Biology subjects

Verdin, E.

Publications and source records attributed to Verdin, E..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Small-molecule ketone esters treat brain network abnormalities in an Alzheimer's disease mouse model

Altered brain network activity and the resulting hypersynchrony are important for the pathogenesis of cognitive decline in Alzheimers disease (AD) mouse models. Treatments that reduce epileptiform discharges (EDs) or network hyperactivity improve cognition in AD models and humans. We first show that ketogenic diet, but not fasting, rapidly and persistently reduced EDs in the hAPPJ20 Alzheimers mouse model over timescales of hours to months. Then, to identify the specific mechanism of the pleiotropic ketogenic diet, we developed small molecule ketone esters to deliver ketone bodies pharmacologically. Two ketone esters recapitulate ED suppression without other dietary manipulation, over time scales of minutes to one week. This small molecule rescue was associated with reduced low-frequency oscillatory activity similar to the recently reported mechanism of an NMDA receptor modulator molecule in this model. Long-term KD resulted in cognitive improvement and in a sex-stratified analysis also improved survival in the more severely affected hAPPJ20 males. Agents that deliver ketone bodies via small molecules or act on downstream targets may hold therapeutic promise in AD through the mechanism of improved network function and reduced epileptiform activity.

neuroscience↗

bioRxiv2022

In-depth Analysis of the Sirtuin 5-regulated Mouse Brain Acylome using Library-free Data-Independent Acquisitions

Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry-based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data-independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach that combines antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock-out mice, and discovered and quantified 466 malonylated and 2,211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 171 malonylated and 640 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome. Statement of significance of the studyPost-translational modifications (PTMs) are key regulators of protein structure, functions, and interactions. A great variety of PTMs have been discovered, including lysine acylation, such as acetylation, malonylation, and succinylation. Lysine acylation is understudied, particularly in the brain, and analysis by mass spectrometry-based proteomics faces significant challenges. In this study, we present a robust and efficient workflow to investigate proteome-wide PTM remodeling combining affinity PTM enrichment and a novel spectral library-free data-independent acquisition (DIA) approach. The strength of label-free DIA becomes evident with the collection of comprehensive information by tandem mass spectrometry for all detectable precursor ions of all biological samples, and the highly accurate quantitative information that can subsequently be retrieved with time-efficient and straightforward library-free strategies. More importantly, this enables confident identification of PTM sites and differentiation of PTM isomers. We applied this workflow to decipher the malonylome and succinylome remodeling and cross-talk in brains from wild-type and Sirt5(-/-) mice, taking advantage of the demalonylase and desuccinylase activities of SIRT5, a nicotinamide adenine dinucleotide (NAD+)-dependent sirtuin. Interestingly, 10 malonylated proteins and 33 succinylated proteins targeted by SIRT5 are involved in the Parkinsons disease pathway, including subunit beta of the calcium/calmodulin-dependent protein kinase type II (Camk2b) and protein DJ-1 (Park7).

cell biology↗

bioRxiv2022

Regulation of urea cycle by reversible high stoichiometry lysine succinylation

The post-translational modification, lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. In the present study, using stable isotope labeling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in urea cycle, are regulated by SIRT5. Mutation of the high stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance and reduces locomotor and exploratory activity in male mice upon high-ammonium diet feeding. Therefore, lysine succinylation is functionally important in ammonia metabolism.

molecular biology↗

bioRxiv2022

Histone malonylation is regulated by SIRT5 and KAT2A

The posttranslational modification lysine malonylation is found in many proteins, including histones. However, it remains unclear whether histone malonylation is regulated or functionally relevant. Here, we report that availability of malonyl-co-enzyme A (malonyl-CoA), an endogenous provider of malonyl groups, affects lysine malonylation, and that the deacylase SIRT5 selectively reduces malonylation of histones. To determine if histone malonylation is enzymatically catalyzed, we knocked down each of the 22 lysine acetyltransferases (KATs) to test their malonyltransferase potential. KAT2A knockdown in particular reduced histone malonylation levels. By mass spectrometry, H2B_K5 was highly malonylated and significantly regulated by SIRT5 in mouse brain and liver. Acetyl-CoA carboxylase (ACC), the malonyl-CoA producing enzyme, was partly localized in the nucleolus, and histone malonylation increased nucleolar area and ribosomal RNA expression. Levels of global lysine malonylation and ACC expression were higher in older mouse brains than younger mice. These experiments highlight the role of histone malonylation in ribosomal gene expression.

molecular biology↗

bioRxiv2022

Coenzyme A binding sites induce proximal acylation across protein families

Lysine N{varepsilon}-acylations, such as acetylation or succinylation, are post-translational modifications that regulate protein function. In mitochondria, lysine acylation is predominantly non-enzymatic, and only a specific subset of the proteome is acylated. Coenzyme A (CoA) can act as an acyl group carrier via a thioester bond, but what controls the acylation of mitochondrial lysines remains poorly understood. Using published datasets, here we found that proteins with a CoA-binding site are more likely to be acetylated, succinylated, and glutarylated. Using computational modeling, we show that lysine residues near the CoA-binding pocket are highly acylated compared to those farther away. We hypothesized that acyl-CoA binding enhances acylation of nearby lysine residues. To test this hypothesis, we co-incubated enoyl-CoA hydratase short chain 1 (ECHS1), a CoA-binding mitochondrial protein, with succinyl-CoA and CoA. Using mass spectrometry, we found that succinyl-CoA induced widespread lysine succinylation and that CoA competitively inhibited ECHS1 succinylation. CoA-induced inhibition at a particular lysine site correlated inversely with the distance between that lysine and the CoA-binding pocket. Our study indicated that CoA acts as a competitive inhibitor of ECHS1 succinylation by binding to the CoA-binding pocket. Together, this suggests that proximal acylation at CoA-binding sites is a primary mechanism for lysine acylation in the mitochondria.

biochemistry↗

bioRxiv2022

Human Elp3/Kat9 is a mitochondrial tRNA modifying enzyme

Post-translational modifications, such as lysine acetylation, regulate the activity of diverse proteins across many cellular compartments. Protein deacetylation in mitochondria is catalyzed by the enzymatic activity of the NAD+-dependent deacetylase sirtuin 3 (SIRT3), however it remains unclear whether corresponding mitochondrial acetyltransferases exist. We used a bioinformatics approach to search for mitochondrial proteins with an acetyltransferase catalytic domain, and identified a novel splice variant of ELP3 (mt-ELP3) of the elongator complex, which localizes to the mitochondrial matrix in mammalian cells. Unexpectedly, mt-ELP3 does not mediate mitochondrial protein acetylation but instead induces a post-transcriptional modification of mitochondrial-transfer RNAs (mt-tRNAs). Overexpression of mt-ELP3 leads to the protection of mt-tRNAs against the tRNA-specific RNase angiogenin, increases mitochondrial translation, and furthermore increases expression of OXPHOS complexes. This study thus identifies mt-ELP3 as a non-canonical mt-tRNA modifying enzyme.

molecular biology↗

bioRxiv2022

SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

microbiology↗

bioRxiv2021

A Proposed Unified Mitotic Chromosome Architecture

A molecular architecture is proposed for an example mitotic chromosome, human Chromosome 10. This architecture is built on a previously described interphase chromosome structure based on Cryo-EM cellular tomography (1), thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle, for mitotic chromosomes, is specific coiling of the 11-nm nucleosome fiber into large scale approximately 200 nm structures (a Slinky (2, motif cited in 3) in interphase, and then further modification and subsequent additional coiling for the final structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well known cytological configurations. In addition, proof is experimentally provided, by digital PCR technology, that G1 T-cell nuclei are diploid, thus one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils. Significance StatementThe significance of this proposed mitotic chromosome architecture is that a specific, sequenced chromosome, human Chromosome 10, can be built into a specific architecture that accounts for the dimensional values and cytological descriptions, a first time result. Since this molecular architecture is an extension of the interphase chromosome structure, a coiling of the 11-nm nucleosome fiber with further coiling, a unifying molecular structure motif is present throughout the entire mitotic cycle, interphase through mitosis.

cell biology↗

bioRxiv2021

T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses should be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of an enhancer specific CBP/P300-inhibitor on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac, efficiently recruited Tat. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.

microbiology↗

bioRxiv2021

Deep Learning Shows Cellular Senescence Is a Barrier to Cancer Development

Cellular senescence is a critical component of aging and many age-related diseases, but understanding its role in human health is challenging in part due to the lack of exclusive or universal markers. Using neural networks, we achieve high accuracy in predicting senescence state and type from the nuclear morphology of DAPI-stained human fibroblasts, murine astrocytes and fibroblasts derived from premature aging diseases in vitro. After generalizing this approach, the predictor recognizes an increasing rate of senescent cells with age in H&E-stained murine liver tissue and human dermal biopsies. Evaluating corresponding medical records reveals that individuals with increased senescent cells have a significantly decreased rate of malignant neoplasms, lending support for the protective role of senescence in limiting cancer development. In sum, we introduce a novel predictor of cellular senescence and apply it to diagnostic medical images, indicating cancer occurs more frequently for those with a lower rate of senescence.

cell biology↗

bioRxiv2021

Targeting evolutionary conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, and especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wildtype viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. Here, we analyze in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses dont stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications.

microbiology↗

bioRxiv2020

FOXO1 promotes HIV Latency by suppressing ER stress in T cells

Quiescence is a hallmark of CD4+ T cells latently infected with HIV-1. While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. Here we report that FOXO1, a key regulator of T-cell quiescence, promotes latency and suppresses productive HIV infection. In resting T cells, FOXO1 inhibition induces ER stress and activates two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of PKR-like endoplasmic reticulum kinase (PERK), a known link between ER stress and ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppress HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a novel link between FOXO1, ER stress, and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.

immunology↗

bioRxiv2019

Viral gene drive in herpesviruses

Herpesviruses are ubiquitous pathogens in need of novel therapeutic solutions. Current engineered gene drive strategies rely on sexual reproduction, and are thought to be restricted to sexual organisms. Here, we report on the design of a novel gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Our results indicate that viral gene drives can be used to suppress a viral infection and may represent a novel therapeutic strategy against herpesviruses.

microbiology↗

bioRxiv2019

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

cell biology↗

bioRxiv2019

Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication. Initial findings in immortalized cells were validated using CD4+ cells from donor blood. Tracking clonal behavior by proviral \"zip codes\" indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in the nef open reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP-cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in genes sense orientation were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP-population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.\n\nSummaryVery few HIV-1 infected cells persist in patients for more than a couple days, but those that do pose life-long health risks. Strategies designed to eliminate these cells have been based on assumptions about what viral properties allow infected cell survival. However, such approaches for HIV-1 eradication have not yet shown therapeutic promise, possibly because much of the research underlying assumptions about virus persistence has been focused on a limited number of infected cell types, the averaged behavior of cells in diverse populations, or snapshot views. Here, we developed a high-throughput approach to study hundreds of distinct HIV-1 infected cells and their progeny over time in an unbiased way. This revealed that each virus established its own pattern of gene expression that, upon infected cell division, was stably transmitted to all progeny cells. Expression patterns consisted of alternating waves of activity and inactivity, with the extent of activity differing among infected cell families over a 1000-fold range. The dynamics and variability among infected cells and within complex populations that the work here revealed has not previously been evident, and may help establish more accurate correlates of persistent HIV-1 infection.

microbiology↗

bioRxiv2019

Aging-related inflammation driven by cellular senescence enhances NAD consumption via activation of CD38+ macrophages

Decline in tissue NAD levels during aging is linked to aging and its associated diseases. However, the mechanism for aging-associated NAD decline remains unclear. Here we report that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues including visceral white adipose tissue and the liver during aging. Remarkably, these M1-like macrophages highly express the NAD consuming enzyme CD38 and have enhanced CD38-dependent NADase activity. We also find that senescent cells progressively accumulate in visceral white adipose tissue during aging and that inflammatory cytokines found in the supernatant from senescent cells (Senescence associated secretory proteins, SASP) induce macrophages to proliferate and to express CD38. These results highlight a new causal link between visceral tissue senescence and tissue NAD decline during aging and represent a novel therapeutic opportunity targeting maintenance of NAD levels during aging.

immunology↗

bioRxiv2018

Multi-Omic Profiling Reveals the Opposing Forces of Excess Dietary Sugar and Fat on Liver Mitochondria Protein Acetylation and Succinylation

Dietary macronutrient composition alters metabolism through several mechanisms, including post-translational modification (PTM) of proteins. To connect diet and molecular changes, here we performed short- and long-term feeding of mice with standard chow diet (SCD) and high-fat diet (HFD), with or without glucose or fructose supplementation, and quantified liver metabolites, 861 proteins, and 1,815 protein level-corrected mitochondrial acetylation and succinylation sites. Nearly half the acylation sites were altered by at least one diet; nutrient-specific changes in protein acylation sometimes encompass entire pathways. Although acetyl-CoA is an intermediate in both sugar and fat metabolism, acetyl-CoA had a dichotomous fate depending on its source; chronic feeding of dietary sugars induced protein hyperacetylation, whereas the same duration of HFD did not. Instead, HFD resulted in citrate accumulation, anaplerotic metabolism of amino acids, and protein hypo-succinylation. Together, our results demonstrate novel connections between dietary macronutrients, protein post-translational modifications, and regulation of fuel selection in liver.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/263426_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (53K):\norg.highwire.dtl.DTLVardef@1419c64org.highwire.dtl.DTLVardef@82a874org.highwire.dtl.DTLVardef@16023org.highwire.dtl.DTLVardef@4ed44e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract\n\nC_FIG

systems biology↗

bioRxiv2018

Chromatin Functional States Correlate with HIV Latency Reversal in Infected Primary CD4+ T Cells

Human immunodeficiency virus (HIV) infection cannot be cured due to a small reservoir of latently infected CD4+ T cells in treated patients. The \"shock and kill\" approach proposes to eliminate the reservoir by inducing its activation and the direct or indirect killing of infected cells. Current latency reversing agents (LRAs) do not reduce the viral reservoir in treated patients.\n\nWe use a novel dual-fluorescent HIV reporter to identify and purify latent cells, and to determine the fraction of latent cells that undergo viral reactivation after infection of primary CD4+ T cells. Unexpectedly, LRAs reactivate less than 5% of latent proviruses. Analysis of HIV integration sites from induced and non-induced latent populations reveals distinct provirus integration sites between these two populations in terms of chromatin functional states.\n\nThese findings challenge \"shock and kill\", and suggest the need of more potent LRAs in combination with immunomodulatory approaches to eradicate HIV reservoir.

microbiology↗