scholarly journals Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase

2021 ◽  
Author(s):  
Zhejian Ji ◽  
Hao Li ◽  
Daniele Peterle ◽  
Joao A Paulo ◽  
Scott B Ficarro ◽  
...  
Keyword(s):  
Polyubiquitin Chain ◽  
Polyubiquitin Chains ◽  
Macromolecular Complexes ◽  
Protein Substrates ◽  
Double Ring ◽  
Isopeptide Bond ◽  
C Terminus ◽  
Ubiquitin Molecule ◽  
Central Pore

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

scholarly journals Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae

1991 ◽  
Vol 273 (3) ◽  
pp. 615-620 ◽  
Author(s):  
N Agell ◽  
C Ryan ◽  
M J Schlesinger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Partial Purification ◽  
Histone H2a ◽  
Polyubiquitin Chain ◽  
Ubiquitinated Protein ◽  
Protein Conjugates ◽  
Isopeptide Bond ◽  
Isopeptide Bonds ◽  
Ubiquitin Molecule ◽  
Intracellular Proteolysis

A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.

scholarly journals Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

2016 ◽  
Vol 36 (11) ◽  
pp. 1720-1732 ◽  
Author(s):  
Spencer Hill ◽  
Joseph S. Harrison ◽  
Steven M. Lewis ◽  
Brian Kuhlman ◽  
Gary Kleiger
Keyword(s):  
Protein Complexes ◽  
Structural Basis ◽  
Polyubiquitin Chain ◽  
Chain Formation ◽  
Polyubiquitin Chains ◽  
Computational Docking ◽  
C Terminus ◽  
Ubiquitin Conjugating Enzyme ◽  
Covalently Linked ◽  
Conjugating Enzyme

Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and Asp 143 on Ube2R1/2. The validity of this interaction was confirmed through biochemical experiments. Since structural examples involving Arg 54 in protein-ubiquitin complexes are exceedingly rare, these results provide additional insight into how ubiquitin-protein complexes can be stabilized. We discuss how these findings relate to how other ubiquitin-conjugating enzymes direct the lysine specificity of polyubiquitin chains.

scholarly journals A novel polyubiquitin chain linkage formed by viral Ubiquitin prevents cleavage by deubiquitinating enzymes

2019 ◽  
Author(s):  
Hitendra Negi ◽  
Pothula Puroshotham Reddy ◽  
Chhaya Patole ◽  
Ranabir Das
Keyword(s):  
Biochemical Properties ◽  
Autographa Californica ◽  
Polyubiquitin Chain ◽  
Deubiquitinating Enzymes ◽  
Polyubiquitin Chains ◽  
Antiviral Responses ◽  
Binding Domains ◽  
Central Helix ◽  
Ubiquitin Binding ◽  
The Stability

ABSTRACTThe Baculoviridae family of viruses encode a viral Ubiquitin gene. Although the viral Ubiquitin is homologous to eukaryotic Ubiquitin (Ub), preservation of this gene in the viral genome indicates a unique function that is absent in the host eukaryotic Ub. We report the structural, biophysical, and biochemical properties of the viral Ubiquitin from Autographa Californica Multiple Nucleo-Polyhedrosis Virus (AcMNPV). The structure of viral Ubiquitin (vUb) differs from Ub in the packing of the central helix α1 to the beta-sheet of the β-grasp fold. Consequently, the stability of the fold is lower in vUb compared to Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54). The K54-linked polyubiquitin chains are neither effectively cleaved by deubiquitinating enzymes, nor are they targeted by proteasomal degradation. We propose that modification of proteins with the viral Ubiquitin is a mechanism to counter the host antiviral responses.

scholarly journals The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells

2019 ◽  
Vol 116 (27) ◽  
pp. 13293-13298 ◽  
Author(s):  
Ian R. Kelsall ◽  
Jiazhen Zhang ◽  
Axel Knebel ◽  
J. Simon C. Arthur ◽  
Philip Cohen
Keyword(s):  
Ubiquitin Ligase ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
De Novo ◽  
E3 Ligase ◽  
Toll Like Receptor ◽  
Signaling Mechanism ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Physiological Substrates

The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1–generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism.

scholarly journals RPAP3 C-Terminal Domain: A Conserved Domain for the Assembly of R2TP Co-Chaperone Complexes

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1139 ◽  
Author(s):  
Carlos F. Rodríguez ◽  
Oscar Llorca
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphatidylinositol 3 Kinase ◽  
Macromolecular Complexes ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Human Proteins ◽  
Chaperone Complex

The Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex is a co-chaperone complex that works together with HSP90 in the activation and assembly of several macromolecular complexes, including RNA polymerase II (Pol II) and complexes of the phosphatidylinositol-3-kinase-like family of kinases (PIKKs), such as mTORC1 and ATR/ATRIP. R2TP is made of four subunits: RuvB-like protein 1 (RUVBL1) and RuvB-like 2 (RUVBL2) AAA-type ATPases, RNA polymerase II-associated protein 3 (RPAP3), and the Protein interacting with Hsp90 1 (PIH1) domain-containing protein 1 (PIH1D1). R2TP associates with other proteins as part of a complex co-chaperone machinery involved in the assembly and maturation of a growing list of macromolecular complexes. Recent progress in the structural characterization of R2TP has revealed an alpha-helical domain at the C-terminus of RPAP3 that is essential to bring the RUVBL1 and RUVBL2 ATPases to R2TP. The RPAP3 C-terminal domain interacts directly with RUVBL2 and it is also known as RUVBL2-binding domain (RBD). Several human proteins contain a region homologous to the RPAP3 C-terminal domain, and some are capable of assembling R2TP-like complexes, which could have specialized functions. Only the RUVBL1-RUVBL2 ATPase complex and a protein containing an RPAP3 C-terminal-like domain are found in all R2TP and R2TP-like complexes. Therefore, the RPAP3 C-terminal domain is one of few components essential for the formation of all R2TP and R2TP-like co-chaperone complexes.

scholarly journals Function of the p97–Ufd1–Npl4 complex in retrotranslocation from the ER to the cytosol

2003 ◽  
Vol 162 (1) ◽  
pp. 71-84 ◽  
Author(s):  
Yihong Ye ◽  
Hemmo H. Meyer ◽  
Tom A. Rapoport
Keyword(s):  
Atp Hydrolysis ◽  
Bound State ◽  
Polyubiquitin Chain ◽  
Polyubiquitin Chains ◽  
Evolutionarily Conserved ◽  
The Family ◽  
Complex Functions ◽  
Ubiquitin Binding ◽  
Subsequent Degradation ◽  
Er Membrane

Amember of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1–Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97–Ufd1–Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.

scholarly journals Pseudo-DUBs as allosteric activators and molecular scaffolds of protein complexes

2018 ◽  
Vol 46 (2) ◽  
pp. 453-466 ◽  
Author(s):  
Miriam Walden ◽  
Safi Kani Masandi ◽  
Krzysztof Pawłowski ◽  
Elton Zeqiraj
Keyword(s):  
Cell Biology ◽  
Protein Complexes ◽  
Cell Signalling ◽  
Control Cell ◽  
Mechanisms Of Action ◽  
Molecular Scaffolds ◽  
Polyubiquitin Chains ◽  
Macromolecular Complexes ◽  
Cellular Life ◽  
Almost All

The ubiquitin (Ub) proteasome system and Ub signalling networks are crucial to cell biology and disease development. Deubiquitylases (DUBs) control cell signalling by removing mono-Ub and polyubiquitin chains from substrates. DUBs take part in almost all processes that regulate cellular life and are frequently dysregulated in disease. We have catalogued 99 currently known DUBs in the human genome and sequence conservation analyses of catalytic residues suggest that 11 lack enzyme activity and are classed as pseudo-DUBs. These pseudoenzymes play important biological roles by allosterically activating catalytically competent DUBs as well as other active enzymes. Additionally, pseudoenzymes act as assembly scaffolds of macromolecular complexes. We discuss how pseudo-DUBs have lost their catalytic activity, their diverse mechanisms of action and their potential as therapeutic targets. Many known pseudo-DUBs play crucial roles in cell biology and it is likely that unstudied and overlooked pseudo-DUB genes will have equally important functions.

scholarly journals Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

2007 ◽  
Vol 282 (49) ◽  
pp. 35787-35795 ◽  
Author(s):  
Guennadi Kozlov ◽  
Long Nguyen ◽  
Tong Lin ◽  
Gregory De Crescenzo ◽  
Morag Park ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Damage Repair ◽  
The Family ◽  
Uba Domain ◽  
Ordered Water

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85Å crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices α1 and α3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

scholarly journals T-cell regulator RNF125/TRAC-1 belongs to a novel family of ubiquitin ligases with zinc fingers and a ubiquitin-binding domain

2008 ◽  
Vol 410 (1) ◽  
pp. 101-111 ◽  
Author(s):  
Ana Lucia Giannini ◽  
Yifang Gao ◽  
Marie-José Bijlmakers
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Zinc Fingers ◽  
Ubiquitin Ligases ◽  
Ring Domain ◽  
Hiv Replication ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Ubiquitin Binding Domain

The recently identified RNF125 [RING (really interesting new gene) finger protein 125], or TRAC-1 (T-cell RING protein in activation 1), is unique among ubiquitin ligases in being a positive regulator of T-cell activation. In addition, TRAC-1 has been shown to down-modulate HIV replication and to inhibit pathogen-induced cytokine production. However, apart from the presence of an N-terminal C3HC4 (Cys3-His-Cys4) RING domain, the TRAC-1 protein remains uncharacterized. In the present paper, we report novel interactions and modifications for TRAC-1, and elucidate its domain organization. Specifically, we determine that TRAC-1 associates with membranes and is excluded from the nucleus through myristoylation. Our data are further consistent with a crucial role for the C-terminus in TRAC-1 function. In this region, novel domains were recognized through the identification of three closely related proteins: RNF114, RNF138 and RNF166. TRAC-1 and its relatives were found to contain, apart from the RING domain, a C2HC (Cys2-His-Cys)- and two C2H2 (Cys2-His2)-type zinc fingers, as well as a UIM (ubiquitin-interacting motif). The UIM of TRAC-1 binds Lys48-linked polyubiquitin chains and is, together with the RING domain, required for auto-ubiquitination. As a consequence of auto-ubiquitination, the half-life of TRAC-1 is shorter than 30 min. The identification of these novel modifications, interactions, domains and relatives significantly widens the contexts for investigating TRAC-1 activity and regulation.

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.

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