Person: Finley, Daniel
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Finley
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Finley, Daniel
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Publication Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals(The Rockefeller University Press, 2017) Quirós, Pedro M.; Prado, Miguel A.; Zamboni, Nicola; D’Amico, Davide; Williams, Robert W.; Finley, Daniel; Gygi, Steven; Auwerx, JohanMitochondrial stress activates a mitonuclear response to safeguard and repair mitochondrial function and to adapt cellular metabolism to stress. Using a multiomics approach in mammalian cells treated with four types of mitochondrial stressors, we identify activating transcription factor 4 (ATF4) as the main regulator of the stress response. Surprisingly, canonical mitochondrial unfolded protein response genes mediated by ATF5 are not activated. Instead, ATF4 activates the expression of cytoprotective genes, which reprogram cellular metabolism through activation of the integrated stress response (ISR). Mitochondrial stress promotes a local proteostatic response by reducing mitochondrial ribosomal proteins, inhibiting mitochondrial translation, and coupling the activation of the ISR with the attenuation of mitochondrial function. Through a trans–expression quantitative trait locus analysis, we provide genetic evidence supporting a role for Fh1 in the control of Atf4 expression in mammals. Using gene expression data from mice and humans with mitochondrial diseases, we show that the ATF4 pathway is activated in vivo upon mitochondrial stress. Our data illustrate the value of a multiomics approach to characterize complex cellular networks and provide a versatile resource to identify new regulators of mitochondrial-related diseases.Publication Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins(eLife Sciences Publications, Ltd, 2017) Whiteley, Alexandra; Prado, Miguel A; Peng, Ivan; Abbas, Alexander R; Haley, Benjamin; Paulo, Joao; Reichelt, Mike; Katakam, Anand; Sagolla, Meredith; Modrusan, Zora; Lee, Dong Yun; Roose-Girma, Merone; Kirkpatrick, Donald S; McKenzie, Brent S; Gygi, Steven; Finley, Daniel; Brown, Eric JUbiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1−/− mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1−/− B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.Publication Reconfiguration of the proteasome during chaperone-mediated assembly(2013) Park, Soyeon; Li, Xueming; Kim, Ho Min; Singh, Chingakham Ranjit; Tian, Geng; Hoyt, Martin A.; Lovell, Scott; Battaile, Kevin P.; Zolkiewski, Michal; Coffino, Philip; Roelofs, Jeroen; Cheng, Yifan; Finley, DanielThe proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring1–4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5–10. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes2–4, it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.Publication K63 polyubiquitination is a new modulator of the oxidative stress response(2014) Silva, Gustavo M.; Finley, Daniel; Vogel, ChristineUbiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking, and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, which has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional K63-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae subjected to peroxides in a highly sensitive and regulated manner. We demonstrated that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2 leading to accumulation of K63 conjugates assembled by the Rad6-Bre1 ubiquitin conjugase and ligase, respectively. Using linkage-specific isolation methods and SILAC-based quantitative proteomics, we identified >100 new K63 polyubiquitinated targets, which were significantly enriched in ribosomal proteins. Finally, we demonstrated that impairment of K63 ubiquitination during oxidative stress impacts polysome stability and protein expression, rendering cells more sensitive to stress, revealing a new redox-regulatory role for this modification.Publication USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites(Springer Nature, 2016) Lee, Byung-Hoon; Lu, Ying; Prado, Miguel A.; Shi, Yuan; Tian, Geng; Sun, Shuangwu; Elsasser, Suzanne; Gygi, Steven; King, Randall; Finley, DanielUSP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes1-9. Its effects on protein turnover are substrate specific, for unknown reasons. We report that USP14 shows a dramatic preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14’s catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin chain architecture.Publication Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation(Nature Publishing Group, 2016) Choi, Won Hoon; de Poot, Stefanie A. H.; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min JaeWhen in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells.Publication Components of the Ubiquitin-Proteasome Pathway Compete for Surfaces on Rad23 Family Proteins(BioMed Central, 2008) Goh, Amanda M; Walters, Kylie J; Elsasser, Suzanne; Verma, Rati; Deshaies, Raymond J; Finley, Daniel; Howley, PeterBackground: The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain. Results: In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome.Conclusion: We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting.Publication Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome(Springer Nature, 2018-11-12) Dong, Yuanchen; Zhang, Shuwen; Wu, Zhaolong; Li, Xuemei; Wang, Wei; Zhu, Yanan; Stoilova-McPhie, Svetla; Lu, Ying; Finley, Daniel; Mao, YoudongThe proteasome is an ATP-dependent, 2.5-megadalton molecular machine that is responsible for selective protein degradation in eukaryotic cells. Here we present cryo-electron microscopy structures of the substrate-engaged human proteasome in seven conformational states at 2.8–3.6 Å resolution, captured during breakdown of a polyubiquitylated protein. These structures illuminate a spatiotemporal continuum of dynamic substrate–proteasome interactions from ubiquitin recognition to substrate translocation, during which ATP hydrolysis sequentially navigates through all six ATPases. There are three principal modes of coordinated hydrolysis, featuring hydrolytic events in two oppositely positioned ATPases, in two adjacent ATPases and in one ATPase at a time. These hydrolytic modes regulate deubiquitylation, initiation of translocation and processive unfolding of substrates, respectively. Hydrolysis of ATP powers a hinge-like motion in each ATPase that regulates its substrate interaction. Synchronization of ATP binding, ADP release and ATP hydrolysis in three adjacent ATPases drives rigid-body rotations of substrate-bound ATPases that are propagated unidirectionally in the ATPase ring and unfold the substrate.Publication Localization to the Proteasome is Sufficient for Degradation(Elsevier BV, 2004-05-14) Janse, Daniel M.; Crosas, Bernat; Finley, Daniel; Church, GeorgeThe majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.