Broas, S. | bioRxiv Science

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↗