Biology subjects

Demir, M.

Publications and source records attributed to Demir, M..

13 recordsLinked to original sources

bioRxiv2026

Structural Basis for Nucleobase Activation by the Adenine DNA Glycosylase MutY

MutY excises adenine (A) from 8-oxo-guanine:adenine (OG:A) lesions in DNA to initiate base excision repair (BER) and thereby prevent mutations. A catalytic Glu, found at position 43 in the enzyme from Geobacillus stearothermophilus (Gs MutY), protonates the nucleobase at N7 to labilize the N-glycosidic bond. The resulting oxocarbenium ion transition state is stabilized by a covalent DNA-enzyme intermediate and resolved by nucleophilic attack to yield the beta-anomer abasic AP site product. The retaining SN1 mechanism for MutY posits deprotonation of the nucleophile by the catalytic Glu. Here we tested kinetic and structural consequences of Glu replacement and found that E43Q and E43S substitution variants were severely impaired, retained measurable activity, but engage the substrate nucleobase in an anti conformation, rotated by 180 {degrees} from the syn conformation seen in previous substrate complexes. The enzyme-generated AP product is observed in its alpha-anomer configuration for these Glu-replacement variants. Comparison with inverting adenine glycosylases that act on RNA or nucleosides shows that MutYs mechanism is uniquely reliant on one catalytic residue for both leaving group and nucleophile activation, a situation that may serve to ensure only rare adenines paired with OG are excised.

biochemistry↗

bioRxiv2026

Uncompetitive Allosteric Inhibitor of Mitochondrial Creatine Kinase Prevents Binding and Release of Creatine by Stabilization of Loop Closure

Mitochondrial creatine kinase (MtCK) is a key enzyme in energy buffering and homeostasis in cells. It catalyzes transfer of phosphoryl group from ATP to creatine. Overexpression of MtCK occurs in many cancer cells to meet elevated energy demands, which is associated with poor prognosis. This suggests that MtCK may be a promising target for cancer therapeutics. We sought to discover first-in-class selective inhibitors of MtCK with diverse mechanisms of action using high-throughput screening, biochemical characterization and cryo-EM studies. Through these studies, we identified diverse types of compounds that modulate activity of MtCK in vitro, including fast-equilibrium and time-dependent orthosteric and allosteric inhibitors. Select hits were subjected to in vitro enzymatic and binding assays to assess MtCK inhibition and binding. A subset of inhibitors was advanced into structural studies using cryo-EM resulting in the molecular structure of MtCK with and without bound substrates in complex with an allosteric uncompetitive inhibitor that we discovered. These studies identified compounds unique binding pocket on MtCK and established the molecular steps manifesting in the apparent uncompetitive mode of inhibition. We demonstrate that the compound stabilizes an active site loop in a closed conformation restricting access of the creatine substrate to and the release of phosphocreatine product from its binding pocket. These findings establish chemical tools suitable for validation of MtCK as a promising breast cancer target with high therapeutic potential and build a foundation for future structure-guided optimization of the hit compounds of MtCK we identified and de novo rational design of novel MtCK inhibitors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/696683v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1fd9870org.highwire.dtl.DTLVardef@230e31org.highwire.dtl.DTLVardef@178d8fdorg.highwire.dtl.DTLVardef@edeca6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Hepatocyte Circadian Clocks Control Cholesterol Metabolism and Protect From Metabolic Dysfunction-Associated Steatohepatitis (MASH)

The circadian clock synchronizes physiological processes with the 24-hour light-dark cycle. Clock disruption contributes to metabolic disorders, including metabolic dysfunction-associated steatohepatitis (MASH). Here, we investigated the role of the hepatocyte clock in MASH using hepatocyte-specific Bmal1 deletion (Hep-Bmal1KO) mice. Hep-Bmal1KO mice showed faster MASH progression with increased hepatic cholesterol, inflammation, and fibrosis. Transcriptomic and lipidomic analyses revealed dysregulated cholesterol metabolism in Hep-Bmal1KO mice, marked by reduced expression and disrupted rhythmicity of key cholesterol-related genes. Bioinformatic analyses identified Chrebp as a potential co-regulator of these transcriptional changes. In an in vitro model with palmitate exposure and gene silencing, we found that Bmal1, but not Chrebp, regulated cholesterol accumulation, indicating Bmal1s specific role in hepatic cholesterol metabolism. Translating our findings to a human patient cohort revealed a significantly shifted circadian phase, despite no marked effect on hepatic cholesterol levels in the livers of patients with more advanced liver disease (i.e., MASH) compared to simple steatosis. Taken altogether, our findings offer a roadmap to understand the hepatocyte clocks role in MASH and its potential as a therapeutic target.

pathology↗

bioRxiv2024

Identification of methylation-sensitive human transcription factors using meSMiLE-seq

Transcription factors (TFs) are key players in eukaryotic gene regulation, but the DNA binding specificity of many TFs remains unknown. Here, we assayed 284 mostly poorly characterized, putative human TFs using selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq), revealing 72 new DNA binding motifs. To investigate whether some of the 158 TFs for which we did not find motifs preferably bind epigenetically modified DNA (i.e. methylated CG dinucleotides), we developed methylation-sensitive SMiLE-seq (meSMiLE-seq). This microfluidic assay simultaneously probes the affinity of a protein to methylated and unmethylated DNA, augmenting the capabilities of the original method to infer methylation-aware binding sites. We assayed 114 TFs with meSMiLE-seq and identified DNA-binding models for 48 proteins, including the known methylation-sensitive binding modes for POU5F1 and RFX5. For 11 TFs, binding to methylated DNA was preferred or resulted in the discovery of alternative, methylation-dependent motifs (e.g. PRDM13), while aversion towards methylated sequences was found for 13 TFs (e.g. USF3). Finally, we uncovered a potential role for ZHX2 as a putative binder of Z-DNA, a left-handed helical DNA structure which is adopted more frequently upon CpG methylation. Altogether, our study significantly expands the human TF codebook by identifying DNA binding motifs for 98 TFs, while providing a versatile platform to quantitatively assay the impact of DNA modifications on TF binding.

genomics↗

bioRxiv2024

Structure of human MUTYH and functional profiling of cancer-associated variants reveal an allosteric network between its cluster cofactor and active site required for DNA repair

MUTYH is a clinically important DNA glycosylase that thwarts mutations by initiating base-excision repair at 8-oxoguanine (OG):A lesions. The roles for its [4Fe-4S] cofactor in DNA repair remain enigmatic. Functional profiling of cancer-associated variants near the [4Fe-4S] cofactor revealed that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal center and bound substrate DNA tightly but were completely inactive. We solved the crystal structure of human MUTYH bound to a transition state mimic and this revealed that Arg241 and Asn238 build an H-bond network connecting the [4Fe-4S] cluster to the catalytic Asp236 that mediates base excision. The structure of the bacterial MutY variant R149Q, along with molecular dynamics simulations of the human enzyme, show the cofactor functions to position and activate the catalytic Asp. These results suggest that allosteric cross-talk between the DNA binding [4Fe-4S] cofactor and the base excision site regulate DNA repair.

biochemistry↗

bioRxiv2024

Structural Basis for Substrate Binding, Catalysis and Inhibition of Breast Cancer Target Mitochondrial Creatine Kinase by Covalent Inhibitor via Cryo-EM

Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. High levels of MtCK observed in Her2+ breast cancer and inhibition of breast cancer cell growth by substrate analog, cyclocreatine, indicate dependence of cancer cells on the energy shuttle for cell growth and survival. Hence, understanding the key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present the mutational and structural investigation on understudied ubiquitous mitochondrial creatine kinase (uMtCK). Our cryo-EM structures and biochemical data on uMtCK showed closure of the loop comprising residue His61 is specific to and relies on creatine binding and the reaction mechanism of phosphoryl transfer depends on electrostatics in the active site. In addition, the previously identified covalent inhibitor CKi showed inhibition in breast cancer BT474 cells, however our biochemical and structural data indicated that CKi is not a potent inhibitor for breast cancer due to strong dependency on the covalent link formation and inability to induce conformational changes upon binding.

biochemistry↗

bioRxiv2023

Adaptive Artificial Intelligence to Teach Interactive Molecular Dynamics in the Context of Human-Computer Interaction

Artificial Intelligence (AI) can be easily integrated into virtual education to drive adaptive instruction and real-time constructive feedback to students, offering a possible conduit for fostering discovery curiosity in learners. This study examines and characterizes Human-AI-Teaming (HAT) coordination dynamics to monitor the inception of discovery curiosity in online laboratories of interactive molecular dynamics (IMD). We used molecular physics measures (kinetic/ potential energy and action) obtained from simple and complex examples of simulated mouse tracking datasets in IMD log files as a proxy for understanding the context of molecular sciences and developing novel interactions for inquiry. These measures are good features of our HAT context because kinetic energy reflects the systems atoms overall motion regarding the individual atoms speed. While kinetic energy represents if a learner applies artificial forces to the task, potential energy can be AIs response to these forces. The action is a systems-level reaction to the changes during the task. By applying nonlinear dynamical systems methods to the physics measures, we extracted the Largest Lyapunov Exponent and Determinism metrics as HATs coordination stability and predictability, respectively. The findings underline that while the more complex IMD task required less stable and predictable HAT coordination dynamics, the simple task is more. One explanation is that AI needs to anticipate the learner by providing feedback at the right time and place during the more complex IMD task to initiate and sustain the learners discovery curiosity. In IMD, future HAT design should consider coordination dynamics for fostering discovery curiosity and practical learning.

bioengineering↗

bioRxiv2023

Non-alcoholic steatohepatitis disrupts diurnal liver transcriptome rhythms in mice

Background & AimsThe liver ensures organismal homeostasis through modulation of physiological functions over the course of the day. How liver diseases such as non-alcoholic steatohepatitis (NASH) affects daily transcriptome rhythms in the liver remains elusive. To start closing this gap, we evaluated the impact of NASH on the diurnal regulation of the liver transcriptome in mice. Along this, we investigated how stringent consideration of circadian rhythmicity affects the outcomes of NASH transcriptome analyses. Approach & ResultsComparative rhythm analysis of the liver transcriptome from diet-induced NASH and control mice revealed an almost 3h phase advance in global gene expression rhythms. Rhythmically expressed genes associated with DNA repair and cell cycle regulation showed increased overall expression and circadian amplitude. In contrast, lipid and glucose metabolism associated genes showed loss of circadian amplitude, reduced overall expression, and phase advances in NASH livers. Comparison of NASH-induced liver transcriptome responses between published studies revealed little overlap (12%) in differentially expressed genes (DEGs). However, by controlling for sampling time and using circadian analytical tools, a 7-fold increase in DEG detection was achieved compared to methods without time control. ConclusionsNASH had a strong effect on circadian liver transcriptome rhythms with phase- and amplitude-specific effects for key metabolic and cell repair pathways, respectively. Accounting for circadian rhythms in NASH transcriptome studies markedly improves DEGs detection and enhances reproducibility.

physiology↗

bioRxiv2021

Odor motion sensing enables complex plume navigation

Studies dating back a century (Flugge, 1934) have stressed the critical role of the wind as the primary directional cue in odor plume navigation. Here, we show that Drosophila shape their navigational decisions using a second directional cue - the direction of motion of odors - which they detect from the temporal correlations of the odor signal between their two antennae. Using a high-resolution virtual reality paradigm to deliver spatiotemporally complex fictive odors to freely-walking flies, we demonstrate that such odor direction sensing is computationally equivalent to motion detection algorithms underlying motion detection in vision. Simulations and theoretical analysis of turbulent plumes reveal that odor motion contains valuable directional information absent from the airflow; indeed, this information is used by both Drosophila and virtual agents to navigate naturalistic odor environments. The generality of our findings suggests that odor direction sensing is likely used throughout the animal kingdom, and could significantly improve olfactory robot navigation in harsh chemical environments.

neuroscience↗

bioRxiv2020

Walking Drosophila navigate complex plumes using stochastic decisions biased by the timing of odor encounters

Insects find food, mates, and egg-laying sites by tracking odor plumes swept by complex wind patterns. Previous studies have shown that moths and flies localize plumes by surging upwind at odor onset and turning cross- or downwind at odor offset. Less clear is how, once within the expanding cone of the odor plume, insects use their brief encounters with individual odor packets, whose location and timing are random, to progress towards the source. Experiments and theory have suggested that the timing of odor encounters might assist navigation, but connecting behaviors to individual encounters has been challenging. Here, we imaged complex odor plumes simultaneous with freely-walking flies, allowing us to quantify how behavior is shaped by individual odor encounters. Combining measurements, dynamical models, and statistical inference, we found that within the plume cone, individual encounters did not trigger reflexive surging, casting, or counterturning. Instead, flies turned stochastically with stereotyped saccades, whose direction was biased upwind by the timing of prior odor encounters, while the magnitude and rate of saccades remained constant. Odor encounters did not strongly affect walking speed. Instead, flies used encounter timing to modulate the rate of transitions between walks and stops. When stopped, flies initiated walks using information from multiple odor encounters, suggesting that integrating evidence without losing position was part of the strategy. These results indicate that once within the complex odor plume, where odor location and timing are unpredictable, animals navigate with biased random walks shaped by the entire sequence of encounters.

neuroscience↗

bioRxiv2019

Controlling and measuring dynamic odorant stimuli in the laboratory

Animals experience complex odorant stimuli that vary widely in composition, intensity and temporal properties. However, stimuli used to study olfaction in the laboratory are much simpler. This mismatch arises from the challenges in measuring and controlling them precisely and accurately. Even simple pulses can have diverse kinetics that depend on their molecular identity. Here, we introduce a model to describe how stimulus kinetics depend on the molecular identity of the odorant and the geometry of the delivery system. We describe methods to deliver dynamic odorant stimuli of several types, including broadly distributed stimuli that reproduce some of the statistics of naturalistic plumes, in a reproducible and precise manner. Finally, we introduce a method to calibrate a Photo-Ionization Detector to any odorant it can detect, using no additional components. Our approaches are affordable and flexible and can be used to advance our understanding of how olfactory neurons encode real-world odor signals.

neuroscience↗

bioRxiv2018

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

Mutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.\n\nOne Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

cell biology↗

bioRxiv2017

Identification and dynamics of the DHHC16-DHHC6 palmitoylation cascade

S-Palmitoylation is the only reversible post-translational lipid modification. Knowledge about the DHHC family of palmitoyltransferases is very limited. Here we show that mammalian DHHC6, which modifies key proteins of the endoplasmic reticulum, is controlled by an upstream palmitoyltransferase, DHHC16, revealing the first palmitoylation cascade. Combination of site specific mutagenesis of the three DHHC6 palmitoylation sites, experimental determination of kinetic parameters and data-driven mathematical modelling allowed us to obtain detailed information on the 8 differentially palmitoylated DHHC6 species. We found that species rapidly interconvert through the action of DHHC16 and the Acyl Protein Thioesterase APT2, that each species varies in terms of turnover rate and activity, altogether allowing the cell to robustly tune its DHHC6 activity.

cell biology↗