiently decoded and transcripts levels negatively correlated with the requirement of HB-rare AAs for protein synthesis, which are consistent with poor codon optimality causing co-translational mRNA decay. Intriguingly, proliferation-related genes have evolved to require a high level of HB-rare AAs in their encoded proteins, thereby allowing the parasite to control its proliferation by repressing protein synthesis of these genes during nutrient stress. We conclude that the parasite modulates translation elongation by maintaining a discordant tRNA profile as a mechanism to exploit variations in AA-composition among genes as an adaptation strategy.

microbiology↗

bioRxiv2022

Mass spectrometry of RNA-binding proteins during liquid-liquid phase separation reveals distinct assembly mechanisms and droplet architectures

Phase separation of heterogeneous ribonucleoproteins (hRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility-mass spectrometry, and molecular dynamics simulations. We used a phase separation-compatible spider silk domain and pH changes to control the self-assembly of the hRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with phase separation. We find that NT*-FUS monomers undergo an unfolded-to-globular transition, whereas NT*-TDP-43 oligomerizes into partially disordered dimers and trimers. NT*-hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over phase separation. The divergent assembly mechanisms result in structurally distinct complexes, indicating differences in RNA processing and translation depending on biological context.

biophysics↗

bioRxiv2022

A grappling hook interaction balances self-assembly and chaperone activity of Nucleophosmin 1

How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers which provide the scaffold for ribosome assembly but also prevent protein aggregation as part of the cellular stress response. Examining the relationship between the self-assembly and chaperone activity of NPM1, we find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning and cryo-electron microscopy reveal fuzzy interactions between the disordered region and the C-terminal nucleotide-binding domain that cross-link NPM1 pentamers into oligomers. Ribosomal peptides mediate in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. We conclude that NPM1 uses a "grappling hook" interaction to form a network-like structure whose chaperone activity is tuned by basic proteins, suggesting a regulatory mechanism for the nucleolar stress response.

biophysics↗

bioRxiv2021

A spindle and thread-mechanism unblocks translation of N-terminally disordered proteins

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular "spindle and thread" mechanism can overcome poor translation efficiency of partially disordered proteins.

biochemistry↗