Formylation facilitates the reduction of oxidized initiator methionines.

Within a cell, protein-bound methionines can be chemically or enzymatically oxidized, 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 α-amine group by formylation or acetylation.

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 the sample preparation and ionization steps of typical proteomic workflows. Isotopically labeled hydrogen peroxide (HO) can be used to block unoxidized methionines and enables accurate measurement of levels of methionine oxidation.

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.

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.

Long lifetime and tissue-specific accumulation of lamin A/C in Hutchinson-Gilford progeria syndrome.

LMNA mutations cause laminopathies that afflict the cardiovascular system and include Hutchinson-Gilford progeria syndrome. The origins of tissue specificity in these diseases are unclear as the lamin A/C proteins are broadly expressed. We show that LMNA transcript levels are not predictive of lamin A/C protein levels across tissues and use quantitative proteomics to discover that tissue context and disease mutation each influence lamin A/C protein's lifetime.

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.

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

Mutations to the 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.

Turnover and replication analysis by isotope labeling (TRAIL) reveals the influence of tissue context on protein and organelle lifetimes.

The lifespans of proteins range from minutes to years within mammalian tissues. Protein lifespan is relevant to organismal aging, as long-lived proteins accrue damage over time. It is unclear how protein lifetime is shaped by tissue context, where both cell turnover and proteolytic degradation contribute to protein turnover.

Accurate Proteomewide Measurement of Methionine Oxidation in Aging Mouse Brains.

The oxidation of methionine has emerged as an important post-translational modification of proteins. A number of studies have suggested that the oxidation of methionines in select proteins can have diverse impacts on cell physiology, ranging from detrimental effects on protein 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 hampered by technical limitations.

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 given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation.

Comprehensive Structure-Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C-H Functionalization.

The plant-derived sesquiterpene lactone micheliolide was recently found to possess promising antileukemic activity, including the ability to target and kill leukemia stem cells. Efforts toward improving the biological activity of micheliolide and investigating its mechanism of action have been hindered by the paucity of preexisting functional groups amenable for late-stage derivatization of this molecule. Here, we report the implementation of a probe-based P450 fingerprinting strategy to rapidly evolve engineered P450 catalysts useful for the regio- and stereoselective hydroxylation of micheliolide at two previously inaccessible aliphatic positions in this complex natural product.

Interspecies Differences in Proteome Turnover Kinetics Are Correlated With Life Spans and Energetic Demands.

Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover, and energetic demand remains unclear.

MicroRNA-574 regulates FAM210A expression and influences pathological cardiac remodeling.

Aberrant expression of mitochondrial proteins impairs cardiac function and causes heart disease. The mechanism of regulation of mitochondria encoded protein expression during cardiac disease, however, remains underexplored. Here, we show that multiple pathogenic cardiac stressors induce the expression of miR-574 guide and passenger strands (miR-574-5p/3p) in both humans and mice.

Redox-mediated regulation of an evolutionarily conserved cross-β structure formed by the TDP43 low complexity domain.

A methionine-rich low complexity (LC) domain is found within a C-terminal region of the TDP43 RNA-binding protein. Self-association of this domain leads to the formation of labile cross-β polymers and liquid-like droplets. Treatment with HO caused phenomena of methionine oxidation and droplet melting that were reversed upon exposure of the oxidized protein to methionine sulfoxide reductase enzymes.

Methionine oxidation within the prion protein.

Prion diseases are characterized by the self-templated misfolding of the cellular prion protein (PrP) into infectious aggregates (PrP). The detailed molecular basis of the misfolding and aggregation of PrP remains incompletely understood. It is believed that the transient misfolding of PrP into partially structured intermediates precedes the formation of insoluble protein aggregates and is a critical component of the prion misfolding pathway.

Publisher Correction: Global analysis of protein degradation in prion infected cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Global analysis of protein degradation in prion infected cells.

Prion diseases are rare, neurological disorders caused by the misfolding of the cellular prion protein (PrP) into cytotoxic fibrils (PrP). Intracellular PrP 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 PrP aggregates has the potential to globally influence protein degradation kinetics within an infected cell.

Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome.

The oxidation of methionine is an important post-translational 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.

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.

JNK modifies neuronal metabolism to promote proteostasis and longevity.

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear.

Developmentally regulated H2Av buffering via dynamic sequestration to lipid droplets in embryos.

Regulating nuclear histone balance is essential for survival, yet in early embryos many regulatory strategies employed in somatic cells are unavailable. Previous work had suggested that lipid droplets (LDs) buffer nuclear accumulation of the histone variant H2Av. Here, we elucidate the buffering mechanism and demonstrate that it is developmentally controlled.

Increased Degradation Rates in the Components of the Mitochondrial Oxidative Phosphorylation Chain in the Cerebellum of Old Mice.

Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy.

Cross-species Comparison of Proteome Turnover Kinetics.

The constitutive process of protein turnover plays a key role in maintaining cellular homeostasis. Recent technological advances in mass spectrometry have enabled the measurement of protein turnover kinetics across the proteome. However, it is not known if turnover kinetics of individual proteins are highly conserved or if they have evolved to meet the physiological demands of individual species.

Proteome-wide modulation of degradation dynamics in response to growth arrest.

In dividing cells, cytoplasmic dilution is the dominant route of clearance for long-lived proteins whose inherent degradation is slower than the cellular growth rate. Thus, as cells transition from a dividing to a nondividing state, there is a propensity for long-lived proteins to become stabilized relative to short-lived proteins, leading to alterations in the abundance distribution of the proteome. However, it is not known if cells mount a compensatory response to counter this potentially deleterious proteostatic disruption.