Biology subjects

Welle, K. A.

Publications and source records attributed to Welle, K. A..

13 recordsLinked to original sources

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↗

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↗

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↗

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↗

bioRxiv2019

Quantitative analysis of in vivo methionine oxidation of the human proteome

The oxidation of methionine is an important posttranslational modification of proteins with numerous roles in physiology and pathology. However, the quantitative analysis of methionine oxidation on a proteome-wide scale has been hampered by technical limitations. Methionine is readily oxidized in vitro during sample preparation and analysis. In addition, there is a lack of enrichment protocols for peptides that contain an oxidized methionine residue; making the accurate quantification of methionine oxidation difficult to achieve on a global scale. Herein, we report a methodology to circumvent these issues by isotopically labeling unoxidized methionines with 18O labeled hydrogen peroxide and quantifying the relative ratios of 18O and 16O oxidized methionines. We validate our methodology using artificially oxidized proteomes made to mimic varying degrees of methionine oxidation. Using this method, we identify and quantify a number of novel sites of in vivo methionine oxidation in an unstressed human cell line.

biochemistry↗

bioRxiv2019

Global analysis of protein degradation in prion infected cells

Prion diseases are rare neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc). Intracellular PrPSc aggregates primarily accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrPSc aggregates have the potential to globally influence protein degradation kinetics within an infected cell. We analyzed the proteome-wide effect of prion infection on protein degradation rates in N2a neuroblastoma cells by dynamic stable isotopic labeling with amino acids in cell culture (dSILAC) and bottom-up proteomics. The analysis quantified the degradation rates of more than 4,700 proteins in prion-infected and uninfected cells. As expected, the degradation rate of the prion protein is significantly decreased upon aggregation in infected cells. In contrast, the degradation kinetics of the remainder of the N2a proteome generally increases upon prion infection. This effect occurs concurrently with increases in the cellular activities of autophagy and lysosomal hydrolases. The resulting enhancement in proteome flux may play a role in the survival of N2a cells upon prion infection.

biochemistry↗

bioRxiv2018

Global analysis of methionine oxidation provides a census of folding stabilities for the human proteome

The stability of proteins influences their tendency to aggregate, undergo degradation or become modified in cells. Despite their significance to understanding protein folding and function, quantitative analyses of thermodynamic stabilities have been mostly limited to soluble proteins in purified systems. We have used a highly multiplexed proteomics approach, based on analyses of methionine oxidation rates, to quantify stabilities of ~10,000 unique regions within ~3,000 proteins in human cell extracts. The data identify lysosomal and extracellular proteins as the most stable ontological subsets of the proteome. We show that the stability of proteins impacts their tendency to become oxidized and is globally altered by the osmolyte trimethylamine-N-oxide (TMAO). We also show that most proteins designated as intrinsically disordered retain their unfolded structure in the complex environment of the cell. Together, the data provide a census of the stability of the human proteome and validate a methodology for global quantitation of folding thermodynamics.

biochemistry↗