Biology subjects

Saha, P.

Publications and source records attributed to Saha, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

circEPSTI1 Enables Dengue Innate Immune Evasion by Sponging miR-942-5p, a Repressor of AKT1 and the Viral Genome

Circular RNAs (circRNAs) have recently been identified as crucial regulators of gene expression in various diseases. Their role in dengue remains unexplored. In this study, circEPSTI1 induction was observed in peripheral blood mononuclear cells (PBMCs) and plasma of dengue patients. The induction of circEPSTI1 is interferon-dependent and enhances DENV infection by sponging the expression of miR-942-5p. The antiviral role of miR-942-5p is mediated by bidirectional inhibition through targeting conserved genomic sequences of the DENV genome of different subtypes across NS1, NS3, and NS5 and the host AKT1 signaling pathway. RNA-seq analysis of DENV-infected circEPSTI1 knockdown A549 cells identified SERPINE1 and AKT1 signaling pathways to be dysregulated significantly. circEPSTI1 relieves the inhibition of miR-942-5p over DENV genomic RNA and the host SERPINE1 to activate AKT1 signaling. The activation of AKT1 signaling facilitates cell survival and enhances DENV replication. The expression of SERPINE1 and circEPSTI1 was upregulated in Dengue patients, and pharmaceutical inhibition of SERPINE1 through Tiplaxtinin inhibits the DENV replication by reducing the expression of circEPSTI1. Overall, our result demonstrates the therapeutic potential of Tiplaxtinin by targeting the circ-EPSTI1-miR-942-5p-SERPINE1-AKT axis in dengue treatment.

immunology↗

bioRxiv2025

Beige fat appearance during lipoatrophy reveals trade-offs between metabolic homeostasis and female fertility

White adipose tissue (WAT) meets the energetic demands required for estrous cycle regularity, ovulation, and reproductive success in women. Here, we report WAT loss and subsequent female-specific compensatory beige fat recruitment impact metabolic and reproductive outcomes in mice. Female mice that undergo progressive lipoatrophy ( fat-less) displayed disrupted estrous cycles, reduced ovarian reserve, and subfertility. These effects were attributable to an accumulation of thermogenic beige fat cells in residual subcutaneous WAT depots and greater energy expenditure. Conversely, high-fat diet lowered energy expenditure and rescued estrous cycle regularity among fat-less mice compared to littermate controls, despite profound insulin resistance and metabolic dysfunction. Together, these findings provide evidence of trade-offs between the maintenance of metabolic homeostasis and reproduction, where increased energy expenditure and beige fat cell emergence compensate for fat loss but occur at the expense of fecundity in female mice.

cell biology↗

bioRxiv2025

Targeting anemia-induced CD71+ reticulocytes protects mice from Plasmodium infection

Malaria, caused by Plasmodium spp., is a global health concern linked to anemia and increased mortality. Compensatory erythropoiesis seen during acute anemia results in an increased circulating reticulocyte count (i.e., immature RBC) a key factor in understanding the relationship between pre-existing anemia and Plasmodium burden. Reticulocytes in mice are marked by transferrin receptor (CD71+) and glycophorin A-associated protein (Ter119+). To model acute anemia with increased reticulocytes, C57BL/6 mice were either bled (i.e. phlebotomized) or administered phenylhydrazine, before being infected with Plasmodium yoelii (P. yoelii), a mouse-specific strain with a preference for reticulocytes. In P. yoelii-infected anemic mice, we observed heightened parasitemia and significant body weight loss compared with non-anemic P. yoelii-infected mice. Additionally, serum inflammatory cytokines, erythropoietin, and liver injury markers, along with hemozoin deposition significantly increased in anemic P. yoelii-infected mice. RBC transfusion from healthy normal donors to P. yoelii-infected anemic recipient mice ameliorated anemia by reducing overall reticulocyte count and increasing mature RBC count. RBC transfusion rescued body weight loss, decreased parasitemia, and reduced serum erythropoietin levels. Finally, to confirm the role of reticulocytes in P. yoelii infection, reticulocytes were depleted using anti-CD71 monoclonal antibody in P. yoelii-infected mice. We observed improvement in hematologic parameters and stark reduction in parasitemia in both pre-existing anemic and non-anemic P. yoelii-infected mice. Collectively, our results suggest that pre-existing anemia may increase the risk of Plasmodium infection due to the greater reticulocytes population. Anti-CD71 treatment in Plasmodium infection may offer a novel therapeutic strategy to combat Plasmodium infection and malaria. Grant supportThis work was supported by grants from the Crohns and Colitis Foundation (CCF) and American Heart Association (AHA) Career Development Award (854385 and 855256 respectively) to Piu Saha; grant from the National Institutes of Health (NIH) to Matam Vijay-Kumar (DK134053) and Liver Scholar Award from American liver Foundation to Beng San Yeoh.

pathology↗

bioRxiv2024

The ketone body β-Hydroxybutyrate mediated epigenetic chromatin β-hydroxybutyrylation protects kidneys

Starvation, intermittent fasting and exercise, all of which are recommended lifestyle modifiers share a common metabolic signature, ketogenesis to generate the ketone bodies, predominantly {beta}-hydroxybutyrate. {beta}-hydroxybutyrate exerts beneficial effects across various contexts, preventing or mitigating disease. We hypothesized that these dynamic health benefits of {beta}-hydroxybutyrate might stem from its ability to regulate genome architecture through chromatin remodeling via histone {beta}-hydroxybutyrylation, thereby influencing the transcriptome. Focusing on the kidney, which is an end organ protected by {beta}-hydroxybutyrate, we examined histone {beta}-hydroxybutyrylation-mediated chromatin remodeling. Notably, regions of the genome associated with lipid catabolism were predominantly in an open chromatin configuration, leading to active transcription and translation. Significant {beta}-hydroxybutyrylation was observed in the kidneys and the most highly upregulated gene actively transcribed and translated was 3-hydroxy-3-methyglutaryl CoA Synthase 2 (Hmgcs2), a gene responsible for the biosynthesis of {beta}-hydroxybutyrate in mitochondria. In contrast, regions with more compact chromatin structures were enriched with genes related to immune function such as protein tyrosine phosphatase receptor type C (Ptprc) and lymphocyte cytosolic protein 1 (Lcp1), which exhibited reduced transcription and translation. These results reveal that renal epigenetic histone {beta}-hydroxybutyrylation is a novel mechanism by which transcriptional regulation of both energy metabolism and immune function occur concomitantly to protect kidneys and lower hypertension. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/628574v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@f16cdorg.highwire.dtl.DTLVardef@a22427org.highwire.dtl.DTLVardef@f3a6d3org.highwire.dtl.DTLVardef@4d705d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

physiology↗

bioRxiv2024

Discovery of alternative stable states in a synthetic human gut microbial community

AbstractSeveral human-associated microbial communities exist in multiple configurations and can change their composition in response to perturbations, remaining in an altered state even after the perturbation ends. Multistability has been previously proposed to explain this behavior for gut microbiota in particular, but has not been clearly demonstrated experimentally. Here, we first investigated the life history strategies of three common human gut bacteria to identify mechanisms driving alternative states. We then used this data to build and parameterize a kinetic model, which predicted that alternative states emerge due to phenotype switching between subpopulations of the same species. Perturbation experiments supported these predictions, and confirmed the existence of alternative states. Finally, simulations showed that phenotype switching can also explain alternative states in larger communities. Thus, a transient perturbation combined with metabolic flexibility is sufficient for alternative communities to emerge, implying that they are not necessarily explained by differences between individuals. One-Sentence SummaryWe demonstrate the existence of alternative states in a human gut microbial community and propose phenotype switching as a mechanism explaining their emergence.

microbiology↗

bioRxiv2024

Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts

Pediatric malignancies frequently harbor chromosomal translocations that induce expression of fusion oncoproteins. The EWSR1-FLI1 fusion oncoprotein acts as a neomorphic transcription factor and is the dominant genetic driver of Ewings sarcoma. Interrogation of the mechanisms by which EWSR1-FLI1 drives tumorigenesis has been limited by a lack of model systems to precisely and selectively control its expression in patient-derived cell lines and xenografts. Here, we report the generation of a panel of patient-derived EWS cell lines in which inducible protein degrons were engineered into the endogenous EWSR1-FLI1 locus. These alleles enabled rapid and efficient depletion of EWSR1-FLI1. Complete suppression of EWSR1-FLI1 induced a reversible cell cycle arrest at the G1-S checkpoint, and we identified a core set of transcripts downstream of EWSR1-FLI1 across multiple cell lines and degron systems. Additionally, depletion of EWSR1-FLI1 potently suppressed tumor growth in xenograft models validating efforts to directly target EWSR1-FLI1 in Ewings sarcoma.

cancer biology↗

bioRxiv2024

Predictive and Experimental Motif Interaction Analysis Identifies Functions of the WNK-OSR1/SPAK Pathway

The WNK-OSR1/SPAK protein kinase signaling pathway regulates ion homeostasis and cell volume, but its other functions are poorly understood. To uncover undefined signaling functions of the pathway we analyzed the binding specificity of the conserved C-terminal (CCT) domains of OSR1 and SPAK to find all possible interaction motifs in human proteins. These kinases bind the core consensus sequences R-F-x-V/I and R-x-F-x-V/I. Motifs were ranked based on sequence, conservation, cellular localization, and solvent accessibility. Out of nearly 3,700 motifs identified, 90% of previously published motifs were within the top 2% of those predicted. Selected candidates (TSC22D1, CAVIN1, ATG9A, NOS3, ARHGEF5) were tested. Upstream kinases WNKs 1-4 and their close relatives, the pseudokinases NRBP1/2, contain CCT-like domains as well. We identified additional distinct motif variants lacking the conserved arginine previously thought to be required, and found that the NRBP1 CCT-like domain binds TSC22D1 via the same motif as OSR1 and SPAK. Our results further highlight the rich and diverse functionality of CCT and CCT-like domains in connecting WNK signaling to cellular processes.

biochemistry↗

bioRxiv2024

Improving Olfactory Receptor Structure Modeling via Hybrid Methods

Understanding the structure of Olfactory Receptors (ORs) is pivotal in deciphering the molecular complexities of smell, a sense crucial for health, and survival, and holding immense therapeutic potential. However, the scarcity of detailed experimental data on ORs has hindered progress, demanding inventive approaches. This study explores various structure prediction methods tailored to ORs based on their phylogenetic and structural characteristics, using OR51E2 as a reference. We employed a hybrid homology modeling approach, utilizing AlphaFold structures, yielding models with lower RMSD (1.019 {degrees}A across pruned/significant pairs and 2.33 {degrees}A over-all) and enhanced structural metrics compared to standalone AlphaFold (RMSD - 2.5 {degrees}A) predictions. Our pipeline successfully replicated experimental findings for OR51E2 and was applied to homologous ORs: OR51E1, OR51D1, and OR51G2. Various tools were also used to predict potential binding sites for each receptor. Molecular dynamics simulations validated the stability of these OR models in a lipid bilayer environment, with biophysical analyses revealing that AlphaFold models exhibit relatively less ideal behavior compared to the Hybrid Model. Our study presents a targeted approach to investigate and generate optimum OR structures for further conformational analyses.

bioinformatics↗

bioRxiv2024

Crude extract of Ruellia tuberosa L. flower induces intracellular ROS, promotes DNA damage and apoptosis in Triple Negative Breast Cancer Cells

Ethnophamacological relevanceIn the traditional folklore medicine system, the primary uses of Ruellia tuberosa L. include as a diuretic, anti-hypertensive, antipyretic, anti-diabetic, analgesic, and gastroprotective agent. Some reports also demonstrated that it has been used to treat gonorrhea-like diseases. PurposeExploring the anti-cancer potential of the methanolic extract of Ruellia tuberosa L. flower (RTME) with special emphasis on human triple-negative breast cancer (TNBC) and investigating the possible signaling networks and regulatory pathways underlying it. MethodsPreparation of RTME and identifying the possible phytochemicals through GC-MS analysis. The anti-cancer potential of RTME was executed through in-vitro cytotoxicity assay, clonogenic assay, wound healing assay, ROS generation assay, cell cycle arrest, apoptotic nuclear morphology study, cellular apoptosis study, mitochondrial membrane potential (MMP) alteration study, protein and gene expressions alteration study. Apart from this, toxicological status and in-silico molecular docking studies were also conducted. ResultsFrom this study, it was obvious that several phytochemicals within RTME have the potential to act as anti-cancerous agents. RTME was found to exhibit significant in-vitro cytotoxicity along with a reduction in colony formation and inhibition of cell migratory potential in MDA-MB-231 cells. RTME also induced intracellular ROS, promoted G0/G1 cell cycle arrest, caused mitochondrial membrane potential (MMP) alteration, and promoted cell death. From the pro- and anti-apoptotic marker study through the western blot and the qRT-PCR analysis, it was revealed that RTME promoted the intrinsic pathway of apoptosis. Furthermore, blood parameters and histological analysis revealed that RTME doesnt exhibit any toxic effect on female Balb/C mice. Finally, an in-silico molecular docking study revealed that the three identified lead phytochemicals in RTME show strong receptor-ligand interactions with the anti-apoptotic Bcl-2 and give a clue to the possible molecular mechanism of the RTME extract. ConclusionFrom the findings, it was concluded that RTME has a significant therapeutic potential against TNBC which could be an alternative option for anti-cancer drug development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/586749v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@72780corg.highwire.dtl.DTLVardef@1276029org.highwire.dtl.DTLVardef@1a2d91org.highwire.dtl.DTLVardef@a5cb44_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPreparation of methanolic extract of Ruellia tuberosa L. flower, C_LIO_LIIdentification of phytochemicals from the methanolic extract of Ruellia tuberosa L. flower, C_LIO_LIMethanolic extract of Ruellia tuberosa L. (RTME) flower exhibited significant anti-cancer potential in triple-negative breast cancer (TNBC) cells, MDA-MB-231 through induction of intracellular ROS, G0/G1 cell cycle arrest, and apoptosis, C_LIO_LIToxicological assessments of RTME on female Balb/C mice, C_LIO_LIIn-silico assessments of lead phytochemicals with the target anti-apoptotic protein, bcl-2 C_LI

cancer biology↗

bioRxiv2024

Modulation of SARS-CoV-2 spike binding to ACE2 throughconformational selection

The first step of SARS-CoV-2 infection involves the interaction between the trimeric viral spike protein (S) and the host angiotensin-converting enzyme 2 (ACE2). The receptor binding domain (RBD) of S adopts two conformations: open and closed, respectively, accessible and inaccessible to ACE2. Therefore, RBD motions are suspected to affect ACE2 binding; yet a quantitative description of the underlying mechanism has been elusive. Here, using single-molecule approaches, we visualize RBD opening and closing and probe the S/ACE2 interaction. Our results show that RBD dynamics affect ACE2 binding but not unbinding. The resulting modulation is quantitatively predicted by a conformational selection model in which each protomer behaves independently. Our work reveals a general molecular mechanism affecting binding affinity without altering binding strength, helping to understand coronavirus infection and immune evasion.

biophysics↗

bioRxiv2024

NRP1-expressing myeloid cells promote arteriogenesis in the ischemic limb

Peripheral arterial disease can cause limb threatening blood flow restriction, requiring amputation in up to a third of patients despite contemporary treatments. Stimulating new blood vessel growth in ischemic limbs using growth factors such as VEGF-A or supplying mixed cell populations via autologous transplantation has been investigated as a therapy for surgically intractable cases, but to date has proven ineffective in randomised controlled clinical studies. To identify more potent pro-arteriogenic cell populations, we have investigated VEGF-A responsive myeloid cell populations in a mouse model of severe hindlimb ischemia. We found that NRP1 ablation from monocytes and macrophages impaired arteriogenesis in the adductor muscle and flow recovery in the injured limb. Vice versa, the exogenous delivery of NRP1-expressing (but not NRP1-negative) macrophages enhanced arteriogenesis in the adductor and flow recovery in the ischemic limb in a VEGF signalling-dependent manner. Further, patient-derived NRP1-expressing monocytes promoted vascular morphogenesis ex vivo. As the levels of circulating NRP1-expressing monocytes were raised in patients with limb ischemia, the therapeutic delivery of autologous NRP1-expressing monocytes could be explored as a treatment for critical limb ischemia.

pathology↗

bioRxiv2023

Sex-specific role for the long noncoding RNA Pnky in mouse behavior

The human brain expresses thousands of different long noncoding RNAs (lncRNAs), and aberrant expression of specific lncRNAs has been associated with cognitive and psychiatric disorders. While a growing number of lncRNAs are now known to regulate neural cell development and function, relatively few have been shown to underlie animal behavior, particularly with genetic strategies that establish lncRNA function in trans. Pnky is an evolutionarily conserved, neural lncRNA that regulates brain development. Using mouse genetic strategies, we show that Pnky has sex-specific roles in mouse behavior and that this lncRNA underlies specific behavior by functioning in trans. Male Pnky-knockout (KO) mice have deficits in cued fear recall, a type of Pavlovian associative memory. In female Pnky-KO mice, the acoustic startle response (ASR) is increased and accompanied by a decrease in prepulse inhibition (PPI), both of which are behaviors altered in affective disorders. Remarkably, expression of Pnky from a bacterial artificial chromosome (BAC) transgene reverses the ASR phenotype of female Pnky-KO mice, demonstrating that Pnky underlies specific animal behavior by functioning in trans. More broadly, these data provide genetic evidence that a lncRNA gene and its function in trans can play a key role in the behavior of adult mammals, contributing fundamental knowledge to our growing understanding of the association between specific lncRNAs and disorders of cognition and mood.

neuroscience↗

bioRxiv2023

Myeloperoxidase activity predicts atherosclerotic plaque disruption and atherothrombosis

BackgroundUnstable atherosclerotic plaque is characterized by increased myeloperoxidase (MPO) activity. As unstable plaque is vulnerable to disruption and ensuing thrombosis, we examined whether plaque MPO activity predicts atherothrombosis in a pre-clinical model and correlates with ruptured human atheroma. MethodsTo assess if plaque MPO activity predicts atherothrombosis, rabbits were subjected to aortic endothelial denudation, cholesterol feeding, in vivo magnetic resonance imaging (MRI) of MPO activity using MPO-Gd (gadolinium), followed by pharmacological triggering of atherothrombosis, histology, and MPO activity determined by liquid chromatography tandem mass spectrometry (LC-MSMS) by quantifying the MPO-specific product of hydroethidine, 2-chloroethidium. To correlate plaque MPO activity to ruptured human atheroma, ex vivo determination of MPO activity by MPO-Gd retention in carotid endarterectomy (CEA) specimens was correlated with in vivo MRI plaque phenotyping in patients, histology, and MPO activity determined by LC-MSMS. ResultsIn rabbits, pre-trigger in vivo MPO activity, validated by LC-MSMS and histology, was higher in thrombosis-prone than thrombosis-resistant plaques and lesion-free segments (R1 relaxation rate = 2.2 {+/-} 0.4 versus 1.6 {+/-} 0.2 and 1.5 {+/-} 0.2 s-1, respectively, p<0.0001), and it predicted atherothrombosis. In CEA specimens, MPO-Gd retention was greater in histologic and MRI-graded American Heart Association (AHA) type VI than types III, IV and V plaques ({Delta}R1 relaxation rate from baseline = 48 {+/-} 6 versus 16 {+/-} 7, 17 {+/-} 8 and 23 {+/-} 8%, respectively, p<0.0001). This association was confirmed by comparing AHA grade to MPO activity determined by LC-MSMS (277 {+/-} 338 versus 7 {+/-} 6, 11 {+/-} 12 and 42 {+/-} 39 pmol 2-chloroethidium/mg protein for type VI versus type III-V plaques, respectively, p=0.0008). ConclusionsMPO activity is elevated in thrombosis-prone rabbit and ruptured human atheroma. Non-invasive molecular imaging of MPO activity predicts atherothrombosis, highlighting the potential of arterial MPO activity to detect vulnerable, destabilized atherosclerosis.

biochemistry↗

bioRxiv2023

PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

Adaptation to hypothermic stress is important for skeletal muscle cells, but a comprehensive knowledge of molecular mediators is lacking. We show that adaptation to mild hypothermia (320C) improves the ability of skeletal muscle myoblasts to differentiate into myotubes in vitro. We performed proteomic analysis of mouse myoblasts exposed to mild hypothermia for various time points and identified dynamic changes in mitochondrial metabolism and proteostasis. This revealed that RBM3, an RNA-binding protein, increases progressively with acute and chronic exposure to hypothermic stress, and is necessary for the enhanced differentiation upon hypothermic adaptation. We also demonstrate that overexpression of RBM3 at physiological temperatures is sufficient to (i) enhance mitochondrial metabolism as judged by a decrease in the AMPK energy-sensing pathway, (ii) increase levels of proteins associated with translation and increase levels of 4E-BP1 phosphorylation, (iii) increase stem cell markers (MyoD1, PAX7), and improve differentiation of myoblasts from both young and aged mice.

biochemistry↗

bioRxiv2023

Augmentation of Histone Deacetylase 6 Activity Impairs Mitochondrial Respiratory Complex I in Ischemic/Reperfused Diabetic Hearts

BACKGROUNDDiabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor (TNF) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and {beta}-oxidation. Here we examined how HDAC6 regulates TNF production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts. METHODSHDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNF and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups. RESULTSMyocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNF levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNF with an anti-TNF monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNF levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNF levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSIONSAugmenting HDAC6 activity inhibits mCI activity by increasing TNF levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIschemic heart disease (IHS) is a leading cause of death globally, and its presence in diabetic patients is a grievous combination, leading to high mortality and heart failure. C_LIO_LIDiabetes impairs assembly of mitochondrial complex I (mCI), complex III dimer, and complex IV monomer into the respiratory chain supercomplexes, resulting in electron leak and the formation of reactive oxygen species (ROS). C_LIO_LIBy oxidizing reduced nicotinamide adenine dinucleotide (NADH) and reducing ubiquinone, mCI physiologically regenerates NAD+ to sustain the tricarboxylic acid cycle and {beta}-oxidation. C_LI What New Information Does This Article Contribute?O_LIMyocardial ischemia/reperfusion injury (MIRI) and diabetes as comorbidities augment myocardial HDCA6 activity and generation of tumor necrosis factor (TNF), which inhibit myocardial mCI activity. C_LIO_LIGenetic disruption of histone deacetylase 6 (HDAC6) decreases mitochondrial NADH levels and augments mCI activity in type 1 diabetic mice undergoing MIRI via decreasing TNF production, leading to decreases in MIRI. C_LIO_LIPretreatment of type 2 diabetic db/db mice with a HDAC6 inhibitor, tubastatin A (TSA), decreases mitochondrial NADH levels and augments mCI activity by decreasing TNF levels, leading to improvements in cardiac function. C_LI Patients with diabetes are more susceptible to MIRI than non-diabetics with greater mortality and resultant heart failure. There is an unmet medical need in diabetic patients for the treatment of IHS. Our biochemical studies find that MIRI and diabetes synergistically augment myocardial HDAC6 activity and generation of TNF, along with cardiac mitochondrial fission and low bioactivity of mCI. Intriguingly, genetic disruption of HDAC6 decreases the MIRI-induced increases in TNF levels, concomitant with augmented mCI activity, decreased myocardial infarct size, and ameliorated cardiac dysfunction in T1D mice. Importantly, treatment of obese T2D db/db mice with TSA reduces the generation of TNF and mitochondrial fission and enhances mCI activity during reperfusion after ischemia. Our isolated heart studies revealed that genetic disruption or pharmacological inhibition of HDAC6 reduces mitochondrial NADH release during ischemia and ameliorates dysfunction of diabetic hearts undergoing MIRI. Furthermore, HDAC6 knockdown in cardiomyocytes blocks high glucose- and exogenous TNF-induced suppression of mCI activity in vitro, implying that HDAC6 knockdown can preserve mCI activity in high glucose and hypoxia/reoxygenation. These results demonstrate that HDAC6 is an important mediator in MIRI and cardiac function in diabetes. Selective inhibition of HDAC6 has high therapeutic potential for acute IHS in diabetes.

pharmacology and toxicology↗

bioRxiv2022

Vitamin B2 enables peroxisome proliferator-activated receptor α regulation of fasting glucose availability

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2 deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPAR, including those required for gluconeogenesis. Treatment with the PPAR agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. Overall, these findings reveal PPAR governs metabolic responses to FAD availability and nominate its pharmacologic activation as strategies for organic acidemias.

physiology↗

bioRxiv2022

H3.3 contributes to chromatin accessibility and transcription factor binding at promoter-proximal regulatory elements

BackgroundThe histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood. ResultsWe performed genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and found evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 deposition at promoters reduces chromatin accessibility and transcription factor (TF) footprinting for nearly all TFs expressed in ESCs. H3.3 deletion leads to reduced promoter enrichment of the transcriptional coactivator and histone acetyltransferase, p300. Subsequently, histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the bromodomain-containing protein BRD4, an acetyl lysine reader. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TFs motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate. ConclusionsH3.3 facilitates DNA accessibility, TF binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it is required for TF binding at new promoters during differentiation.

genomics↗

bioRxiv2022

A cell cycle-linked mechanism for the glutamine driven establishment of stem cell fate

The cell cycle offers a unique opportunity for stem cells to sample metabolic and signaling cues to establish cell fate. Molecular pathways that integrate and convey these signals to cell cycle machinery to license cell fate transitions and drive terminal differentiation remain unknown. Here, we describe a signaling role of mitochondrial glutamine metabolism in driving exit from cell cycle-linked self-renewal to generate differentiation competent progenitors. In proliferating stem cells, mitochondrial glutamine metabolism opposes the WDR5-linked self-renewal network via acetylation and nuclear translocation of its upstream regulator, PASK. Nuclear PASK disrupts the mitotic WDR5-anaphase-promoting complex (APC/C) interaction to drive exit from self-renewal. Consistent with these roles, loss of PASK or inhibition of glutamine metabolism preserves stemness in vitro and in vivo during muscle regeneration. Our results suggest a mechanism whereby the proliferative functions of glutamine metabolism are co- opted by stem cells to establish cell fate.

cell biology↗