Regulated protein degradation underlies the timely execution of essential gene expression programs in bacteria. Here, we deployed time-resolved chemoproteomics, text mining of the PubMed and EcoCyc knowledge bases, and machine learning classification to identify proteolytic regulation in exponential and stationary phase Escherichia coli cultures. We experimentally validated the instability of diverse homeostatic and stress response regulators, including the principal cyclic-di-GMP phosphodiesterase PdeH, the N-end rule substrate chaperone ClpS, and all four A-type domain iron–sulfur cluster carriers, IscA, ErpA, NfuA, and SufA. Mutagenesis of the PdeH N-terminal extension abolished ClpXP recognition, thereby impairing stationary phase depletion of PdeH and altering macrocolony biofilm surface morphology. Unstable proteins synthesized in stationary phase such as the morphology regulator BolA, RNA polymerase ω subunit, and the biofilm regulator BssR were implicated in quiescence. Finally, machine learning–assisted substrate identification revealed Lon-mediated degradation of two opposing key regulators of surface adhesion, the RpoS antagonist FliZ and the major biofilm regulator CsgD, suggesting proteolysis may hasten transitions between motility and sessility. Together, these results highlight the role of regulated proteolysis in driving physiological adaptation for this model organism.
", "doi": "10.1073/pnas.2515265123", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences", "publication_date": "2026-03-10", "series_number": "10", "volume": "123", "issue": "10", "pages": "e2515265123" }, { "id": "authors:aq0vs-sxf90", "collection": "authors", "collection_id": "aq0vs-sxf90", "cite_using_url": "https://authors.library.caltech.edu/records/aq0vs-sxf90", "type": "article", "title": "PLAA/UFD-3 regulates P-bodies through its intrinsic disordered domain", "author": [ { "family_name": "Das", "given_name": "Alakananda", "clpid": "Das-Alakananda" }, { "family_name": "Qiu", "given_name": "Yanping", "orcid": "0000-0003-2948-2173", "clpid": "Qiu-Yanping" }, { "family_name": "Wolf", "given_name": "Trevor J.", "clpid": "Wolf-Trevor-J" }, { "family_name": "Brissett", "given_name": "Ella", "clpid": "Brissett-Ella" }, { "family_name": "Cho", "given_name": "Jaehyoung", "clpid": "Cho-Jaehyoung" }, { "family_name": "Park", "given_name": "Heenam", "orcid": "0000-0001-7911-5828", "clpid": "Park-Heenam" }, { "family_name": "Chen", "given_name": "Eugene C.", "clpid": "Chen-Eugene-C" }, { "family_name": "Chou", "given_name": "Tsui-Fen", "orcid": "0000-0003-2410-2186", "clpid": "Chou-Tsui-Fen" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "abstract": "Regulation of proteome homeostasis is crucial for the survival and adaptation to changing environments for all species. In eukaryotes, this process is finely tuned through regulation at the level of transcription, translation, protein modification, and protein degradation. The phospholipase A2 activating protein (PLAA) is present in all eukaryotes and believed to be a key player in ubiquitin-dependent protein sorting and degradation via its interactions with ubiquitin and/or the AAA+ ATPase, valosin-containing protein (VCP/p97). PLAA’s molecular targets and interaction network remain unclear. We used Caenorhabditis elegans and unbiased proteome-scale approaches to investigate neuronal specific interactors of the C. elegans PLAA ortholog UFD-3 (ubiquitin fusion degradation 3), its effect on ubiquitinated proteins, and global protein expression changes in an ufd-3 mutant. We found that PLAA may play a unique role in cytoplasmic messenger ribonucleic acid (mRNA) processing bodies (P-bodies). Using biochemical analysis in vitro and fluorescence imaging in C. elegans, we show that UFD-3 directly interacts with the mRNA decapping complex regulatory subunit DCAP-1. UFD-3's intrinsic disordered region (IDR), which contains conserved amino acid motifs, is important for the recruitment of DCAP-1 to P-bodies. Finally, we show that loss of the IDR does not affect UFD-3's role in sorting ubiquitinated proteins through the multivesicular body pathway. Collectively, our results suggest that UFD-3's role in P-bodies is distinct from its role in the ubiquitin-dependent protein degradation pathway and the IDR is only critical for UFD-3-regulated P-bodies pathways. Thus, PLAA/UFD-3 might regulate the proteome via two distinct pathways: ubiquitinated protein turnover, as well as mRNA regulation through P-bodies.
", "doi": "10.1073/pnas.2427250122", "pmcid": "PMC12232612", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences", "publication_date": "2025-07-01", "series_number": "26", "volume": "122", "issue": "26", "pages": "e2427250122" }, { "id": "authors:n18vd-tcz58", "collection": "authors", "collection_id": "n18vd-tcz58", "cite_using_url": "https://authors.library.caltech.edu/records/n18vd-tcz58", "type": "article", "title": "Optimizing Protein Production in the One-Pot PURE System: Insights into Reaction Composition and Expression Efficiency", "author": [ { "family_name": "Zhang", "given_name": "Yan", "orcid": "0000-0003-0719-5456", "clpid": "Zhang-Yan" }, { "family_name": "Deveikis", "given_name": "Matas", "orcid": "0000-0001-7444-2950" }, { "family_name": "Qiu", "given_name": "Yanping", "orcid": "0000-0003-2948-2173", "clpid": "Qiu-Yanping" }, { "family_name": "Bj\u00f6rn", "given_name": "Lovisa", "orcid": "0009-0007-6983-3341", "clpid": "Bj\u00f6rn-Lovisa" }, { "family_name": "Martinez", "given_name": "Zachary A.", "orcid": "0000-0002-7830-3162", "clpid": "Martinez-Zachary-A" }, { "family_name": "Chou", "given_name": "Tsui-Fen", "orcid": "0000-0003-2410-2186", "clpid": "Chou-Tsui-Fen" }, { "family_name": "Freemont", "given_name": "Paul S.", "orcid": "0000-0002-5658-8486" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "abstract": "The One-Pot PURE (Protein synthesis Using Recombinant Elements) system simplifies the preparation of traditional PURE systems by coculturing and purifying 36 essential proteins for gene expression in a single step, enhancing accessibility and affordability for widespread laboratory adoption and customization. However, replicating this protocol to match the productivity of traditional PURE systems can take considerable time and effort due to uncharacterized variability. In this work, we observed unstable PURE protein expression in the original One-Pot PURE strains, E. coli M15/pREP4 and BL21(DE3), and addressed this issue using glucose-mediated catabolite repression to minimize burdensome background expression. We also identified several limitations making the M15/pREP4 strain unsuitable for PURE protein expression, including coculture incompatibility with BL21(DE3) and uncharacterized proteolytic activity. We showed that consolidating all expression vectors into a protease-deficient BL21(DE3) strain minimized proteolysis, led to more uniform coculture cell growth at the time of induction, and improved the stoichiometry of critical translation initiation factors in the final PURE mixture for efficient cell-free protein production. In addition to optimizing the One-Pot PURE protein composition, we found that variations in commercial energy solution formulations could compensate for suboptimal PURE protein stoichiometry. Notably, altering the source of E. coli tRNAs in the energy solution alone led to significant differences in the expression capacity of cell-free reactions, highlighting the importance of tRNA codon usage in influencing protein expression yield. Taken together, this work systematically investigates the proteome and biochemical factors influencing the One-Pot PURE system productivity, offering insights to enhance its robustness and adaptability across laboratories.
", "doi": "10.1021/acssynbio.4c00779", "issn": "2161-5063", "publisher": "American Chemical Society", "publication": "ACS Synthetic Biology", "publication_date": "2025-05-16", "series_number": "5", "volume": "14", "issue": "5", "pages": "1496-1508" }, { "id": "authors:x44fc-kq819", "collection": "authors", "collection_id": "x44fc-kq819", "cite_using_url": "https://authors.library.caltech.edu/records/x44fc-kq819", "type": "article", "title": "Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples", "author": [ { "family_name": "Pang", "given_name": "Marion", "orcid": "0000-0002-0158-2976", "clpid": "Pang-Marion" }, { "family_name": "Jones", "given_name": "Jeff J.", "orcid": "0000-0002-7142-2222", "clpid": "Jones-Jeff-J" }, { "family_name": "Wang", "given_name": "Ting-Yu", "orcid": "0000-0002-9014-6825", "clpid": "Wang-Ting-Yu" }, { "family_name": "Quan", "given_name": "Baiyi", "orcid": "0000-0001-6313-4274", "clpid": "Quan-Baiyi" }, { "family_name": "Kubat", "given_name": "Nicole J.", "clpid": "Kubat-Nicole-J" }, { "family_name": "Qiu", "given_name": "Yanping", "orcid": "0000-0003-2948-2173", "clpid": "Qiu-Yanping" }, { "family_name": "Roukes", "given_name": "Michael L.", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Chou", "given_name": "Tsui-Fen", "orcid": "0000-0003-2410-2186", "clpid": "Chou-Tsui-Fen" } ], "abstract": "The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.
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