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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.
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...
The Proteasome02:18

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst 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. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
The Proteasome Structure01:17

The Proteasome Structure

The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
The proteasome is an...
Export of Misfolded Proteins out of the ER01:32

Export of Misfolded Proteins out of the ER

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for...

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Related Experiment Video

Updated: May 23, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

A human ubiquitin conjugating enzyme (E2)-HECT E3 ligase structure-function screen.

Yi Sheng1, Jenny H Hong, Ryan Doherty

  • 1Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.

Molecular & Cellular Proteomics : MCP
|April 13, 2012
PubMed
Summary
This summary is machine-generated.

This study reveals key structural features of human ubiquitin E2 conjugating proteins that influence ubiquitin chain building. It also shows that HECT E3 ligase domains dictate ubiquitin chain type, independent of the E2 protein.

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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

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Related Experiment Videos

Last Updated: May 23, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
09:47

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Published on: May 10, 2022

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • Ubiquitin conjugation is a critical post-translational modification.
  • E2 conjugating enzymes and HECT E3 ligases collaborate in ubiquitylation.
  • Understanding E2-E3 interactions is vital for deciphering ubiquitylation pathways.

Purpose of the Study:

  • To systematically analyze the structure-function relationships of human ubiquitin E2 conjugating proteins.
  • To investigate the interactions between E2 proteins and HECT E3 ligase domains.
  • To identify structural determinants of E2 activity in ubiquitin chain formation.

Main Methods:

  • Determination of 15 new high-resolution 3D structures of E2 catalytic domains.
  • Autoubiquitylation assays for 26 Ub-loading E2s against nine HECT E3 ligase domains.
  • Mass spectrometry to characterize autoubiquitylation products.

Main Results:

  • Identified E2 surface properties (charge, acidic trough, basic region) linked to Ub chain building.
  • Found that HECT E3 ligase domains, not E2s, largely determine the type of ubiquitin chain formed.
  • Characterized functional E2-HECT E3 pair interactions and autoubiquitylation products.

Conclusions:

  • Provides the first comprehensive analysis of E2-HECT E3 interactions.
  • Offers a framework for understanding the molecular mechanisms of ubiquitylation.
  • Highlights the distinct roles of E2 and E3 enzymes in ubiquitin chain synthesis.