Biology subjects

Schilling, B.

Publications and source records attributed to Schilling, B..

At least 73 records · Page 4Linked to original sources

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

U1 snRNP-dependent suppression of miRNA biogenesis by alternative intronic polyadenylation in melanoma

Activation of intronic polyadenylation signals results in premature cleavage and polyadenylation (PCPA). The majority of mammalian miRNAs are also located within intronic regions of protein-coding genes and are transcriptionally co-expressed with their host genes. Here we show that U1-dependent PCPA by telescripting dysregulates miRNA biogenesis. When U1 is reduced, miR-211 levels are decreased as a direct consequence of activation of a newly identified alternative intronic polyadenylation signal located upstream of miR-211 within its host gene TRPM1. Various melanoma cell lines revealed decreased U1 levels and a shift from full-length to truncated TRPM1 isoforms with concomitant decreased miR-211 expression. Modulation of TRPM1 alternative polyadenylation (APA) by morpholino oligonucleotides inhibits and potentially restores miR-211 expression to endogenous levels. This mechanism of intronic PCPA and its effects on miRNA biogenesis represents a previously unrecognized layer of gene expression regulation suitable for therapeutic modulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/479622v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@169c2f1org.highwire.dtl.DTLVardef@7d8f34org.highwire.dtl.DTLVardef@144b9d8org.highwire.dtl.DTLVardef@10523f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Proteomic Analysis of Huntington's Disease Medium Spiny Neurons Identifies Alterations in Lipid Droplets

Huntingtons disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin (HTT) gene. The resulting polyglutamine (polyQ) tract alters the function of the HTT protein. Although HTT is expressed in different tissues, the medium spiny projection neurons (MSNs) in the striatum are particularly vulnerable in HD. Thus, we sought to define the proteome of human HD patient-derived MSNs. We differentiated HD72 induced pluripotent stem cells and isogenic controls into MSNs and carried out quantitative proteomic analysis by two approaches. First, using data-dependent acquisitions with FAIMS (FAIMS-DDA) for label-free quantification on the Orbitrap Lumos mass spectrometer, we identified 6,323 proteins with at least two unique peptides (FDR [≤] 0.01). Of these, 901 proteins were significantly altered in the HD72-MSNs, compared to isogenic controls. Second, we quantitatively validated protein candidates by comprehensive data-independent acquisitions on a TripleTOF 6600 mass spectrometer quantifying 3,106 proteins with at least two unique peptides. Functional enrichment analysis identified pathways related to the extracellular matrix, including TGF-y regulation of extracellular matrix, epithelial-mesenchymal transition, DNA replication, senescence, cardiovascular system, organism development, regulation of cell migration and locomotion, aminoglycan glycosaminoglycan proteoglycan, growth factor stimulus and fatty acid processes. Conversely, processes associated with the downregulated proteins included neurogenesis-axogenesis, the brain-derived neurotrophic factor-signaling pathway, Ephrin-A: EphA pathway, regulation of synaptic plasticity, triglyceride homeostasis cholesterol, plasmid lipoprotein particle immune response, interferon-{gamma} signaling, immune system major histocompatibility complex, lipid metabolism and cellular response to stimulus. Moreover, proteins involved in the formation and maintenance of axons, dendrites, and synapses (e.g., Septin protein members) are dysregulated in HD72-MSNs. Importantly, lipid metabolism pathways were altered, and we found that lipid droplets accumulated in the HD72-MSNs, suggesting a deficit in lipophagy. Our proteomics analysis of HD72-MSNs identified relevant pathways that are altered in MSNs and confirm current and new therapeutic targets for HD.

neuroscience↗

bioRxiv2022

A natural variation-based screen in mouse cells reveals USF2 as a regulator of the DNA damage response and cellular senescence

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-kB, p53, and C/EBP{beta}. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory M. musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, USF2, for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program--shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

genomics↗

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

Deletion of the microglial transmembrane immune signaling adaptor TYROBP ameliorates Huntington's disease mouse phenotype

BACKGROUNDHuntingtons disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat in the huntingtin gene. Immune activation is abundant in the striatum of HD patients. Detection of active microglia at presymptomatic stages suggests that microgliosis is a key early driver of neuronal dysfunction and degeneration. Recent studies showed that deletion of Tyrobp, a microglial-enriched protein, ameliorates neuronal function in Alzheimers disease amyloid and tauopathy mouse models while decreasing components of the complement subnetwork, thus raising the possibility that Tyrobp deletion can be beneficial for HD. METHODSTo test the hypothesis that Tyrobp deficiency would be beneficial in a HD model, we placed the Q175 HD mouse model on a Tyrobp-null background. We characterized these mice with a combination of behavioral testing, immunohistochemistry, transcriptomic and proteomic profiling. Further, we evaluated the Q175 microglia-specific gene signature, with and without Tyrobp, by purifying microglia from these mice for transcriptomics. RESULTSComprehensive analysis of publicly available transcriptomic HD human data revealed that TYROBP network is overactivated in HD putamen. The Q175 mice showed morphologic microglial activation, reduced levels of post-synaptic density-95 protein and motor deficits at 6 and 9 months of age, all of which were ameliorated on the Tyrobp-null background. Gene expression analysis revealed that lack of Tyrobp in the Q175 model does not prevent the decrease in the expression of striatal neuronal genes but reduces pro-inflammatory pathways that are specifically active in HD human brain. Integration of transcriptomic and proteomic data identified that astrogliosis and complement system pathway were reduced after Tyrobp deletion. Results were further validated by immunofluorescence analysis. Microglia-specific HD gene dysregulation, characterized by overexpression of neuronal genes, was also not corrected by Tyrobp deletion. CONCLUSIONSOur data provide molecular and functional support demonstrating that Tyrobp deletion prevents many of the abnormalities in the Q175 HD mouse model, in the absence of correction of striatal neuronal gene expression, suggesting that the Tyrobp pathway is a potential therapeutic candidate for Huntingtons disease.

neuroscience↗

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

Dietary restriction and clock delay eye aging to extend lifespan in D. melanogaster

Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. Herein, we demonstrate that dietary restriction extends lifespan in D. melanogaster by promoting circadian homoeostatic processes that protect the visual system from age- and light- associated damage. Disrupting circadian rhythms in the eye by inhibiting the transcription factor, Clock (CLK), or CLK-output genes, accelerated visual senescence, induced a systemic immune response, and shortened lifespan. Flies subjected to dietary restriction were protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extended lifespan in flies reared on a high-nutrient diet. Our findings establish the eye as a diet-sensitive modulator of lifespan and indicate that vision is an antagonistically pleiotropic process that contributes to organismal aging.

neuroscience↗

bioRxiv2021

Comprehensive Proteomic Quantification of Bladder Stone Progression in a Cystinuric Mouse Model Using Data-Independent Acquisitions

Cystinuria is one of various disorders that cause biomineralization in the urinary system, including bladder stone formation in humans. It is most prevalent in children and adolescents and more aggressive in males. There is no cure, and only limited disease management techniques help to solubilize the stones. Recurrence, even after treatment, occurs frequently. Other than a buildup of cystine, little is known about factors involved in the formation, expansion, and recurrence of these stones. This study sought to define the growth of bladder stones, guided by micro-computed tomography imaging, and to profile dynamic stone proteome changes in a cystinuria mouse model. After bladder stones developed in vivo, they were harvested and separated into four developmental stages (sand, small, medium and large stone), based on their size. Data-dependent and data-independent acquisitions allowed deep profiling of stone proteomics. The proteomic signatures and pathways illustrated major changes as the stones grew. Stones initiate from a small nidus, grow outward, and show major enrichment in ribosomal proteins and factors related to coagulation and platelet degranulation, suggesting a major dysregulation in specific pathways that can be targeted for new therapeutic options.

cell biology↗

bioRxiv2021

Proteome plasticity in response to persistent environmental change

Temperature is a variable component of the environment and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses resulting in the refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here, we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to the reduction of temperature sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and importantly, cellular functions. We postulate that in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

molecular biology↗

bioRxiv2021

Comprehensive Profiling of Plasma Exosomes Using Data Independent Acquisitions, New Tools for Aging Cohort Studies

Aging is a complex biological process associated with progressive loss of physiological function and susceptibility to several diseases, such as cancer and neurodegeneration. Exosomes are involved in many cellular signaling pathways, and their cargo may serve as promising disease or aging biomarkers. These membrane-bound extracellular vesicles facilitate the transport of intracellular contents to proximal and distal cells in the body. Here, we investigated two omics approaches for exosome analysis. To overcome the challenges of plasma exosome contamination with abundant soluble plasma proteins, we developed a high-throughput method to isolate highly purified exosomes from human plasma by sequential size-exclusion chromatography and ultrafiltration. First, we used data-dependent acquisitions from offline high-pH reversed-phase fractions of exosome lysate to generate a deep spectral library comprising [~]2,300 exosome proteins. Second, in a pilot aging study, we used comprehensive data-independent acquisitions to compare plasma exosomes from young (20-26 yrs) and old (60-66 yrs) individuals. We quantified 1,318 exosome proteins, and levels of 144 proteins were significantly different in young and old plasma groups (Q<0.05 and >1.5-fold change). We also analyzed exosome miRNA cargo and detected 331 miRNAs. Levels of several were significantly different in young and old individuals. In addition, 88 and 17 miRNAs were unique to old and young individuals, respectively. Plasma exosome biomarkers have great potential for translational studies investigating biomarkers of aging and age-related diseases and to monitor therapeutic aging interventions.

cell biology↗

bioRxiv2020

Differential effects of PD-1 and CTLA-4 blockade on the melanoma-reactive CD8 T cell response

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral. blood. If activation of circulating tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses towards 71 melanoma associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the melanoma-reactive CD8 T cell response in peripheral blood was unaltered upon PD-1 blockade. In contrast, a broadening of the melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. On the basis of these results, we conclude that PD-1 and CTLA-4 blockade impact the tumor-reactive CD8 T cell response in a distinct manner. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site. One sentence summaryWe demonstrate that, contrary to CTLA-4 blockade, PD-1 blockade in melanoma patients does not lead to an increase in the breadth or magnitude of the melanoma-reactive CD8 T cell response in peripheral blood, thereby arguing for a dominant effect of PD-1 blockade at the tumor site.

immunology↗

bioRxiv2020

Improved SILAC quantification with data independent acquisition to investigate bortezomib-induced protein degradation

Stable isotope labeling by amino acids in cell culture (SILAC) coupled to data-dependent acquisition (DDA) is a common approach to quantitative proteomics with the desirable benefit of reducing batch effects during sample processing and data acquisition. More recently, using data-independent acquisition (DIA/SWATH) to systematically measure peptides has gained popularity for its comprehensiveness, reproducibility, and accuracy of quantification. The complementary advantages of these two techniques logically suggests combining them. Here, we develop a SILAC-DIA-MS workflow using free, open-source software. We determine empirically that using DIA achieves similar peptide detection numbers as DDA and that DIA improves the quantitative accuracy and precision of SILAC by an order of magnitude. Finally, we apply SILAC-DIA-MS to determine protein turnover rates of cells treated with bortezomib, a 26S proteasome inhibitor FDA-approved for multiple myeloma and mantle cell lymphoma. We observe that SILAC-DIA produces more sensitive protein turnover models. Of the proteins determined differentially degraded by both acquisition methods, we find known ubiquitin-proteasome degrands such as HNRNPK, EIF3A, and IF4A1/EIF4A-1, and a slower turnover for CATD, a protein implicated in invasive breast cancer. With improved quantification from DIA, we anticipate this workflow making SILAC-based experiments like protein turnover more sensitive.

biochemistry↗

bioRxiv2020

Translational control of methionine and serine metabolic pathways underpin the paralog-specific phenotypes of Rpl22 ribosomal protein mutants in cell division and replicative longevity

A long-standing problem is how cells that lack one of the highly similar ribosomal proteins (RPs) often display distinct phenotypes. Some may reflect general effects due to lower growth rate and ribosome levels, but a number of diverse phenotypes cannot be explained through this mechanism. Yeast and other organisms live longer when they lack specific ribosomal proteins, especially of the large 60S subunit of the ribosome. However, longevity is neither associated with the generation time of RP deletion mutants nor with bulk inhibition of protein synthesis. Here, we comprehensively queried actively dividing RP paralog mutants through the cell cycle. Our data link transcriptional, translational, and metabolic changes to phenotypes associated with the loss of paralogous RPs. We uncovered specific translational control of transcripts encoding enzymes of methionine and serine metabolism, which are part of one-carbon (1C) pathways. Cells lacking Rpl22Ap, which are long-lived, have lower levels of metabolites associated with 1C metabolism. Loss of 1C enzymes, such as the serine hydroxymethyltransferase Shm2p increased the longevity of wild type cells. These results provide a molecular basis for paralog-specific phenotypes in ribosomal mutants and underscore the significance of 1C metabolic pathways in mechanisms of cell division and cellular aging. 1C pathways exist in all organisms, including humans, and targeting the relevant enzymes could represent longevity interventions.

molecular biology↗

bioRxiv2020

A Simultaneous Methylome and Proteome Quantitative Workflow for Complex Samples

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and tri-methylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated non-alcoholic steatohepatitis (NASH) mouse models. We report altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

systems biology↗