Biology subjects

Ghaemmaghami, S.

Publications and source records attributed to Ghaemmaghami, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Mass spectrometric analysis of free methionine oxidation levels in E. coli

Oxidation of free methionine plays important roles in cellular redox homeostasis, yet its accurate quantification has been hindered by methodological challenges. Here, we introduce free Methionine Oxidation by Blocking (fMObB), a mass spectrometry-based method that enables accurate measurement of the fractional oxidation of free methionines. Applying fMObB to Escherichia coli, we quantify free methionine oxidation under basal and oxidative stress conditions, and in strains lacking methionine sulfoxide reductases. We find that during oxidative stress, free methionines exhibit higher oxidation levels than protein-bound methionines and that methionine sulfoxide reductases play a central role in maintaining reduced free methionine pools. Together, this work establishes fMObB as a generalizable strategy for probing free methionine redox states in cellular systems.

biochemistry↗

bioRxiv2026

Long-lived mammals contain more phosphorylation sites in the SIRT6 C-terminus that enhance PARP1 interaction and resistance to oxidative stress

Sirtuin 6 (SIRT6) is a protein deacetylase and ribosyltransferase that is a vital hub for maintaining epigenetic homeostasis, regulating the transcriptome, and repairing DNA double stranded breaks (DSBs). Comprehensive proteomic profiling of the SIRT6 post-translational landscape, however, remains elusive. The SIRT6 C-terminal domain contains multiple phosphorylation sites. We find that the presence and use of these sites is strongly correlated with maximum lifespan across mammals. Subsequent biochemical and in silico analyses revealed that SIRT6 hyperphosphorylation enhances its interaction with PARP1. Mutating the T294 phosphorylation site in human fibroblasts led to decreased survival after oxidative stress in the phospho-null T294A and improved oxidative stress resistance in the phospho-mimetic T294E. Together, these results suggest SIRT6 C-terminal phosphorylation increases stress resistance through interaction with PARP1, and that this effect has been enhanced by evolution in long-lived species.

molecular biology↗

bioRxiv2026

Protein entanglement misfolding determines divergent fates: proteasomal degradation or persistence in near-native misfolded states

A novel class of protein misfolding, involving changes in entanglement status, occurs across the cytosolic proteome of a bacterium and likely occurs in many other organisms. Here, we examine if this class of misfolding has measurable downstream consequences for protein homeostasis. Specifically, we test the hypothesis that proteins that misfold in this way are more likely to be degraded by the ubiquitin-proteasome system immediately after synthesis. We do this by cross-referencing protein structural information with ubiquitin mass spectrometry (Ubq-MS) data from human fibroblast cells. Ubq-MS identifies proteins that have been covalently modified with ubiquitin in a particular pattern and is a cellular signal for that protein to be degraded by the proteasome. We find that nascent proteins with native entanglements, which were previously shown to be twice as likely to misfold, are 93% (95% Confidence Interval: [44%, 160%]) more likely to be tagged with ubiquitin and targeted to the proteasome compared to proteins that do not contain such entanglements. Simulating the folding of these proteins using a coarse-grained model, we find that the ubiquitin-tagged proteins containing native entanglements are four times more likely to misfold than the non-ubiquitinated proteins that are devoid of entanglements. These results indicate that entanglement misfolding, primarily involving a failure to form native entanglements, leads to an increased likelihood that those proteins will be degraded in human cells. Finally, we estimate that approximately one-third of the globular proteome likely misfolds in this way but bypasses proteasomal degradation because their misfolded states are structurally similar to their native ensemble. These consequences for protein degradation are likely common across organisms as entanglement misfolding is inherent to the polymeric nature of proteins.

biophysics↗

bioRxiv2025

Structural proteomics of the human ubiquitinome

The proteasome maintains the integrity of eukaryotic proteomes by selectively degrading ubiquitinated protein substrates. Ubiquitination targets a wide range of substrates for degradation, including translationally stalled nascent chains, misfolded proteins, and properly folded but short-lived proteins destined for regulatory degradation. Distinct structural features and ubiquitination patterns across these classes of substrates remain largely undefined. In this study, we combine structural proteomics and time-resolved isotopic labeling to profile the modification sites, dynamics, and conformational properties of the human ubiquitinome. We show that proteins undergoing rapid proteasomal degradation are ubiquitinated at lysine residues that are normally buried within structured regions of their native conformations. We provide proteome-wide evidence that this high-flux subset of the ubiquitinome is enriched in newly synthesized proteins that have non-native conformations. Together, our findings demonstrate how the lack of structural integrity of misfolded nascent proteins influences their ubiquitination patterns and ensures proper proteasomal degradation. Significance StatementProtein degradation by the ubiquitin-proteasome system (UPS) is central to maintaining cellular protein quality control, yet the structural and kinetic determinants that govern which proteins are targeted for degradation remain poorly defined. Using deep-coverage structural proteomics combined with metabolic labeling, we show that ubiquitination events can be categorized into two broad classes with distinct properties: those at buried lysines within nascent misfolded proteins that lead to rapid proteasomal degradation, and those at exposed lysines in mature proteins that are associated with slower turnover or regulatory functions. This proteome-wide partitioning provides structural insights into how the UPS targets defective nascent and mature proteins for proteasomal clearance.

molecular biology↗

bioRxiv2025

Antibiotic treatment modulates Escherichia coli-derived bacterial extracellular vesicle (BEV) production and their capacity to upregulate ICAM-1 in human endothelial cells

Antibiotic treatment is often necessary to eliminate life-threatening bacterial infections. However, these treatments can alter production of bacterial extracellular vesicles (BEVs), which often contain pro-inflammatory biomolecules. In this study, we examined how the clinically-relevant antibiotics meropenem, tobramycin, and ciprofloxacin impacted BEV production from a urinary tract infection-associated Escherichia coli strain (CFT073 [WAM2267]) and a meningitis-associated strain (K1 RS218). BEVs from both strains caused a dose-dependent increase in expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells, priming the endothelium for interactions with immune cells. Blockade of toll-like receptor 4 revealed that this receptor was responsible for BEV-endothelial interactions. Treatment with meropenem, a {beta}-lactam antibiotic, increased production of BEVs from strain K1 RS218. Furthermore, meropenem treatment caused strain CFT073 [WAM2267] to produce BEVs with heightened stimulatory capacity, possibly by amplifying the content of lipoprotein Lpp in these BEVs as measured by mass spectrometry. To our knowledge, this is the first study examining the interplay between antibiotic treatment and the effects of the resulting BEVs on endothelial ICAM-1 expression. Our results indicate treatment risks of certain antibiotics against specific strains of E. coli and could help identify therapeutic targets to reduce BEV-mediated endothelial stimulation during infection.

cell biology↗

bioRxiv2025

O-GlcNAc modifications regulate lamin A tail processing

Lamin A processing is highly regulated, and necessary for proper assembly of the nuclear lamina facilitating its role in nuclear structure and chromatin organization. Pre-lamin A is first farnesylated, and then a short C-terminal peptide is cleaved to produce mature lamin A. O-GlcNAc Transferase (OGT), a glucose sensitive post-translational modification enzyme, is a potential regulator for lamin A processing. To explore the role of OGT in lamin A biogenesis, we examined the effects of OGT levels and OGT inhibition. Variation in OGT dose or inhibition of its activity did not alter endogenous lamin A abundance or distribution. To more directly test the regulatory effects of O-GlcNAcylation on lamin A, we adapted a tail cleavage assay. Mutation of an OGT binding motif and O-GlcNAc modification sites reduced tail cleavage efficiency, suggesting that O-GlcNAcylation promotes lamin A processing. Our findings add to the understanding of the regulation of lamin A cleavage and identify a potential link between glucose metabolism and lamina biogenesis.

cell biology↗

bioRxiv2024

Pervasive divergence in protein thermostability is mediated by both structural changes and cellular environments

Temperature is a universal environmental constraint and organisms have evolved diverse mechanisms of thermotolerance. A central feature of thermophiles relative to mesophiles is a universal shift in protein stability, implying that it is a major constituent of thermotolerance. However, organisms have also evolved extensive buffering systems, such as those that disaggregate and refold denatured proteins and enable survival of heat shock. Here, we show that both cellular and protein structural changes contribute to divergence in protein thermostability between two closely related Saccharomyces species that differ by 8{degrees}C in their thermotolerance. Using thermal proteomic profiling we find that 85% of S. cerevisiae proteins are more stable than their S. uvarum homologs and there is an average shift of 1.6{degrees}C in temperature induced protein aggregation. In an interspecific hybrid of the two species, S. cerevisiae proteins retain their thermostability, while the thermostability of their S. uvarum homologs is enhanced, indicating that cellular context contributes to protein stability differences. By purifying orthologous proteins we show that amino acid substitutions underlie melting temperature differences for two proteins, Guk1 and Aha1. Amino acid substitutions are also computationally predicted to contribute to stability differences for most of the proteome. Our results imply that coordinated changes in protein thermostability impose a significant constraint on the time scales over which thermotolerance can evolve.

evolutionary biology↗

bioRxiv2024

Enzymatic synthesis of isotopically labeled hydrogen peroxide for mass spectrometry-based applications

Methionine oxidation is involved in multiple biological processes including protein misfolding and enzyme regulation. However, it is often challenging to measure levels of methionine oxidation by mass spectrometry, in part due to the prevalence of artifactual oxidation that occurs during sample preparation and ionization steps of typical proteomic workflows. Isotopically labeled hydrogen peroxide (H218O2) can be used to block unoxidized methionines and enable accurate measurement of in vivo levels of methionine oxidation. However, H218O2 is an expensive reagent that can be difficult to obtain from commercial sources. Here, we report a method for synthesizing H218O2 in house. Glucose oxidase catalyzes the oxidation of {beta}-D-glucose and produces hydrogen peroxide in the process. We took advantage of this reaction to enzymatically synthesize H218O2 from 18O2 and assessed its concentration, purity, and utility in measuring methionine oxidation levels by mass spectrometry.

biochemistry↗

bioRxiv2024

Formylation facilitates the reduction of oxidized initiator methionines

Within a cell, protein-bound methionines can be oxidized by reactive oxygen species (ROS) or monooxygenases, and subsequently reduced by methionine sulfoxide reductases (Msrs). Methionine oxidation can result in structural damage or be the basis of functional regulation of enzymes. In addition to participating in redox reactions, methionines play an important role as the initiator residue of translated proteins where they are commonly modified at their -amine group by formylation or acetylation. Here, we investigated how formylation and acetylation of initiator methionines impact their propensity for oxidation and reduction. We show that in vitro, N-terminal methionine residues are particularly prone to chemical oxidation, and that their modification by formylation or acetylation greatly enhances their subsequent enzymatic reduction by MsrA and MsrB. Concordantly, in vivo ablation of methionyl-tRNA formyltransferase (MTF) in E. coli increases the prevalence of oxidized methionines within synthesized proteins. We show that oxidation of formylated initiator methionines is detrimental in part because it obstructs their ensuing deformylation by peptide deformylase (PDF) and hydrolysis by methionyl aminopeptidase (MAP). Thus, by facilitating their reduction, formylation mitigates the misprocessing of oxidized initiator methionines. Classification: Biological Sciences; Biochemistry

biochemistry↗

bioRxiv2023

Proteome birthdating reveals age-selectivity of protein ubiquitination

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed "proteome birthdating" that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics without the requirement for multiple kinetic time points. As a first use case of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identified a smaller subset of very old ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways. Significance StatementCellular proteins have widely different ages - whereas some have been recently synthesized, others have existed in the cell for days or even years. How a proteins age influences its functions and interactions is largely unknown because it is difficult to globally differentiate proteins based on their time of synthesis. To address this challenge, we developed an analytical method named "proteome birthdating" that can partition cellular proteins into multiple discernible age groups. As an example application, we used proteome birthdating to examine the protein age-selectivity of the ubiquitin proteasome system, a major protein degradation pathway in eukaryotes. Our results show that proteins destined for degradation by this pathway consist of either particularly young or particularly old proteins, with the former being the predominant population. Together, our results establish proteome birthdating as a useful approach for analyzing the turnover of proteins and investigating the functional consequences of their age.

biochemistry↗

bioRxiv2023

Methionine Alkylation as an Approach to Quantify Methionine Oxidation Using Mass Spectrometry

Post-translational oxidation of methionine residues can destabilize proteins or modify their functions. Although levels of methionine oxidation can provide important information regarding the structural integrity and regulation of proteins, their quantitation is often challenging as analytical procedures in and of themselves can artifactually oxidize methionines. Here, we develop a mass spectrometry-based method called Methionine Oxidation by Blocking with Alkylation (MObBa) that quantifies methionine oxidation by selectively alkylating and blocking unoxidized methionines. Thus, alkylated methionines can be used as a stable proxy for unoxidized methionines. Using proof of concept experiments, we demonstrate that MObBa can be used to measure methionine oxidation levels within individual synthetic peptides and on proteome-wide scales. MObBa may provide a straightforward experimental strategy for mass spectrometric quantitation of methionine oxidation. Significance StatementOver time, cellular proteins can become oxidatively damaged by reactive oxygen species (ROS). A residue that is particularly prone to oxidative damage is methionine. Here, we develop and validate a methodology for detecting and quantifying levels of methionine oxidation by mass spectrometry. This approach has a number of practical advantages over methods currently available for analysis of methionine oxidation. The ability to accurately quantify methionine oxidation will provide important insights into factors that influence protein homeostasis within a cell.

biochemistry↗

bioRxiv2023

Long lifetime and selective accumulation of the A-type lamins accounts for the tissue specificity of Hutchinson-Gilford progeria syndrome

Mutations to the LMNA gene cause laminopathies including Hutchinson-Gilford progeria syndrome (HGPS) that severely affect the cardiovascular system. The origins of tissue specificity in these diseases are unclear, as the A-type Lamins are abundant and broadly expressed proteins. We show that A-type Lamin protein and transcript levels are uncorrelated across tissues. As protein-transcript discordance can be caused by variations in protein lifetime, we applied quantitative proteomics to profile protein turnover rates in healthy and progeroid tissues. We discover that tissue context and disease mutation each influence A-type Lamin protein lifetime. Lamin A/C has a weeks-long lifetime in the aorta, heart, and fat, where progeroid pathology is apparent, but a days-long lifetime in the liver and gastrointestinal tract, which are spared from disease. The A-type Lamins are insoluble and densely bundled in cardiovascular tissues, which may present an energetic barrier to degradation and promote long protein lifetime. Progerin is even more long-lived than Lamin A/C in the cardiovascular system and accumulates there over time. Progerin accumulation interferes broadly with protein homeostasis, as hundreds of abundant proteins turn over more slowly in progeroid tissues. These findings indicate that potential gene therapy interventions for HGPS will have significant latency and limited potency in disrupting the long-lived Progerin protein. Finally, we reveal that human disease alleles are significantly over-represented in the long-lived proteome, indicating that long protein lifetime may influence disease pathology and present a significant barrier to gene therapies for numerous human diseases. Significance statementMany human diseases are caused by mutations to broadly expressed proteins, yet disease mysteriously manifests only in specific tissues. An example of this is Hutchinson-Gilford progeria syndrome (HGPS), which is caused by a mutation to the Lamin A/C protein. We show that this mutation slows the turnover of Lamin A/C proteins in disease-afflicted tissues, causing the mutant "Progerin" protein to accumulate over time and interfere with the normal turnover of hundreds of other proteins. Because Progerin is a long-lived protein, effective therapies for this disease will need to attack the protein and not just the gene that encodes it.

cell biology↗

bioRxiv2022

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry

The folding of most proteins occurs during the course of their translation while their tRNA-bound C-termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurements sub-global folding stabilities of ribosome nascent chains (RNCs) within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (DHFR, CheY and DinB) in soluble and ribosome-bound states. The data indicated that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

biochemistry↗

bioRxiv2022

Combined measurements of proteome and cell turnover reveal the influences of tissue context and pathological aging on protein and organelle lifetimes

The lifespans of proteins can range from minutes to years within mammalian tissues. Protein lifespan is relevant to organismal aging, as long-lived proteins can accrue damage over time. It is unclear how protein lifetime is shaped by tissue context, where both cell division and proteolytic degradation contribute to protein turnover. Here, we develop turnover and replication analysis by 15N isotope labeling (TRAIL) to quantify both protein and cell lifetimes with high precision and no toxicity over a 32-day labeling period across 4 mammalian tissues. We report that cell division promotes non-selective protein turnover in proliferative tissues, while physicochemical features such as hydrophobicity, charge, and intrinsic disorder exert a significant influence on protein turnover only in non-proliferative tissues. Protein lifetimes vary non-randomly across tissues after correcting for differences in cell division rate. Multiprotein complexes such as the ribosome have highly consistent lifetimes across tissues, while mitochondria, peroxisomes, and lipid droplets have variable lifetimes. These data indicate that cell turnover, sequence-encoded features, and other environmental factors modulate protein lifespan in vivo. In the future, TRAIL can be used to explore how environment, aging, and disease affect tissue homeostasis.

systems biology↗

bioRxiv2022

Accurate proteome-wide measurement of methionine oxidation in aging mouse brains

The oxidation of methionine side chains has emerged as an important posttranslational modification of proteins. A diverse array of low-throughput and targeted studies have suggested that the oxidation of methionine residues in select proteins can have diverse impacts on cell physiology, ranging from detrimental effects on protein structure and stability to functional roles in cell signaling. Despite its importance, the large-scale investigation of methionine oxidation in a complex matrix, such as the cellular proteome, has been historically hampered by technical limitations. Herein we report a methodology, Methionine Oxidation by Blocking (MobB), that allows for accurate and precise quantification of low levels of methionine oxidation typically observed in vivo. To demonstrate the utility of this methodology, we applied MobB to the brain tissues of young (6 m.o.) and old (20 m.o.) mice and identified over 280 novel sites for in vivo methionine oxidation. We further demonstrated that oxidation stoichiometries for specific methionine residues are highly consistent between individual animals and methionine sulfoxides are enriched in clusters of functionally related gene products including membrane and extracellular proteins. However, we did not detect significant changes in methionine oxidation in brains of old mice. Our results suggest that under normal in vivo conditions methionine oxidation is a biologically regulated process rather than a result of stochastic chemical damage.

biochemistry↗

bioRxiv2021

Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility (SA) have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the E. coli proteome using several proteomic methodologies and globally measured oxidation rates of methionines in the presence and absence of tertiary structure, as well as folding stabilities of methionine containing domains. The data indicate that buried methionines have a wide range of protection factors against oxidation which correlate strongly with folding stabilities. Concordantly, we show that in comparison to E. coli, the proteome of the thermophile T. thermophilus is significantly more stable and thus more resistant to methionine oxidation. These results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and suggest a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures.

biochemistry↗

bioRxiv2020

Interspecies differences in proteome turnover kinetics are correlated with lifespans and energetic demands

Cells continually degrade and replace damaged and old proteins. However, the high energetic demand of protein turnover generates reactive oxygen species (ROS) that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse lifespans including the longest-lives rodent, the naked mole rat and the longest-lived mammal, the bowhead whale. We show that organismal lifespan is negatively correlated with turnover rates of highly abundant proteins. In comparison to mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production and reduced ROS levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

systems biology↗

bioRxiv2020

MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling

Aberrant synthesis of mitochondrial proteins impairs cardiac function and causes heart disease. However, the mechanism of regulation of mitochondria encoded protein expression during cardiac disease remains underexplored. Here, we have shown that multiple pathogenic cardiac stressors induce the expression of miR-574 guide and passenger strands (miR-574-5p/3p) in both humans and mice. miR-574 knockout mice exhibit severe cardiac disorder under heart disease-triggering stresses. miR-574-5p/3p mimics that are delivered systematically using nanoparticles reduce cardiac pathogenesis under disease insults. Transcriptome analysis of miR-574-null hearts uncovers FAM210A as a common target mRNA for both strands of miR-574. The interactome capture and translational state analyses suggest that FAM210A interacts with mitochondrial translation factors and regulates the protein expression of mitochondrial encoded electron transport chain genes. Using a human cardiomyocyte cell culture system, we discover that miR-574 regulates FAM210A expression and modulates mitochondrial encoded protein expression, which influences cardiac remodeling in heart failure.

pathology↗