Structure and function of the AAA+ ATPase p97/Cdc48p.
Ontology highlight
ABSTRACT:
SUBMITTER: Xia D
PROVIDER: S-EPMC4821690 | biostudies-literature | 2016 May
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.
| S-EPMC5559722 | biostudies-literature
Project description:High level of the multifunctional AAA-ATPase p97/VCP is often correlated to the development of cancer; however, the underlying mechanism is not understood completely. Here, we report a novel function of p97/VCP in actin regulation and cell motility. We found that loss of p97/VCP promotes stabilization of F-actin, which cannot be reversed by actin-destabilizing agent, Cytochalasin D. Live-cell imaging demonstrated reduced actin dynamics in p97/VCP-knockdown cells, leading to compromised cell motility. We further examined the underlying mechanism and found elevated RhoA protein levels along with increased phosphorylation of its downstream effectors, ROCK, LIMK, and MLC upon the knockdown of p97/VCP. Since p97/VCP is indispensable in the ubiquitination-dependent protein degradation pathway, we investigated if the loss of p97/VCP hinders the protein degradation of RhoA. Knockdown of p97/VCP resulted in a higher amount of ubiquitinated RhoA, suggesting p97/VCP involvement in the proteasome-dependent protein degradation pathway. Finally, we found that p97/VCP interacts with FBXL19, a molecular chaperone known to guide ubiquitinated RhoA for proteasomal degradation. Reduction of p97/VCP may result in the accumulation of RhoA which, in turn, enhances cytoplasmic F-actin formation. In summary, our study uncovered a novel function of p97/VCP in actin regulation and cell motility via the Rho-ROCK dependent pathway which provides fundamental insights into how p97/VCP is involved in cancer development.
| S-EPMC6967774 | biostudies-literature
Project description:p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97-p47 complex is in regulation of membrane fusion events. Mono-ubiquitin recognition by p47 has also been shown to be crucial in the p97-p47-mediated Golgi membrane fusion events. Here, we describe the high-resolution solution structures of the N-terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three-helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA-ubiquitin complex. The p47 SEP domain adopts a novel fold with a betabetabetaalphaalphabeta secondary structure arrangement, where beta4 pairs in a parallel fashion to beta1. Based on biophysical studies, we demonstrate a clear propensity for the self-association of p47. Furthermore, p97 N binding abolishes p47 self-association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.
| S-EPMC391063 | biostudies-literature
Project description:Exploratory SAR studies of a new phenyl indole chemotype for p97 inhibition revealed C-5 indole substituent effects in the ADPGlo assay that did not fully correlate with either electronic or steric factors. A focused series of methoxy-, trifluoromethoxy-, methyl-, trifluoromethyl-, pentafluorosulfanyl-, and nitro-analogues was found to exhibit IC50s from low nanomolar to double-digit micromolar. Surprisingly, we found that the trifluoromethoxy-analogue was biochemically a better match of the trifluoromethyl-substituted lead structure than a pentafluorosulfanyl-analogue. Moreover, in spite of their almost equivalent strongly electron-depleting effect on the indole core, pentafluorosulfanyl- and nitro-derivatives were found to exhibit a 430-fold difference in p97 inhibitory activities. Conversely, the electronically divergent C-5 methyl- and nitro-analogues both showed low nanomolar activities.
| S-EPMC4677369 | biostudies-literature
Project description:p97/VCP, a member of the AAA-ATPase super family, has been associated with a wide variety of essential cellular protein pathways com prising: (i) nuclear envelope reconstruction, (ii) cell cycle, (iii) Golgi reassembly, (iv) suppression of apoptosis and (v) DNA-damage response [1-6]. In addition, vasolin-containing protein (VCP) dislodges the ubiquitinated proteins from the endoplasmic reticulum (ER) and chaperones them to the cytosol for proteasomal degradation by endoplasmic reticulum-associated degradation (ERAD) [7]. The interactions of VCP in the endoplasmic reticulum-associated degradation (ERAD) pathway determine the substrate selection for proteasomal degradation. Moreover, the interaction with VCP is also required for the ubiquitination of substrate. VCP is phosphorylated by the master cellular kinase, Akt as a mechanism to regulate ERAD [8]. These multiple interactions in protein degradation pathways points to central role of VCP in misfolded protein degradation. VCP has a polyglutamine and ubiquitin-binding capacity and is involved in proteasomal degradation, cytosolic aggregation and processing of polyQ and polyUb aggregates in neurodegenerative and other misfolded protein diseases [9, 10]. Mutations in VCP gene are also linked to a protein deposition disorder, IBMFD [11]. We propose VCP as a therapeutic target for diseases caused by cytosolic protein aggregation or degradation of misfolded protein. We predict that selective interference of VCP interaction(s) with aberrant protein or its ERAD function will be an effective therapeutic site to rescue functional misfolded protein in diseases like cystic fibrosis and alpha-1-trypsin deficiency. The control of VCP expression is also proposed to be a potential therapeutic target in ex-polyQ-induced neurodegenerative diseases [12]. The further functional characterization of VCP and associated proteins in these diseases will help in designing of selective therapeutics.
| S-EPMC4514128 | biostudies-literature
Project description:The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.
| S-EPMC1456939 | biostudies-literature
Project description:Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.
| S-EPMC6231190 | biostudies-literature
Project description:Multimeric AAA ATPases represent a structurally homologous yet functionally diverse family of proteins. The essential and highly abundant hexameric AAA ATPase p97 is perhaps the best studied AAA protein, playing an essential role in various important cellular activities. During ATP-hydrolysis process, p97 undergoes dramatic conformational changes, and these changes are initiated in the C-terminal ATPase domain and transmitted across the entire length of the molecule to the N-terminal effector domain. However, the detailed mechanism of the motion transmission remains unclear. Here, we report an interprotomer motion-transmission mechanism to explain this process: The nucleotide-dependent motion transmission between the two ATPase domains of one protomer is mediated by its neighboring protomer. This finding reveals a strict requirement for interprotomer coordination of p97 during the motion-transmission process and may shed light on studies of other AAA ATPases.
| S-EPMC3309748 | biostudies-literature
Project description:Protein complexes of the Type II AAA+ (ATPases associated with diverse cellular activities) family are typically hexamers of 80-150 kDa protomers that harbor two AAA+ ATPase domains. They form double ring assemblies flanked by associated domains, which can be N-terminal, intercalated or C-terminal to the ATPase domains. Most prominent members of this family include NSF (N-ethyl-maleimide sensitive factor), p97/VCP (valosin-containing protein), the Pex1/Pex6 complex and Hsp104 in eukaryotes and ClpB in bacteria. Tremendous efforts have been undertaken to understand the conformational dynamics of protein remodeling type II AAA+ complexes. A uniform mode of action has not been derived from these works. This review focuses on p97/VCP and the Pex1/6 complex, which both structurally remodel ubiquitinated substrate proteins. P97/VCP plays a role in many processes, including ER- associated protein degradation, and the Pex1/Pex6 complex dislocates and recycles the transport receptor Pex5 from the peroxisomal membrane during peroxisomal protein import. We give an introduction into existing knowledge about the biochemical and cellular activities of the complexes before discussing structural information. We particularly emphasize recent electron microscopy structures of the two AAA+ complexes and summarize their structural differences.
| S-EPMC5447069 | biostudies-literature
Project description:The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.
| S-EPMC3971404 | biostudies-literature