scholarly journals Toward an understanding of the Cdc48/p97 ATPase

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1318 ◽  
Author(s):  
Nicholas Bodnar ◽  
Tom Rapoport
Keyword(s):  
Atp Hydrolysis ◽  
Critical Role ◽  
Ring Structure ◽  
Cellular Systems ◽  
Aaa Atpase ◽  
Double Ring ◽  
Terminal Domain ◽  
D2 Domain ◽  
Central Pore

A conserved AAA+ ATPase, called Cdc48 in yeast and p97 or VCP in metazoans, plays an essential role in many cellular processes by segregating polyubiquitinated proteins from complexes or membranes. For example, in endoplasmic reticulum (ER)-associated protein degradation (ERAD), Cdc48/p97 pulls polyubiquitinated, misfolded proteins out of the ER and transfers them to the proteasome. Cdc48/p97 consists of an N-terminal domain and two ATPase domains (D1 and D2). Six Cdc48 monomers form a double-ring structure surrounding a central pore. Cdc48/p97 cooperates with a number of different cofactors, which bind either to the N-terminal domain or to the C-terminal tail. The mechanism of Cdc48/p97 action is poorly understood, despite its critical role in many cellular systems. Recent in vitro experiments using yeast Cdc48 and its heterodimeric cofactor Ufd1/Npl4 (UN) have resulted in novel mechanistic insight. After interaction of the substrate-attached polyubiquitin chain with UN, Cdc48 uses ATP hydrolysis in the D2 domain to move the polypeptide through its central pore, thereby unfolding the substrate. ATP hydrolysis in the D1 domain is involved in substrate release from the Cdc48 complex, which requires the cooperation of the ATPase with a deubiquitinase (DUB). Surprisingly, the DUB does not completely remove all ubiquitin molecules; the remaining oligoubiquitin chain is also translocated through the pore. Cdc48 action bears similarities to the translocation mechanisms employed by bacterial AAA ATPases and the eukaryotic 19S subunit of the proteasome, but differs significantly from that of a related type II ATPase, the NEM-sensitive fusion protein (NSF). Many questions about Cdc48/p97 remain unanswered, including how it handles well-folded substrate proteins, how it passes substrates to the proteasome, and how various cofactors modify substrates and regulate its function.

From the common molecular basis of the AAA protein to various energy-dependent and -independent activities of AAA proteins

2008 ◽  
Vol 36 (1) ◽  
pp. 68-71 ◽  
Author(s):  
Teru Ogura ◽  
Yuka Matsushita-Ishiodori ◽  
Ai Johjima ◽  
Masayo Nishizono ◽  
Shingo Nishikori ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Facilitated Diffusion ◽  
Independent Manner ◽  
Aromatic Residue ◽  
Aaa Atpase ◽  
Central Pore ◽  
Aaa Protein

AAA (ATPase associated with various cellular activities) proteins remodel substrate proteins and protein complexes upon ATP hydrolysis. Substrate remodelling is diverse, e.g. proteolysis, unfolding, disaggregation and disassembly. In the oligomeric ring of the AAA protein, there is a conserved aromatic residue which lines the central pore. Functional analysis indicates that this conserved residue in AAA proteases is involved in threading unfolded polypeptides. Katanin and spastin have microtubule-severing activity. These AAA proteins also possess a conserved aromatic residue at the central pore, suggesting its importance in their biological activity. We have constructed pore mutants of these AAA proteins and have obtained in vivo and in vitro results indicating the functional importance of the pore motif. Degradation of casein by the Escherichia coli AAA protease, FtsH, strictly requires ATP hydrolysis. We have constructed several chimaeric proteases by exchanging domains of FtsH and its homologues from Caenorhabditis elegans mitochondria, and examined their ATPase and protease activities in vitro. Interestingly, it has been found that some chimaeras are able to degrade casein in an ATP-independent manner. The proteolysis is supported by either ATP[S] (adenosine 5′-[γ-thio]triphosphate) or ADP, as well as ATP. It is most likely that substrate translocation in these chimaeras occurs by facilitated diffusion. We have also investigated the roles of C. elegans p97 homologues in aggregation/disaggregation of polyglutamine repeats, and have found that p97 prevents filament formation of polyglutamine proteins in an ATP-independent fashion.

scholarly journals Fundamental Characteristics of AAA+ Protein Family Structure and Function

Archaea ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Justin M. Miller ◽  
Eric J. Enemark
Keyword(s):  
Atp Hydrolysis ◽  
Molecular Machines ◽  
Ring Structure ◽  
Structure And Function ◽  
Central Channel ◽  
Aaa Atpase ◽  
Cellular Events ◽  
Critical Components ◽  
And Function ◽  
Aaa Protein

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

scholarly journals Coordination of Substrate Binding and ATP Hydrolysis in Vps4-Mediated ESCRT-III Disassembly

2010 ◽  
Vol 21 (19) ◽  
pp. 3396-3408 ◽  
Author(s):  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
Johanna Payne ◽  
Anna Shestakova ◽  
Bruce F. Horazdovsky ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Multivesicular Body ◽  
Virus Budding ◽  
Aaa Atpase ◽  
Enveloped Virus ◽  
Dynamic Assembly

ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

scholarly journals Galectin-3 mediates oligomerization of secreted hensin using its carbohydrate-recognition domain

2013 ◽  
Vol 305 (1) ◽  
pp. F90-F99 ◽  
Author(s):  
Soundarapandian Vijayakumar ◽  
Hu Peng ◽  
George J. Schwartz
Keyword(s):  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Critical Role ◽  
Rabbit Kidney ◽  
Intercalated Cells ◽  
Carbohydrate Recognition ◽  
Terminal Domain ◽  
Galectin 3 ◽  
Number Of Cells

A multidomain, multifunctional 230-kDa extracellular matrix (ECM) protein, hensin, regulates the adaptation of rabbit kidney to metabolic acidosis by remodeling collecting duct intercalated cells. Conditional deletion of hensin in intercalated cells of the mouse kidney leads to distal renal tubular acidosis and to a significant reduction in the number of cells expressing the basolateral chloride-bicarbonate exchanger kAE1, a characteristic marker of α-intercalated cells. Although hensin is secreted as a monomer, its polymerization and ECM assembly are essential for its role in the adaptation of the kidney to metabolic acidosis. Galectin-3, a unique lectin with specific affinity for β-galactoside glycoconjugates, directly interacts with hensin. Acidotic rabbits had a significant increase in the number of cells expressing galectin-3 in the collecting duct and exhibited colocalization of galectin-3 with hensin in the ECM of microdissected tubules. In this study, we confirmed the increased expression of galectin-3 in acidotic rabbit kidneys by real-time RT-PCR. Galectin-3 interacted with hensin in vitro via its carbohydrate-binding COOH-terminal domain, and the interaction was competitively inhibited by lactose, removal of the COOH-terminal domain of galectin-3, and deglycosylation of hensin. Galectin-9, a lectin with two carbohydrate-recognition domains, is also present in the rabbit kidney; galectin-9 partially oligomerized hensin in vitro. Our results demonstrate that galectin-3 plays a critical role in hensin ECM assembly by oligomerizing secreted monomeric hensin. Both the NH2-terminal and COOH-terminal domains are required for this function. We suggest that in the case of galectin-3-null mice galectin-9 may partially substitute for the function of galectin-3.

Nitric oxide inhibits the ATPase activity of the chaperone-like AAA+ ATPase CDC48, a target for S-nitrosylation in cryptogein signalling in tobacco cells

2012 ◽  
Vol 447 (2) ◽  
pp. 249-260 ◽  
Author(s):  
Jéremy Astier ◽  
Angélique Besson-Bard ◽  
Olivier Lamotte ◽  
Jean Bertoldo ◽  
Stéphane Bourque ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Oxidative Modification ◽  
Tobacco Cells ◽  
No Donors ◽  
Defence Reaction ◽  
Aaa Atpase ◽  
Proteomic Approach ◽  
D2 Domain

NO has important physiological functions in plants, including the adaptative response to pathogen attack. We previously demonstrated that cryptogein, an elicitor of defence reaction produced by the oomycete Phytophthora cryptogea, triggers NO synthesis in tobacco. To decipher the role of NO in tobacco cells elicited by cryptogein, in the present study we performed a proteomic approach in order to identify proteins undergoing S-nitrosylation. We provided evidence that cryptogein induced the S-nitrosylation of several proteins and identified 11 candidates, including CDC48 (cell division cycle 48), a member of the AAA+ ATPase (ATPase associated with various cellular activities) family. In vitro, NtCDC48 (Nicotiana tabacum CDC48) was shown to be poly-S-nitrosylated by NO donors and we could identify Cys110, Cys526 and Cys664 as a targets for S-nitrosylation. Cys526 is located in the Walker A motif of the D2 domain, that is involved in ATP binding and was previously reported to be regulated by oxidative modification in Drosophila. We investigated the consequence of NtCDC48 S-nitrosylation and found that NO abolished NtCDC48 ATPase activity and induced slight conformation changes in the vicinity of Cys526. Similarly, substitution of Cys526 by an alanine residue had an impact on NtCDC48 activity. More generally, the present study identified CDC48 as a new candidate for S-nitrosylation in plants facing biotic stress and further supports the importance of Cys526 in the regulation of CDC48 by oxidative/nitrosative agents.

scholarly journals Cryo-EM study on the homo-oligomeric ring formation of yeast TRiC/CCT subunits reveals TRiC ring assembly mechanism

2021 ◽  
Author(s):  
Caixuan Liu ◽  
Huping Wang ◽  
Mingliang Jin ◽  
Wenyu Han ◽  
Shutian Wang ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Complex Form ◽  
Ring Structure ◽  
Cellular Protein ◽  
Ring Closure ◽  
Strategy Development ◽  
Structural Basis ◽  
Double Ring ◽  
Ring Assembly ◽  
Assembly Mechanism

AbstractThe complex eukaryotic chaperonin TRiC/CCT helps maintain cellular protein homeostasis, however, its assembly mechanism remains largely unknown. To address the subunit specificity in TRiC assembly, we express each of the individual yeast TRiC subunit in E. coli. Our cryo-EM structural study and biochemical analyses demonstrate that CCT1/2/6 can form TRiC-like homo-oligomeric double ring (HR) complex, however ATP-hydrolysis cannot trigger their ring closure; after deletion of the long N-terminal extension, CCT5 can form the closed double-ring structure; while CCT3/4/7/8 cannot form the HRs. It appears that CCT1 forms a HR in a unique spiral configuration, and ATP-hydrolysis can drive it to re-assemble with an inserted extra subunit-pair. Our data suggest that CCT5 could be the leading subunit in ATP-hydrolysis-driven TRiC ring closure. Moreover, we demonstrate that ADP is sufficient to trigger the assembly of the HRs and TRiC from the assembly intermediate micro-complex form. Our study reveals that through evolution, the more ancestral subunits may have evolved to take more responsibilities in TRiC ring assembly, and we propose a possible assembly mechanism of TRiC involving subunit-pair insertion. Collectively, our study gives hints on the structural basis of subunit specificity in TRiC assembly and cooperativity, beneficial for future TRiC-related therapeutic strategy development.

scholarly journals Rad52 oligomeric N-terminal domain stabilizes Rad51 nucleoprotein filaments and contributes to their protection against Srs2

2021 ◽  
Author(s):  
Emilie Ma ◽  
Laurent Maloisel ◽  
Lea Le Falher ◽  
Raphael Guerois ◽  
Eric Coic
Keyword(s):  
Genome Stability ◽  
Ring Structure ◽  
Homologous Sequence ◽  
Filament Formation ◽  
Repair Synthesis ◽  
Terminal Domain ◽  
Mediator Protein ◽  
Dna Repair Synthesis

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells without disturbing Rad52 mediator and pairing activity, both in vivo and in vitro. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, our findings indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

scholarly journals Inducible alpha-synuclein overexpression affects human Neural Stem Cells behavior

2018 ◽  
Author(s):  
J Zasso ◽  
M Ahmed ◽  
A Cutarelli ◽  
L Conti
Keyword(s):  
Cell Model ◽  
Critical Role ◽  
Alpha Synuclein ◽  
Cellular Systems ◽  
Neuronal Cultures ◽  
Wild Type ◽  
Gene Encoding ◽  
Human Neural Stem Cells ◽  
Cell Model System

AbstractConverging evidence suggest that levels of alpha-Synuclein (aSyn) expression play a critical role in Parkinson’s disease (PD). Several mutations of the SNCA gene, encoding for aSyn have been associated to either the familial or the sporadic forms of PD. Nonetheless, the mechanism underlying wild type aSyn-mediated neurotoxicity in neuronal cells as well as its specific driving role in PD pathogenesis has yet to be fully clarified. In this view, the development of proper in vitro cellular systems is a crucial step.Here we present a novel human Tet-on hNSC cell line, in which aSyn timing and level of expression can be tightly experimentally tuned. Induction of aSyn in self-renewing hNSCs leads to progressive formation of aSyn aggregates and impairs their proliferation and cell survival. Furthermore, aSyn induction during the neuronal differentiation process results in impaired neurogenic potential due to enhanced refractoriness to exit self-renewal and to increase of gliogenic vs neurogenic competence. Finally, acute aSyn induction in hNSC-derived dopaminergic neuronal cultures results in cell toxicity.This novel conditional in vitro cell model system may be a valuable tool for dissecting of aSyn pathogenic effects in hNSCs and neurons and in developing new potential therapeutic strategies.

The structure and evolution of eukaryotic chaperonin-containing TCP-1 and its mechanism that folds actin into a protein spring

2018 ◽  
Vol 475 (19) ◽  
pp. 3009-3034 ◽  
Author(s):  
Keith Robert Willison
Keyword(s):  
Atp Hydrolysis ◽  
Critical Role ◽  
Tight Binding ◽  
Size Control ◽  
Folding Energy ◽  
Control Networks ◽  
Double Ring ◽  
Sequential Binding ◽  
Initial Binding

Actin is folded to its native state in eukaryotic cytosol by the sequential allosteric mechanism of the chaperonin-containing TCP-1 (CCT). The CCT machine is a double-ring ATPase built from eight related subunits, CCT1–CCT8. Non-native actin interacts with specific subunits and is annealed slowly through sequential binding and hydrolysis of ATP around and across the ring system. CCT releases a folded but soft ATP-G-actin monomer which is trapped 80 kJ/mol uphill on the folding energy surface by its ATP-Mg2+/Ca2+ clasp. The energy landscape can be re-explored in the actin filament, F-actin, because ATP hydrolysis produces dehydrated and more compact ADP-actin monomers which, upon application of force and strain, are opened and closed like the elements of a spring. Actin-based myosin motor systems underpin a multitude of force generation processes in cells and muscles. We propose that the water surface of F-actin acts as a low-binding energy, directional waveguide which is recognized specifically by the myosin lever-arm domain before the system engages to form the tight-binding actomyosin complex. Such a water-mediated recognition process between actin and myosin would enable symmetry breaking through fast, low energy initial binding events. The origin of chaperonins and the subsequent emergence of the CCT–actin system in LECA (last eukaryotic common ancestor) point to the critical role of CCT in facilitating phagocytosis during early eukaryotic evolution and the transition from the bacterial world. The coupling of CCT-folding fluxes to the cell cycle, cell size control networks and cancer are discussed together with directions for further research.

scholarly journals Mg2+ binding triggers rearrangement of the IM30 ring structure, resulting in augmented exposure of hydrophobic surfaces competent for membrane binding

2018 ◽  
Vol 293 (21) ◽  
pp. 8230-8241 ◽  
Author(s):  
Jennifer Heidrich ◽  
Benedikt Junglas ◽  
Natalia Grytsyk ◽  
Nadja Hellmann ◽  
Kristiane Rusitzka ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding ◽  
Hydrophobic Surface ◽  
Ring Structure ◽  
Membrane Fusion Protein ◽  
Double Ring ◽  
Quaternary Structures ◽  
And Function

The “inner membrane–associated protein of 30 kDa” (IM30), also known as “vesicle-inducing protein in plastids 1” (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+. However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of ∼1 mm. Mg2+ binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation in vitro because of increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.

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