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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...
Protein Modifications in the RER01:26

Protein Modifications in the RER

Modification of secretory and transmembrane proteins entering the rough ER begins in the ER lumen. These modifications aid in protein folding and stabilize the acquired tertiary structure. Protein modifications in the rough ER co-occur at different stages of protein folding.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
Receptor Downregulation in MVBs01:15

Receptor Downregulation in MVBs

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal growth factor (EGF) and its receptor (EGFR) form a complex that can be internalized through endocytosis, sorted into an MVB, and later degraded.
The EGFR can initiate signaling pathways that  lead to cell proliferation, migration, and differentiation. Overexpression of EGFR  stimulates cells to proliferate. Excessive  EGFR activation may...

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Related Experiment Video

Updated: May 27, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

E1-E2 interactions in ubiquitin and Nedd8 ligation pathways.

Zeynep Tokgöz1, Thomas J Siepmann1, Frederick Streich2

  • 1Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.

The Journal of Biological Chemistry
|November 10, 2011
PubMed
Summary
This summary is machine-generated.

Researchers investigated ubiquitin conjugation by studying E1-catalyzed E2 transthiolation. They found a conserved Helix 1 motif and a β-grasp domain that direct E2 binding and ensure specificity in ubiquitin signaling.

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Detection of Protein Ubiquitination
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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Signaling

Background:

  • Ubiquitin conjugation is a critical post-translational modification regulating numerous cellular processes.
  • The ubiquitin conjugation pathway involves a cascade of enzymes, including E1 (activating enzyme) and E2 (conjugating enzyme) enzymes.
  • Understanding the specificity and mechanism of E1-E2 interactions is crucial for deciphering cellular signaling.

Purpose of the Study:

  • To probe the mechanism of ubiquitin transfer between E1 and E2 enzymes.
  • To identify the molecular determinants governing E1-E2 binding specificity.
  • To elucidate the role of specific domains in E1-E2 interactions and ubiquitin conjugation fidelity.

Main Methods:

  • Utilized E1-catalyzed E2 transthiolation as a reporter function to measure ubiquitin transfer rates.
  • Performed functional surveys of human E2 paralogs with the human Uba1 E1 enzyme.
  • Employed sequence analysis and site-directed mutagenesis to identify key residues in E2 proteins.
  • Investigated the role of the E1 enzyme's β-grasp domain through truncation and domain exchange experiments.

Main Results:

  • Eleven human E2 paralogs exhibited conserved binding affinities (Km) and ubiquitin transfer rates (kcat) to the Uba1 ternary complex.
  • A conserved binding motif in Helix 1 of the E2 core domain was identified, responsible for general E2 binding.
  • Truncation of the Uba1 β-grasp domain significantly impaired cognate E2 binding and ubiquitin transfer.
  • The β-grasp domain acts as a specificity filter, preventing non-cognate E2 conjugation.

Conclusions:

  • A conserved Helix 1 motif and the E1 β-grasp domain are essential for general E2 binding and specificity in ubiquitin conjugation.
  • The β-grasp domain plays a critical role in ensuring the fidelity of ubiquitin signaling by excluding non-cognate E2 paralogs.
  • These findings provide insights into the molecular mechanisms underlying the specificity of the ubiquitin conjugation pathway.