{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/364007"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/364007","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Mechanisms of TRIM21-directed intracellular degradation","abstract":"The cytosolic antibody receptor TRIM21 has been studied for more than a decade for its role in mediating antibody-dependent neutralization of nonenveloped viruses. The presence of antibodies in the cytosol is abnormal—by binding them and initiating inflammatory signaling cascades, TRIM21 acts as a danger sensor and intracellular effector of humoral immunity. In addition to antibody binding, TRIM21 is a ubiquitin E3 ligase, an enzyme which catalyzes the formation of polymers of the protein ubiquitin around target substrates. When viruses coated in non-neutralizing antibodies escape into the cytosol, TRIM21 binds and decorates the complex with these polyubiquitin chains. Polyubiquitin is a highly-conserved protein post- translational modification with a number of important roles, including the initiation of degradative processes and antiviral genetic programs. Polyubiquitin-coated TRIM21:antibody:virus complexes are degraded by the cell, thus neutralizing viral infection. Its ability to bind generic immunoglobulins has allowed TRIM21 to be developed into the targeted protein degradation technology Trim-Away, whose intracellular mechanism is thought to mirror that of antibody-coated virions. Prior to the work presented here, the specific mechanistic steps following TRIM21-antibody binding had been established in some detail, and were believed to involve the sequential action of ubiquitin E2 conjugating enzymes UBE2W and UBE2N, which cooperated with TRIM21 to catalyze the formation of a specific type of linear polyubiquitin chain linked through lysine 63 on ubiquitin. This was thought to lead to the recruitment of the segregase p97/VCP and the 26S proteosome which together disassembled and degraded the encapsidated virion. In this thesis, I describe my efforts to define the sequential enzymatic mechanism of TRIM21 degradative signaling in greater detail. I report data from a series of biochemical and in-cell experiments which are not consistent with a role for UBE2W in polyubiquitin chain priming on TRIM21 during adenoviral neutralization. Then, I detail my development of two proximity-proteomics screens and a whole-genome CRISPR/Cas9 knockout screen for TRIM21 interacting partners, in both Trim-Away and adenovirus neutralization contexts. Collectively, the data from these experiments led me to hypothesize that the TRIM21 mechanism is plastic, and sensitive to substrate features and intracellular context. The whole-genome screen and follow up validatory experiments were consistent with a new model for TRIM21-adenoviral neutralization—rather than processing through the proteasome, intracellular adenoviruses coated with antibodies are degraded by selective autophagy following polyubiquitin deposition by TRIM21. Taken together, this work represents a clear advancement in understanding how TRIM21 targets substrates for proteolysis, and opens several new lines of inquiry into how the protein affects adaptive immunity at the cellular level.","abstract_html":"The cytosolic antibody receptor TRIM21 has been studied for more than a decade for its role in mediating antibody-dependent neutralization of nonenveloped viruses. The presence of antibodies in the cytosol is abnormal—by binding them and initiating inflammatory signaling cascades, TRIM21 acts as a danger sensor and intracellular effector of humoral immunity. In addition to antibody binding, TRIM21 is a ubiquitin E3 ligase, an enzyme which catalyzes the formation of polymers of the protein ubiquitin around target substrates. When viruses coated in non-neutralizing antibodies escape into the cytosol, TRIM21 binds and decorates the complex with these polyubiquitin chains. Polyubiquitin is a highly-conserved protein post- translational modification with a number of important roles, including the initiation of degradative processes and antiviral genetic programs. Polyubiquitin-coated TRIM21:antibody:virus complexes are degraded by the cell, thus neutralizing viral infection. Its ability to bind generic immunoglobulins has allowed TRIM21 to be developed into the targeted protein degradation technology Trim-Away, whose intracellular mechanism is thought to mirror that of antibody-coated virions. Prior to the work presented here, the specific mechanistic steps following TRIM21-antibody binding had been established in some detail, and were believed to involve the sequential action of ubiquitin E2 conjugating enzymes UBE2W and UBE2N, which cooperated with TRIM21 to catalyze the formation of a specific type of linear polyubiquitin chain linked through lysine 63 on ubiquitin. This was thought to lead to the recruitment of the segregase p97/VCP and the 26S proteosome which together disassembled and degraded the encapsidated virion. In this thesis, I describe my efforts to define the sequential enzymatic mechanism of TRIM21 degradative signaling in greater detail. I report data from a series of biochemical and in-cell experiments which are not consistent with a role for UBE2W in polyubiquitin chain priming on TRIM21 during adenoviral neutralization. Then, I detail my development of two proximity-proteomics screens and a whole-genome CRISPR/Cas9 knockout screen for TRIM21 interacting partners, in both Trim-Away and adenovirus neutralization contexts. Collectively, the data from these experiments led me to hypothesize that the TRIM21 mechanism is plastic, and sensitive to substrate features and intracellular context. The whole-genome screen and follow up validatory experiments were consistent with a new model for TRIM21-adenoviral neutralization—rather than processing through the proteasome, intracellular adenoviruses coated with antibodies are degraded by selective autophagy following polyubiquitin deposition by TRIM21. Taken together, this work represents a clear advancement in understanding how TRIM21 targets substrates for proteolysis, and opens several new lines of inquiry into how the protein affects adaptive immunity at the cellular level.","abstract_has_math":false,"creators":["Rhinesmith, Tyler"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["James, Leo"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-11-20","date_published":"2023-11-20","updated_at":"2026-07-22T22:24:23Z","subjects":["antibody","autophagy","cell autonomous immunity","ubiquitin"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cc85db44-e906-45fa-893b-e2815c7111d6/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.105834","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["James, Leo"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["MRC Laboratory of Molecular Biology Cambridge Trust"]},{"key":"dc:creator","label":"Author","values":["Rhinesmith, Tyler"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-11-20"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/364007"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["antibody","autophagy","cell autonomous immunity","ubiquitin"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cc85db44-e906-45fa-893b-e2815c7111d6/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.105834"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3837b7c9-ff21-40c6-88ce-7ca1a586378b/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The cytosolic antibody receptor TRIM21 has been studied for more than a decade for its role in mediating antibody-dependent neutralization of nonenveloped viruses. 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Its ability to bind generic immunoglobulins has allowed TRIM21 to be developed into the targeted protein degradation technology Trim-Away, whose intracellular mechanism is thought to mirror that of antibody-coated virions. Prior to the work presented here, the specific mechanistic steps following TRIM21-antibody binding had been established in some detail, and were believed to involve the sequential action of ubiquitin E2 conjugating enzymes UBE2W and UBE2N, which cooperated with TRIM21 to catalyze the formation of a specific type of linear polyubiquitin chain linked through lysine 63 on ubiquitin. This was thought to lead to the recruitment of the segregase p97/VCP and the 26S proteosome which together disassembled and degraded the encapsidated virion. In this thesis, I describe my efforts to define the sequential enzymatic mechanism of TRIM21 degradative signaling in greater detail. I report data from a series of biochemical and in-cell experiments which are not consistent with a role for UBE2W in polyubiquitin chain priming on TRIM21 during adenoviral neutralization. Then, I detail my development of two proximity-proteomics screens and a whole-genome CRISPR/Cas9 knockout screen for TRIM21 interacting partners, in both Trim-Away and adenovirus neutralization contexts. Collectively, the data from these experiments led me to hypothesize that the TRIM21 mechanism is plastic, and sensitive to substrate features and intracellular context. The whole-genome screen and follow up validatory experiments were consistent with a new model for TRIM21-adenoviral neutralization—rather than processing through the proteasome, intracellular adenoviruses coated with antibodies are degraded by selective autophagy following polyubiquitin deposition by TRIM21. 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Its ability to bind generic immunoglobulins has allowed TRIM21 to be developed into the targeted protein degradation technology Trim-Away, whose intracellular mechanism is thought to mirror that of antibody-coated virions. Prior to the work presented here, the specific mechanistic steps following TRIM21-antibody binding had been established in some detail, and were believed to involve the sequential action of ubiquitin E2 conjugating enzymes UBE2W and UBE2N, which cooperated with TRIM21 to catalyze the formation of a specific type of linear polyubiquitin chain linked through lysine 63 on ubiquitin. This was thought to lead to the recruitment of the segregase p97/VCP and the 26S proteosome which together disassembled and degraded the encapsidated virion. In this thesis, I describe my efforts to define the sequential enzymatic mechanism of TRIM21 degradative signaling in greater detail. I report data from a series of biochemical and in-cell experiments which are not consistent with a role for UBE2W in polyubiquitin chain priming on TRIM21 during adenoviral neutralization. Then, I detail my development of two proximity-proteomics screens and a whole-genome CRISPR/Cas9 knockout screen for TRIM21 interacting partners, in both Trim-Away and adenovirus neutralization contexts. Collectively, the data from these experiments led me to hypothesize that the TRIM21 mechanism is plastic, and sensitive to substrate features and intracellular context. The whole-genome screen and follow up validatory experiments were consistent with a new model for TRIM21-adenoviral neutralization—rather than processing through the proteasome, intracellular adenoviruses coated with antibodies are degraded by selective autophagy following polyubiquitin deposition by TRIM21. 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