Biology subjects

Demir, O.

Publications and source records attributed to Demir, O..

12 recordsLinked to original sources

bioRxiv2025

Microgravity Remodels Longevity Networks in Astronaut PBMCs: Integrated Findings of Telomere Elongation, DNA Repair Responses, and miRNA Suppression

Microgravity provides a unique environment for elucidating the fundamental mechanisms of human aging. In the Microgravity Associated Genetics (MESSAGE) Science Mission--Turkiyes first human space biology experiment--we performed an integrative analysis of telomere dynamics, transcriptomic remodeling, and microRNA regulation in peripheral blood mononuclear cells (PBMCs) collected before launch (L-7day), after suborbital ascent (L+3hrs), and during days 4-10 aboard the International Space Station (ISS). Spaceflight induced a striking early elongation of telomeres, accompanied by transcriptional activation of DNA repair, oxidative stress mitigation, mitochondrial homeostasis, and immune regulatory pathways. Concurrently, microgravity triggered robust suppression of longevity-associated microRNAs, including members of the miR-17-92, miR29, and miR34 families, suggesting coordinated epigenetic reprogramming of genome stability and stress responses. Notably, the adaptor protein gene AP2A1, recently implicated in cellular rejuvenation and mechanotransductive aging processes, emerged as a consistently microgravity-responsive hub, linking cytoskeletal signaling to telomere maintenance and DNA repair networks. Together, these findings reveal that short-duration spaceflight initiates a multi-layered molecular longevity program in human immune cells, characterized by telomere extension, stabilization of genome maintenance pathways, and suppression of aging-associated miRNA regulators. This systems-level view provides foundational insight into how human biology adapts to short-term microgravity exposure and identifies AP2A1-centered networks as promising targets for enhancing astronaut health and performance and ultimately understanding terrestrial aging.

molecular biology↗

bioRxiv2025

Towards In Situ Dynamics of DNA-bound Full-Length p53 Tetramer

p53 is the most important tumor suppressor in humans as well as the most frequently mutated gene found in human cancers with [~]50% of all human tumors bearing p53 missense mutations that leave p53 inactive. Restoring the p53 activity proved to lead to tumor regression even in advanced tumors in mouse models-- and thus, is among the most attractive potential strategies for novel cancer therapy. Full-length p53 (fl-p53) consists of 393 residues and multiple domains; some folded and some disordered. Using crystal structures of folded domains and integrative molecular modelling techniques for disordered domains, we generated the first wild-type fl-p53 tetramer model bound to DNA. When solvated, the system size nears 500K atoms challenging extensive sampling. Using Anton2 supercomputer for microsecond-timescale simulations in explicit solvent and the rigorous Markov state model (MSM) framework, we elucidated the conformational landscape of wild-type p53 as well as two of the p53 hot-spot cancer mutants, Y220C and G245S, in a physiological DNA-bound, full-length tetramer context. In the simulated timescale, DNA-bound fl-p53 tetramer bent DNA and formed a compact complex with interactions between the N-terminal and DNA-binding domains (DBDs), and the C-terminal domains (CTDs) with DNA. WT fl-p53 tetramer also sampled a unique quaternary DBD organization not accessed by the cancer mutants. Free energy landscapes indicated differential dynamics for inner and outer p53 DBDs due to the dimer-dimer interface. The dynamics of the druggable L1/S3 pocket is also closely monitored. Ultimately the MSMs identified an underexplored loop 6 (L6) cryptic pocket and captured the effect of p53 tetramerization and cancer mutations. Significancep53, the most important tumor suppressor in humans, is found inactivated due to single point mutations in almost 50% of all human tumors. As restoring p53 activity is shown to achieve tumor regression even in advanced tumors in mice, there is a lot of interest in finding small molecules to reactivate p53 cancer mutants. p53 has 393 residues and is an intrinsically disordered protein with multiple unstructured and dynamic domains that are known to interact with and help the function of its folded domains. Adding another level of complexity, p53 forms a tetramer to bind DNA and start its transcriptional activity. Due to its high dynamicity, p53 evades full structural characterization leaving many open questions. We generated an integrative model weaving together structures of isolated p53 domains and explored it with molecular dynamics simulations at microsecond timescale. This allowed us to monitor various unresolved aspects with atomistic detail like the dynamics of druggable pockets, the interplay of p53 domains in the physiological complex and how they change in p53 cancer mutants compared to the wild-type p53. Our study provides insights into the structure and dynamics of a key tumor suppressor protein at its physiologically relevant state of full-length tetramer bound to DNA.

biophysics↗

bioRxiv2025

Hypothetical LOC Genes as Biomarkers of Spaceflight Adaptation: A Comparative Study from ISS, Suborbital, and Earth-Based Experiments

Microgravity constitutes one of the most profound environmental stressors encountered by humans during spaceflight, capable of altering fundamental cellular processes and gene regulatory networks. While the effects of spaceflight on well-characterized protein-coding genes have been widely documented, little is known about the behavior of uncharacterized or poorly annotated genomic regions under these conditions. LOC (Locus) genes, often classified as long non-coding RNAs and excluded from conventional analyses, represent a largely unexplored component of the human transcriptome. In this study, we systematically investigated the transcriptional responses of LOC genes as part of the MESSAGE (Microgravity Associated Genetics) Science Mission, Turkiyes first human space biology initiative. Peripheral blood samples were collected from astronauts across five mission phases: pre-launch baseline, post-suborbital flight ([~]100 km), and on International Space Station (ISS) Days 4, 7, and 10 ([~]400 km). RNA-Seq analyses revealed six LOC genes with statistically significant expression changes (p < 0.05, Kruskal-Wallis test), alongside additional transcripts that, while not statistically significant, exhibited biologically meaningful temporal fluctuations. These dynamic profiles included continuous upregulation, transient activation with subsequent return to baseline, and delayed induction at later ISS stages, highlighting the functional diversity of LOC responses. To assess their translational potential, Open Reading Frame (ORF) analyses were performed on significant transcripts, revealing conserved ORF structures--most notably identical ORF33 sequences in LOC124905103 and LOC124900480-- suggesting coding capacity. Phylogenetic analyses further supported evolutionary clustering consistent with expression and ORF similarities. Collectively, these findings challenge the notion of LOC genes as transcriptional noise, instead positioning them as candidate biomarkers and functional elements of microgravity adaptation. By extending space biology research into the "dark genome," this study provides novel insights with potential implications for astronaut health monitoring and therapeutic development in long-duration missions.

molecular biology↗

bioRxiv2025

Assessing Immune Stability in Space: Haematological and T Cell Profiling of Astronauts During the MESSAGE Science Mission in Axiom-3 on the ISS

Microgravity presents unique challenges to human physiology, particularly the immune system, during spaceflight. T lymphocytes, key components of adaptive immunity, play a vital role in immune regulation, yet the effects of microgravity on T-cell function and gene expression remain incompletely understood. This study, conducted as part of the MESSAGE (Microgravity Associated Genetics) Science Mission in the Axiom-3 mission (https://www.nasa.gov/mission/station/research-explorer/investigation/?#id=9100). We aimed to investigate how microgravity influences immune cell responses by analysing blood samples from three astronauts between pre-flight and during their space mission on the ISS (post-launch Day 4, Day 7, and Day 10 in ISS). Hemogram analysis revealed no statistically significant differences in leukocyte, erythrocyte, haemoglobin, and haematocrit levels between pre-flight and in-flight samples, suggesting a stable haematological profile under microgravity conditions. T cell subpopulation analysis indicated fluctuations in effector memory T cells (TemEARLY and TemLATE), but these changes did not reach statistical significance. These findings suggest that, within the short duration of spaceflight, haematological and immune parameters remain largely stable, underscoring the need for further research into long-term immune adaptations in microgravity. As a next step, transcriptomic analysis will be performed to identify microgravity-associated gene candidates, and CRISPR-based knock-out of the genes in T cells will be generated to explore their functional roles in microgravity-induced immune modulation.

molecular biology↗

bioRxiv2024

Regulatory interactions between APOBEC3B N- and C-terminal domains

APOBEC3B (A3B) is implicated in DNA mutations that facilitate tumor evolution. Although structures of its individual N- and C-terminal domains (NTD and CTD) have been resolved through X-ray crystallography, the full-length A3B (fl-A3B) structure remains elusive, limiting understanding of its dynamics and mechanisms. In particular, the APOBEC3B C-terminal domain (A3Bctd) active site is frequently closed in models and structures. In this study, we built several new models of fl-A3B using integrative structural biology methods and selected a top model for further dynamical investigation. We compared dynamics of the truncated (A3Bctd) to the fl-A3B via conventional and Gaussian accelerated molecular dynamics (MD) simulations. Subsequently, we employed weighted ensemble methods to explore the fl-A3B active site opening mechanism, finding that interactions at the NTD-CTD interface enhance the opening frequency of the fl-A3B active site. Our findings shed light on the structural dynamics of fl-A3B, which may offer new avenues for therapeutic intervention in cancer.

biophysics↗

bioRxiv2024

Elucidation of the life cycle of a saprotrophic inoperculate discomycete that is associated with pinesap, using a polyphasic taxonomic approach

This study deals with the elucidation of the life cycle of an inoperculate discomycete that was eventually collected from conifer wood in South Germany and has become famous for the extraordinary diversity of its secondary metabolites when it was studied extensively during the 1990s. It had then been identified as Lachnum papyraceum (Lachnaceae, Helotiales) based on morphological traits, and extracts from its mycelial cultures were found to possess extraordinary nematicidal and antibiotic activities. Over 60 different secondary metabolites were finally identified from this fungus after extensive variation of culture media and scale up of production up to 100 litre scale. Among the main active principles were mycorrhizin A and chloromycorrhizin A, which had first been reported in 1987 from an unnamed "mycorrhizal" fungus of the hemiparasitic plant, Monotropa hypopitys (pinesap) that was isolated in Sweden. We noted that both the Lachnum strain and the original mycorrhizin producer were still available in the public domain, and decided to study them for comparison, using a multilocus phylogeny and also generated secondary metabolite profiles of both strains using analytical high performance liquid chromatography coupled to diode array and mass spectrometric detection (HPLC-DAD/MS). Surprisingly, the sequence data as well as the secondary metabolite profiles of both strains were highly similar, and it was also confirmed by phylogenetic methods that the strains are indeed nested within the genus Lachnum by comparison of their ITS, LSU and RBP2 sequences. The specimen called L. papyraceum in the old publications was tentatively re-identified by Hans-Otto Baral as L. cf. subvirgineum, but substantial further work on the taxonomy of the genus remains to be done, anyway. We conclude that some Lachnum species have a highly complex but all the more interesting life cycle, and the mycorrhizal symbiont partner may invade the host plant, where it may persist as an endophyte and finally turn saprotrophic on the wood of the senescent pine tree. The taxonomy of these fungi should also be further resolved in the future, using a polythetic concept that includes chemotaxonomic data and a multi-locus genealogy.

ecology↗

bioRxiv2024

Development of Allosteric Small Molecule APOBEC3B Inhibitors from In Silico Screening

The APOBEC3 (A3) family of enzymes are zinc metalloenzymes that catalyze the conversion of 2'- deoxycytidine to 2'-deoxyuridine in single stranded DNA. APOBEC3B (A3B), a member of the A3 family, has emerged as a key driver of genomic instability in many cancer types by mutating host DNA to drive tumorigenesis and therapy resistance. Small molecule inhibitors of A3B would extend the durability of current therapies by limiting mutations that promote tumor escape and therapy resistance. Consequently, a computer aided drug discovery (CADD) campaign was employed to identify inhibitors of A3B. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the c-terminal domain of A3B and virtually screened the ChemBridge Diversity Set ([~]110,000 small molecules) against both the active and predicted allosteric sites. High scoring compounds were selected for in vitro testing and triage, resulting in 13 candidate inhibitors. Using cysteine reactive probes, one of the original hit compounds was mapped to the computationally identified allosteric pocket. However, after resynthesis of representative chemotypes and further investigation using in vitro assays, none of the chemotypes retained inhibitory activity. Further analysis revealed that most of the inhibition observed in the primary assay was due to metal contamination in the screening sample, which resulted in the removal of the catalytic zinc from the enzyme active site. Although validated A3B inhibitors were not discovered in this study, we report a ligandable allosteric site on A3B and several cautionary insights for researchers developing small molecule inhibitors of zinc metalloenzymes. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/591187v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b7e577org.highwire.dtl.DTLVardef@13a1a43org.highwire.dtl.DTLVardef@192309eorg.highwire.dtl.DTLVardef@b60a31_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Targeting N-Myc in Neuroblastoma with Selective Aurora Kinase A Degraders

Summary ParagraphMYCN amplification is the most frequent genetic driver in high-risk neuroblastoma (NB) and strongly associated with poor prognosis.1,2 The N-Myc transcription factor, which is encoded by MYCN, is a mechanistically validated, yet challenging target for NB therapy development.3,4 In normal neuronal progenitors, N-Myc undergoes rapid degradation, while in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels.5,6 Allosteric Aurora-A inhibitors that displace N-Myc from binding can promote N-Myc degradation, but with limited efficacy.7-10 Here, we report a chemical approach to decrease N-Myc levels through the targeted protein degradation of Aurora-A. A first-in-class Aurora-A/N-Myc degrader, HLB-0532259 (compound 4), was developed from a novel Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of both Aurora-A and N-Myc with nanomolar potency and excellent selectivity and surpasses the cellular efficacy of established allosteric Aurora-A inhibitors. HLB-0532259 exhibits favorable pharmacokinetics properties and elicits tumor reduction in murine xenograft NB models. More broadly, this study delineates a novel strategy for targeting "undruggable" proteins that are reliant on accessory proteins for cellular stabilization.

cancer biology↗

bioRxiv2021

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-[A] resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

biochemistry↗

bioRxiv2021

Discovery of an IKK2 Site that Allosterically Controls Its Activation

I{kappa}B kinase 2/{beta} (IKK2) is a critical regulator of inflammation which is inducibly activated by a host of stimuli. Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Efforts to prevent these diseases by small-molecule inhibitors of IKK2 activity have not been successful. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. Here we focused on identifying inhibitors to block IKK2 activity from an allosteric site, not the ATP-binding pocket. Using virtual screening, we first identified several candidate allosteric sites and screened for potential small-molecule binders, and then selected candidates inhibitory to IKK2 activity using cell-based functional assays. Hydrogen deuterium exchange coupled to mass-spectrometry (HDX-MS) and MS-MS assays revealed that a class of benzoyl conjugates of pyrrolidinedione covalently bound to a site located at the interface of the kinase domain (KD) and the helical domain (SDD), and inhibited IKK2 activation allosterically by preventing phosphorylation of its activation loop serines. Additionally, this class of inhibitor partially blocks IKK2s catalytic activity by enhancing dynamics within the ATP binding pocket and likely the general active site. Hydrogen deuterium exchange (HDX) experiments further revealed that while binding of substrate ATP perturbs only the local structure surrounding its binding site, binding to ATP-competitive or allosteric inhibitors induces structural perturbations in an expansive area including the helical domain. We propose that these allosteric sites can act as specific targets for the development of novel potent IKK inhibitors. SIGNIFICANCEAberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. By combination of virtual screening and cell-based functional assays, we identified small-molecule binders of the class of benzoyl conjugates of pyrrolidinedione that block IKK2 activity from an allosteric site through covalent attachment and could be specific only for IKK2. HDX-MS and MS-MS assays identified a binding pocket with a Cys-Cys motif for these inhibitors, and revealed specific differences in IKK2 dynamics upon binding to substrate ATP vs ATP-competitive and allosteric inhibitors. Present work provides a framework for the development of allosteric inhibitors to combat IKK2-induced diseases inhibitors.

biochemistry↗

bioRxiv2020

Markov State Models and NMR Uncover an Overlooked Allosteric Loop in p53

The tumor suppressor p53 is the most frequently mutated gene in human cancer, and thus reactivation of mutated p53 is a promising avenue for cancer therapy. Analysis of wildtype p53 and the Y220C cancer mutant long-timescale molecular dynamics simulations with Markov state models and validation by NMR relaxation studies has uncovered the involvement of loop L6 in the slowest motions of the protein. Due to its distant location from the DNA-binding surface, the conformational dynamics of this loop has so far remained largely unexplored. We observe mutation-induced stabilization of alternate L6 conformations, distinct from all experimentally-determined structures, in which the loop is both extended and located further away from the DNA-interacting surface. Additionally, the effect of the L6-adjacent Y220C mutation on the conformational landscape of the functionally-important loop L1 suggests an allosteric role to this dynamic loop and the inactivation mechanism of the mutation. Finally, the simulations reveal a novel Y220C cryptic pocket that can be targeted for p53 rescue efforts. Our approach exemplifies the power of the MSM methodology for uncovering intrinsic dynamic and kinetic differences among distinct protein ensembles, such as for the investigation of mutation effects on protein function.

biophysics↗

bioRxiv2019

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

biochemistry↗