Aaa Atpase
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2021 ◽  
Author(s):  
Miglė Kišonaitė ◽  
Pavel Afanasyev ◽  
Jonida Tafilaku ◽  
Ana Toste Rêgo ◽  
Paula C. A. da Fonseca

SummaryThe 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.


Author(s):  
Natalya Pashkova ◽  
Liping Yu ◽  
Nicholas J. Schnicker ◽  
Chun-Che Tseng ◽  
Lokesh Gakhar ◽  
...  

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have a central ‘V’ domains that non-covalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4 and ubiquitin-binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.


Author(s):  
Jana Riehl ◽  
Ramesh Rijal ◽  
Leonie Nitz ◽  
Christoph S. Clemen ◽  
Andreas Hofmann ◽  
...  

The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384–466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β0-α–1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261–573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261–573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1’ intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.


2021 ◽  
Author(s):  
Mashun Onishi ◽  
Koji Okamoto

Mitophagy removes defective or superfluous mitochondria via selective autophagy. In yeast, the pro-mitophagic protein Atg32 localizes to the mitochondrial surface and interacts with the scaffold protein Atg11 to promote degradation of mitochondria. Although Atg32-Atg11 interactions are thought to be stabilized by Atg32 phosphorylation, how this posttranslational modification is regulated remains obscure. Here we show that cells lacking the guided entry of tail-anchored proteins (GET) pathway exhibit reduced Atg32 phosphorylation and Atg32-Atg11 interactions, which can be rescued by additional loss of the ER-resident Ppg1-Far complex, a multi-subunit phosphatase negatively acting in mitophagy. In GET-deficient cells, Ppg1-Far is predominantly localized to mitochondria. An artificial ER anchoring of Ppg1-Far in GET-deficient cells significantly ameliorates defects in Atg32-Atg11 interactions and mitophagy. Moreover, disruption of GET and Msp1, an AAA-ATPase that extracts non-mitochondrial proteins localized to the mitochondrial surface, elicits synthetic defects in mitophagy. Collectively, we propose that the GET pathway mediates ER targeting of Ppg1-Far, thereby preventing dysregulated suppression of mitophagy activation.


2021 ◽  
pp. 101187
Author(s):  
Brian Caffrey ◽  
Xing Zhu ◽  
Alison Berezuk ◽  
Katharine Tuttle ◽  
Sagar Chittori ◽  
...  

2021 ◽  
Author(s):  
Keith J Mickolajczyk ◽  
Paul Dominic B Olinares ◽  
Brian T Chait ◽  
Shixin Liu ◽  
Tarun M Kapoor

Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying MIDAS-UBL switching between strongly- and weakly-bound states is unknown. Here, we use single-molecule optical tweezers to investigate the force-dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to tenfold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1's MIDAS domain forms catch bonds with more than one UBL-substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.


2021 ◽  
Author(s):  
Brian Caffrey ◽  
Xing Zhu ◽  
Alison Berezuk ◽  
Katharine Tuttle ◽  
Sagar Chittori ◽  
...  

The human AAA ATPase p97, a potential cancer target, plays a vital role in clearing misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083. These structures demonstrate that the mutations affect nucleotide-driven p97 allosteric activation by predominantly interfering with either the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), the inter-protomer interactions (p97A232E), or the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor CB-5083 to the D2 domain prevents conformational changes similar to that seen for mutations that affect coupling between D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations.


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