1. VCP/p97-Mediated Unfolding as a Principle in Protein Homeostasis and Signaling 
Johannes van den Boom, Hemmo Meyer 
Molecular Cell 
Volume 69, Issue 2, Pages (January 2018)
DOI: /j.molcel
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2. Figure 1
The AAA+-Type ATPase VCP/p97 and Its Role in the Ubiquitin System
(A) Structure of human p97 hexamer. Each subunit comprises a globular N domain (green) and the two ATPase domains D1 (cyan) and D2 (blue). Molecular visualization was generated with PyMOL software (PDB: 5ftk).
(B) General model for the function of p97 in the ubiquitin system. A substrate protein (S) is post-translationally modified with ubiquitin (purple) by a cascade of ubiquitinating enzymes (E1, E2, and E3). p97 binds the ubiquitinated substrate with the help of adaptor proteins (yellow) and converts the energy from ATP hydrolysis to structurally remodel the target protein. This can serve to release the substrate from binding partners (B) or other cellular structures (not shown). The substrate protein is then handed over to the proteasome (Pr) for degradation. Alternatively, it may be deubiquitinated by a deubiquitinating enzyme (DUB) for recycling.
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3. Figure 2
Most Prominent Roles of p97 in Protein Homeostasis and Cellular Stress Responses
p97 targets and mobilizes ubiquitinated proteins in different pathways upon proteotoxic, organelle, and genotoxic stress. p97 retrotranslocates misfolded ubiquitinated proteins from the ER into the cytosol for degradation (ER-associated degradation; ERAD). Analogously, it contributes to mitochondria-associated degradation (MAD). It also removes the nascent polypeptide chain from stalled ribosomes during ribosome-associated quality control (RQC) and helps degrade subsets of cytosolic proteins. Moreover, p97 mediates chromatin-associated degradation for protein quality control or in DNA damage responses. Lastly, it drives selective macroautophagy of damaged organelles by regulatory extraction of membrane proteins and affects starvation-induced macroautophagy through unknown mechanisms. Substrates of p97 are depicted in red, ubiquitin chains in violet.
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4. Figure 3
Examples for Chromatin-Associated Regulatory Segregation by p97
(A) The replicative helicase (gray) is released from chromatin by p97. For unloading, the helicase subunit Mcm7 (red) is ubiquitinated (light green). This recruits p97 together with the adaptor complex Ufd1-Npl4 (yellow). Using the energy from ATP hydrolysis, p97 extracts ubiquitinated Mcm7, which causes disassembly of the entire replicative helicase. This event is triggered by replication termination and guarantees restoration of chromatin accessibility.
(B) The heterodimeric ring-shaped DNA repair protein Ku70/80 (orange and red, respectively) becomes sterically trapped on chromatin during DNA repair. Upon ubiquitination of Ku80, p97 is recruited together with adaptor proteins (yellow). Subsequently, p97 hydrolyses ATP to structurally remodel Ku and dissociate it from chromatin, thereby restoring chromatin integrity.
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5. Figure 4 Molecular Motions of p97 and Model for Substrate Processing
(A) Cryo-EM structures of human p97 reveal nucleotide-dependent conformational changes (PDB: 5ftl, 5ftn; Banerjee et al., 2016). For clarity, the three subunits in the front of the hexamer have been omitted. In the ADP state (top), the N domains (green) are in the down-conformation coplanar with the D1 ATPase domain (cyan). Conversely, the N domains are located above the D1 domain (up-conformation) in the ATPγS state (bottom). The histidines at position 317 at the D1 pore entry are depicted in orange. The D2 domain (blue) undergoes significant nucleotide-dependent structural changes in the pore loops 1 and 2 (red and yellow, respectively). In the ADP state, the pore loops provide a polar environment in the D2 pore. In contrast, the ATPγS state features a hydrophobic environment in the D2 pore, with aromatic pore loops reaching into the central channel. In addition, the D2 domains are rotated about an axis perpendicular to the pore. Arrows indicate nucleotide-dependent movements of the N and D2 domains. Pore loops are assigned according to Hänzelmann and Schindelin, 2016.
(B) Model for molecular motions during substrate unfolding. In the ADP state, the N domains are in the down-conformation and p97 is in an inactive state. Structural evidence suggests that ATP binds to the D2 domain first. This facilitates binding of ATP in the D1 domain. ATP binding in the D1 domain brings the N domains in the up-conformation, which is further enforced by substrate binding. In addition, substrate binding increases the ATPase activity in the D2 domain and conversely decreases ATP hydrolysis in the D1 domain to favor the up-conformation. Hydrolysis in the D2 domain is not coupled to hydrolysis in the D1 domain in a one-to-one fashion, which allows repeated cycles of ATP binding and hydrolysis in the D2 domain. Hydrolysis cycles in the D2 domain induce conformational changes in the hydrophobic pore loop 1 residues of D2 (red) and cause an alternating hydrophobicity in the D2 pore. Concomitant global changes lead to a rotation of the D2 domain and trigger an up and down movement of the pore loops along the pore axis. These changes in the D2 domain likely generate the driving force for substrate unfolding. Hydrolysis in both ATPase domains returns p97 to the ADP state. Repeated cycles of hydrolysis in the D1 domain and concomitant up and down movements of the N domain may be required for processive substrate unfolding. Note that the D1ATP-D2ADP conformation has not been structurally captured, but is postulated given that the D2 domain has a higher ATP turnover than the D1 domain.
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6. Figure 5
A Substrate Threading Mechanism Can Explain Diverse Molecular Activities of p97
(A) Threading through the central pore generates a pulling force to extract unfolded substrates (red) out of membranes. In ERAD and MAD, these substrates are targeted to the proteasome.
(B) Threading can unfold and thus preprocess difficult substrates (orange) for proteasomal degradation.
(C) Protein threading can explain segregation of substrates (green) from binding partners (blue) or DNA. The substrate can then be handed over to the proteasome for degradation, which can leave an activated partner behind. Alternatively, the unfolded substrate may be refolded for downstream activity.
Ubiquitin is depicted in purple; p97 adaptor proteins are displayed in yellow.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Hypothetical Alternative Unfolding Mechanisms for Different p97 Cofactor Complexes
(A) Recent data suggest that the p97-Ufd1-Npl4 complex unfolds substrates (S) by threading them through the central pore. The arrow indicates the path of the substrate.
(B) Hypothetical model for substrate processing for p97 in complex with trimeric cofactors (yellow), such as p47. The cofactor may promote incomplete threading and release of partially unfolded substrates (indicated by arrow).
(C) Hypothetical model for substrate processing for p97 in complex with cofactors binding to the C terminus (red and brown), such as UBXD1 and PLAA. This p97-cofactor complex could allow local substrate unfolding in the D2 pore without complete threading (indicated by arrow).
Molecular Cell  , DOI: ( /j.molcel )
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