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Related Concept Videos

Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
Regulated Protein Degradation02:58

Regulated Protein Degradation

It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
Regulated Protein Degradation02:58

Regulated Protein Degradation

It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
The Proteasome01:13

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin...

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Related Experiment Video

Updated: May 26, 2026

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
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In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Published on: January 2, 2026

Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97.

Nils Walter1, Laxmikanta Khamari1, Jingxuan Tang1

  • 1University of Michigan.

Research Square
|May 25, 2026
PubMed
Summary

The VCP/p97 chaperone uses Npl4-Ufd1 to degrade polyubiquitinated proteins, with Npl4 binding strength dependent on ubiquitin chain length. D1 ATP hydrolysis drives Npl4 exchange, gating substrate processing and implicating cofactor cycling in disease.

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In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
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In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Biology

Background:

  • The VCP/p97 chaperone complex is crucial for protein degradation via the proteasome.
  • Understanding how p97 recognizes polyubiquitin chain length and how its ATPase domains (D1 and D2) coordinate substrate processing is essential.

Purpose of the Study:

  • To elucidate the mechanism by which VCP/p97 decodes polyubiquitin chain length.
  • To investigate the roles of D1 and D2 ATPase activities in cofactor exchange and substrate processing.
  • To explore the impact of disease-associated VCP/p97 mutations on its function.

Main Methods:

  • Development and application of single-molecule ubiquitin recognition and dynamics (smUbiRAD) assay.
  • Biochemical analysis of p97-Ufd1-Npl4 complex formation and dynamics.
  • Characterization of wild-type and mutant VCP/p97 variants.

Main Results:

  • A sharp chain-length threshold for Npl4 binding was identified, with preferential long-lived engagement on tetra- and penta-ubiquitin.
  • Ufd1 and p97 stabilize Npl4-ubiquitin complexes by suppressing Npl4 dissociation.
  • D1 ATP hydrolysis, not D2, drives rapid Npl4 exchange in assembled complexes, gating substrate processing.
  • Mutant VCP/p97 variants (R155H, A232E) exhibit increased affinity and accelerated Npl4 exchange, suggesting hyperactive cofactor cycling in disease.

Conclusions:

  • VCP/p97 utilizes a chain-length-dependent mechanism for substrate selection via Npl4.
  • D1 ATPase activity acts as a gatekeeper for cofactor turnover and subsequent D2-mediated substrate processing.
  • Dysregulation of VCP/p97 cofactor cycling, particularly hyperactivation, is linked to multisystem proteinopathy.