Biology subjects

Shishkova, E.

Publications and source records attributed to Shishkova, E..

12 recordsLinked to original sources

bioRxiv2025

IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-simulated gene products, establishing a model for its broad antiviral activity. But, IFIT2 also bound viral mRNAs and enhanced their translation resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here we validate direct interactions between IFIT3 and RNA using electromobility shift assays (EMSAs). RNA-binding site identification (RBS-ID) experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability IFIT3 to promote IAV gene expression and translation efficiency when compared to wild type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers, however the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA-binding and its ability to modulate translation of host and viral mRNAs during IAV infection. ImportanceInfluenza A viruses (IAV) cause considerable morbidity and mortality through sporadic pandemics as well as annual epidemics. Zoonotic IAV strains pose an additional risk of spillover into a naive human population where prior immunity can have minimal effect. In this case, the first line of defense in the host is the innate immune response. Interferon stimulated genes (ISGs) produce a suite of proteins that are front-line effectors of innate immune responses. While ISGs are typically considered antiviral, new work has revealed an emerging trend where viruses co-opt ISGs for pro-viral function. Here, we determine how the ISG IFIT3 is used by IAV as a pro-viral factor, advancing our understanding of IFIT3 function generally as well as specifically in the context of IAV infection.

microbiology↗

bioRxiv2025

Thermodynamics shape the in vivo enzyme burden of glycolytic pathways

Thermodynamically constrained reactions and pathways are hypothesized to impose greater protein demands on cells, requiring higher enzyme amounts to sustain a given flux compared to those with stronger thermodynamics. To test this, we quantified the absolute concentrations of glycolytic enzymes in three bacterial species --Zymomonas mobilis, Escherichia coli, and Clostridium thermocellum-- which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding in vivo metabolic fluxes and{Delta} G measurements, we found that the highly favorable Entner-Doudoroff (ED) pathway in Z. mobilis requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in C. thermocellum, with the Embden-Meyerhof-Parnas (EMP) pathway in E. coli exhibiting intermediate thermodynamic favorability and enzyme demand. Across all three pathways, early reactions with stronger thermodynamic driving forces generally required lower enzyme investment than later, less favorable steps. Additionally, reflecting differences in glycolytic strategies, the highly reversible ethanol fermentation pathway in C. thermocellum requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in Z. mobilis. Thus, thermodynamic driving forces constitute a major in vivo determinant of the enzyme burden in metabolic pathways.

systems biology↗

bioRxiv2024

Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

One-fifth of human proteins are N-glycosylated in the endoplasmic reticulum (ER) by two oligosaccharyltransferases, OST-A and OST-B. Contrary to the prevailing view of N-glycosylation as a housekeeping function, we identified an ER pathway that modulates the activity of OST-A. Genetic analyses linked OST-A to HSP90B1, an ER chaperone for membrane receptors, and CCCD134, an ER protein we identify as the first specificity factor for N-glycosylation. During its translocation into the ER, a N-terminal peptide in HSP90B1 functions as a pseudosubstrate inhibitor of OST-A and templates the assembly of specialized ER translocon complexes containing CCDC134. Unexpectedly, OST-A functions as a scaffold rather than an enzyme in this context, stabilizing HSP90B1 by preventing its hyperglycosylation and degradation. Disruption of this pathway impairs WNT signaling at the cell surface and causes the bone developmental disorder Osteogenesis Imperfecta. Thus, N-glycosylation can be regulated by ER factors to control cell-surface receptor signaling and tissue development. One-Sentence SummaryN-glycosylation of asparagine residues on proteins can be regulated by specificity factors in the endoplasmic reticulum to control cell-surface signaling and tissue development.

cell biology↗

bioRxiv2023

Heme biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue

With age, people tend to accumulate body fat and reduce energy expenditure1. Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles2,3. The activity of brown and beige depots inversely correlates with BMI and age4-11, suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease12-15. Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function16. Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis17. However, BCAAs contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux18. Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.

cell biology↗

bioRxiv2023

One-Hour Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology was benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

systems biology↗

bioRxiv2023

Modulation of hepatic transcription factor EB activity during cold exposure uncovers direct regulation of bis(monoacylglycerol)phosphate lipids by Pla2g15

Cold exposure is a selective environmental stress that elicits a rapid metabolic shift to maintain energy homeostasis. In response to cold exposure, the liver rewires the metabolic state shifting from glucose to lipid catabolism. By probing the liver lipids in cold exposure, we observed that the lysosomal bis(monoacylglycero)phosphate (BMP) lipids were rapidly increased during cold exposure. BMP lipid changes occurred independently of lysosomal abundance but were dependent on the lysosomal transcriptional regulator transcription factor EB (TFEB). Knockdown of TFEB in hepatocytes decreased BMP lipid levels and led to cold intolerance in mice. We assessed TFEB binding sites of lysosomal genes and determined that the phospholipase Pla2g15 regulates BMP lipid catabolism. Knockdown of Pla2g15 in mice increased BMP lipid levels, ablated the cold-induced rise, and improved cold tolerance. Knockout of Pla2g15 in mice and hepatocytes led to increased BMP lipid levels, that were decreased with re-expression of Pla2g15. Mutation of the catalytic site of Pla2g15 ablated the BMP lipid breakdown. Together, our studies uncover TFEB regulation of BMP lipids through Pla2g15 catabolism.

biochemistry↗

bioRxiv2023

Nuclear cytoglobin associates with HMGB2 and regulates DNA damage and genome-wide transcriptional output in the vasculature

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

cell biology↗

bioRxiv2023

Hem25p is a mitochondrial IPP transporter

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor comprised of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing, and targeted uptake assays, we reveal that Hem25p--a mitochondrial glycine transporter required for heme biosynthesis--doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p fail to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enables robust IPP uptake demonstrating that Hem25p is sufficient for IPP transport. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in yeast.

biochemistry↗

bioRxiv2023

Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy

Pptc7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass concomitant with elevation of the mitophagy receptors Bnip3 and Nix. Consistently, Pptc7-/- mouse embryonic fibroblasts (MEFs) exhibit a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs-- including multiple sites on Bnip3 and Nix. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that Pptc7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for Pptc7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.

biochemistry↗

bioRxiv2021

Multi-omics analysis identifies essential regulators of mitochondrial stress response in two wild-type C. elegans strains

The mitochondrial unfolded protein response (UPRmt) is a promising pharmacological target for aging and age-related diseases. However, the integrative analysis of the impact of UPRmt activation on different layers of signaling in animals with a different genetic background is lacking. In this study, we applied systems approaches to investigate the effect of UPRmt induced by administering doxycycline (Dox) on transcriptome, proteome, lipidome, and metabolome in two genetically divergent C. elegans strains. We found that Dox prolongs lifespan of both worm strains through pathways in both shared and strain-specific manners. From the integrated omics datasets, we observed a strong impact of Dox on mitochondrial functions, detected upregulated defense response and lipid metabolism, identified decreased triglycerides and lowered metabolome profiles in both strains. This conserved phenomic footprint has great translational value as it indicates that the beneficial effects of Dox-induced UPRmt on health and lifespan are consistent across different genetic backgrounds.

systems biology↗

bioRxiv2020

Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine auto-modifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

molecular biology↗

bioRxiv2017

Network inference reveals novel connections in pathways regulating growth and defense in the yeast salt response

Cells respond to stressful conditions by coordinating a complex, multi-faceted response that spans many levels of physiology. Much of the response is coordinated by changes in protein phosphorylation. Although the regulators of transcriptome changes during stress are well characterized in Saccharomyces cerevisiae, the upstream regulatory network controlling protein phosphorylation is less well dissected. Here, we developed a computational approach to infer the signaling network that regulates phosphorylation changes in response to salt stress. The method uses integer linear programming (ILP) to integrate stress-responsive phospho-proteome responses in wild-type and mutant strains, predicted phosphorylation motifs on groups of coregulated peptides, and published protein interaction data. A key advance is that by grouping peptides into submodules before inference, the method can overcome missing protein interactions in published datasets to predict novel, stress-dependent protein interactions and phosphorylation events. The network we inferred predicted new regulatory connections between stress-activated and growth-regulating pathways and suggested mechanisms coordinating metabolism, cell-cycle progression, and growth during stress. We confirmed several network predictions with co-immunoprecipitations coupled with mass-spectrometry protein identification and mutant phospho-proteomic analysis. Results show that the cAMP-phosphodiesterase Pde2 physically interacts with many stress-regulated transcription factors targeted by PKA, and that reduced phosphorylation of those factors during stress requires the Rck2 kinase that we show physically interacts with Pde2. Together, our work shows how a high-quality computational network model can facilitate discovery of new pathway interactions during osmotic stress.

systems biology↗