Newly synthesized proteins undergo multiple modifications to ensure proper biogenesis and acquire their functions. N-terminal methionine excision (NME), mediated by the sequential actions of peptide deformylase (PDF) and methionine aminopeptidase (MAP), is an essential and the most prevalent N-terminal protein modification in the bacterial proteome. Despite the extensive studies on enzymatic catalysis, how NME impacts various cellular functions and how the enzymes achieve timing and selectivity under complex cellular conditions have been long-standing puzzles.
\r\n\r\nIn this work, we use a combination of biochemical analyses, computational modeling, and in vivo measurements to investigate the molecular mechanisms and physiological functions of cotranslational NME reactions. We show that the interactions between the ribosome, the nascent chain, the NME enzymes, and other ribosome-associated protein biogenesis factors dramatically remodel the kinetics and specificity of NME reactions under physiological conditions. In addition, we apply time-resolved, system-wide analyses on the translatome and steady-state proteome to study how the inhibition of PDF influences diverse cellular pathways in bacteria. The results unveil the impact of NME on the biogenesis of nascent proteins and highlight the role of the membrane in coupling the biochemical activities of NME enzymes to cellular physiology.
", "doi": "10.7907/qhz7-a383", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:11549", "collection": "thesis", "collection_id": "11549", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282019-090132145", "primary_object_url": { "basename": "BastaDavid2019thesis.pdf", "content": "final", "filesize": 17391825, "license": "other", "mime_type": "application/pdf", "url": "/11549/17/BastaDavid2019thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Genetic Determinants of Growth Arrest Survival in the Bacterial Pathogen Pseudomonas aeruginosa and the Role of Proteases", "author": [ { "family_name": "Basta", "given_name": "David Wagdi", "orcid": "0000-0003-4176-6566", "clpid": "Basta-David-Wagdi" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Growth arrest is the dominant mode of microbial existence on the planet, yet the molecular mechanisms that underpin survival during growth arrest remain far less studied than other growth states. A better understanding of these mechanisms would provide valuable insight into the activity of microbial communities in both biogeochemical and clinical contexts, including the treatment of chronic infections. This thesis investigates the genetic requirements for survival of the bacterium Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen that thrives in diverse environments in which growth arrest is often caused by energy limitation. After reviewing our current knowledge of the strategies used by growth-arrested bacteria to adjust metabolism, regulate transcription and translation, and maintain the chromosome, I perform a functional genomic screen to identify genes that promote fitness of P. aeruginosa during growth arrest caused by carbon or oxygen starvation. I find that P. aeruginosa can survive for days to weeks in these energy-starved conditions by maintaining a reduced steady-state level of ATP, and that many functional classes of genes are required for fitness. Intriguingly, a majority of genetic fitness determinants differ between carbon and oxygen starvation, despite the common endpoint of reduced ATP levels in these two conditions. Among the few genes generally required for fitness are the stress response sigma factor encoded by rpoS and the heat shock protease encoded by ftsH. Using independently-generated deletion strains, I show that mutants in distinct functional categories exhibit temporal fitness dynamics during oxygen starvation: regulatory genes generally manifest a phenotype early during growth arrest, whereas genes involved in cell wall metabolism are required later. Building on these findings, I investigate the functional role of FtsH during growth arrest more deeply and find a surprising negative genetic interaction between ftsH and rpoS, with mutations in rpoS alleviating the fitness defects of \u0394ftsH during growth arrest. I also find that FtsH functions coordinately with the other conserved heat shock proteases to maintain cellular integrity and delay aging of P. aeruginosa during growth arrest. Finally, I investigate the role of FtsH and the other heat shock proteases in a novel N-terminal protein degradation pathway and find that the molecular details of this pathway likely differ between E. coli and P. aeruginosa. Together, these findings uncover essential molecular processes that promote fitness of an important bacterial pathogen during growth and survival.
", "doi": "10.7907/K6X1-GS91", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:8114", "collection": "thesis", "collection_id": "8114", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03072014-090536401", "primary_object_url": { "basename": "lyapina 2001.pdf", "content": "final", "filesize": 18625089, "license": "other", "mime_type": "application/pdf", "url": "/8114/1/lyapina 2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Characterization of the Human SCF Ubiquitin Ligases - Structure, Function, and Regulation", "author": [ { "family_name": "Lyapina", "given_name": "Svetlana Anatol'Evna", "clpid": "Lyapina-Svetlana-Anatol'Evna" } ], "thesis_advisor": [ { "family_name": "Dunphy", "given_name": "William G.", "orcid": "0000-0001-7598-8939", "clpid": "Dunphy-W-G" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Dunphy", "given_name": "William G.", "orcid": "0000-0001-7598-8939", "clpid": "Dunphy-W-G" }, { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A-J" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by\r\ncata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several\r\nevolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein\r\nCDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of\r\nCDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with\r\nhSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover,\r\nhCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin\r\nligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as\r\npart of an SCF ubiquitin ligase complex in human cells. However, purified human SCF\r\ncomplexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional\r\nfactors are required.
\r\n\r\nSubsequently, mammalian SCF ubiquitin ligases were shown to regulate various\r\nphysiological processes by targeting important cellular regulators, like l\u0138B\u03b1, \u03b2-catenin, and p27,\r\nfor ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the\r\nregulation of various SCF complexes. By using sequential immunoaffinity purification and mass\r\nspectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1\r\nin vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF\r\ncomplexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes.\r\nSubsequent work by others demonstrated that these proteins are essential for SCF activity. We\r\nalso discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of\r\nphotomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is\r\nevolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based\r\nubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This\r\nwork sheds light onto an intricate connection that exists between signal transduction pathways\r\nand protein degradation machinery inside the cell and sets stage for gaining further insights into\r\nregulation of protein degradation.
\r\n", "doi": "10.7907/apdy-3d30", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" } ]