RESUMO
The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/química , Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Citidina Trifosfato/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Signal transduction by small ubiquitin-like modifier (SUMO) regulates a myriad of nuclear processes. Here we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery results in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identify the anaphase-promoting complex/cyclosome (APC/C), a master regulator of metaphase to anaphase transition. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation is crucial for accurate progression of cells through mitosis and increases APC/C ubiquitylation activity toward a subset of its targets, including the newly identified target KIF18B. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal how SUMO and ubiquitin cooperate to drive mitosis.
Assuntos
Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Mitose/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Ubiquitinas/metabolismo , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/genética , Linhagem Celular Tumoral , Células HCT116 , Células HeLa , Humanos , Cinesinas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinas/genéticaRESUMO
Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.
Assuntos
Terapia Genética , Microcefalia/genética , Microcefalia/terapia , Células-Tronco Neurais/fisiologia , Proteínas Nucleares/deficiência , Adenoviridae/genética , Animais , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Apoptose/genética , Encéfalo/patologia , Proteínas de Transporte/genética , Moléculas de Adesão Celular/metabolismo , Ciclo Celular , Proliferação de Células , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/patologia , Nestina/genética , Nestina/metabolismo , Neurogênese , Proteínas Nucleares/genética , Sinapsinas/genética , Sinapsinas/metabolismoRESUMO
Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.
