Pulse labeling reveals the tail end of protein folding by proteome profiling

Zhu, Mang; Kuechler, Erich R.; Wong, Ryan W.K.; Calabrese, Gaetano; Sitarik, Ian; Rana, Viraj; Stoynov, Nikolay; O’Brien, Edward P.; Gsponer, Jörg; Mayor, Thibault

doi:10.1016/j.celrep.2022.111096

Cited by 12 publications

(10 citation statements)

References 71 publications

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“…Young proteins can be ubiquitinated and targeted to the proteasome even in cases where the steady-state population of that protein in the cell is relatively long-lived. The results support previous findings indicating that many proteins are rapidly culled during or soon after synthesis, and those that survive become part of a more stable steady-state proteome (35, 38). Functional enrichment analysis identified subunits of protein complexes as one common class of rapidly degraded proteins.…”

Section: Discussionsupporting

confidence: 91%

Proteome birthdating reveals age-selectivity of protein ubiquitination

Meadow,

Broas,

Hoare

et al. 2023

Preprint

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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 protein’s 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.

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Section: Discussionsupporting

confidence: 91%

Proteome birthdating reveals age-selectivity of protein ubiquitination

Meadow,

Broas,

Hoare

et al. 2023

Preprint

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“…To identify features associated with aging and NDs, we collected 38 protein features, similar to properties that we previously assessed when characterizing proteins affected by stress response ( 31 – 33 ). Using principal component analysis (PCA), relevant features were identified by removing features until >80% of the variance was captured in dimension 1.…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with this interpretation, we find that proteins that are enriched in the pellet upon aging have significantly higher supersaturation scores. Interestingly, we recently showed that yeast proteins with similar properties (e.g., high structure content) tend to remain thermo-sensitive for a longer period of time following their synthesis ( 33 ). Therefore, an alternative explanation is that the accumulation of these proteins in the pellet fraction occurs due to a lower folding capacity affecting particular proteins that are being translated.…”

Section: Discussionmentioning

confidence: 99%

Shift of the insoluble content of the proteome in the aging mouse brain

Molzahn,

Kuechler,

Zemlyankina

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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During aging, the cellular response to unfolded proteins is believed to decline, resulting in diminished proteostasis. In model organisms, such as Caenorhabditis elegans, proteostatic decline with age has been linked to proteome solubility shifts and the onset of protein aggregation. However, this correlation has not been extensively characterized in aging mammals. To uncover age-dependent changes in the insoluble portion of a mammalian proteome, we analyzed the detergent-insoluble fraction of mouse brain tissue by mass spectrometry. We identified a group of 171 proteins, including the small heat shock protein α-crystallin, that become enriched in the detergent-insoluble fraction obtained from old mice. To enhance our ability to detect features associated with proteins in that fraction, we complemented our data with a meta-analysis of studies reporting the detergent-insoluble proteins in various mouse models of aging and neurodegeneration. Strikingly, insoluble proteins from young and old mice are distinct in several features in our study and across the collected literature data. In younger mice, proteins are more likely to be disordered, part of membraneless organelles, and involved in RNA binding. These traits become less prominent with age, as an increased number of structured proteins enter the pellet fraction. This analysis suggests that age-related changes to proteome organization lead a group of proteins with specific features to become detergent-insoluble. Importantly, these features are not consistent with those associated with proteins driving membraneless organelle formation. We see no evidence in our system of a general increase of condensate proteins in the detergent-insoluble fraction with age.

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“…misfolding – and/or aggregation. In support of this, several sources report that newly synthesized proteins are more vulnerable to misfolding and aggregation than existing, matured proteins [15, 16], with topologically complex proteins being more at risk [16]. It is also widely understood that premature co-translational misfolding – before a critical nascent chain length is achieved – is indeed detrimental and must be avoided [17].…”

Section: Introductionmentioning

confidence: 99%

Analyzing the implications of protein folding delay caused by translation

Houben,

Duran-Romaña,

Migens

et al. 2024

Preprint

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Because of vectorial protein production, residues that interact in the native protein structure but are distantly separated in the primary sequence are unavailable simultaneously. Instead, there is a temporal delay during which the N-terminal interaction partner is vulnerable to off-pathway, non-native interactions. In this analysis, we introduce "FoldDelay" (FD), a metric that integrates the topological pattern of atomic interactions of the native structure with translation kinetics to quantify such time delays. The FD metric reveals that many proteins, particularly at eukaryotic translation rates, exhibit residues with FDs in the range of tens of seconds. These residues, predominantly in well-structured, buried regions, often coincide with predicted aggregation-prone regions. We show a correlation between FD and co-translational engagement by the yeast Hsp70 chaperone Ssb, suggesting that fold-delayed regions have a propensity to misfold. In support of this, we show that proteins with high FDs are more frequently co-translationally ubiquitinated and prone to aggregate upon Ssb deletion. Finally, we find that FD cannot be adequately reduced through codon optimization, highlighting the importance of co-translational chaperones to shield these vulnerable regions. This work offers insights into co-translational proteostasis and the delicate balance between efficient folding and potential misfolding and aggregation during translation.

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Pulse labeling reveals the tail end of protein folding by proteome profiling

Cited by 12 publications

References 71 publications

Proteome birthdating reveals age-selectivity of protein ubiquitination

Proteome birthdating reveals age-selectivity of protein ubiquitination

Shift of the insoluble content of the proteome in the aging mouse brain

Analyzing the implications of protein folding delay caused by translation

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