Related Experiment Video

Updated: May 8, 2026

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

Caspase-dependent regulation of the ubiquitin-proteasome system through direct substrate targeting

Ting-Chun Yeh1, Shawn B Bratton

  • 1Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.

Proceedings of the National Academy of Sciences of the United States of America
|August 14, 2013
PubMed

Insights

Grim self-associates and is ubiquitinated by DIAP1, leading to its degradation. Caspase cleavage stabilizes Grim, enhancing apoptosis through a feed-forward loop.

Area of Science:

  • Cellular biology
  • Molecular mechanisms of apoptosis
  • Ubiquitin-proteasome system

Background:

  • Drosophila inhibitor of apoptosis 1 (DIAP1) is an E3 ubiquitin ligase regulating apoptosis.
  • IAP antagonists like Grim are thought to induce cell death but can be degraded by DIAP1.

Purpose of the Study:

  • To investigate the regulation of Grim stability and its role in apoptosis.
  • To elucidate the interplay between Grim ubiquitination, caspase activity, and proteasomal turnover.

Main Methods:

  • Cellular assays to study Grim self-association and ubiquitination.
  • Analysis of ubiquitination sites (Lys136) and caspase cleavage sites (Asp132).
  • Investigation of K48-linked ubiquitin chain formation and its dependence on DIAP1's RING domain.

Main Results:

  • Grim self-associates and undergoes DIAP1-mediated ubiquitination at Lys136, leading to rapid turnover.
  • Ubiquitination occurs via K48-linked chains, particularly on BIR2-bound Grim near DIAP1's RING domain.
  • Active caspases cleave Grim at Asp132, removing the ubiquitination site and enhancing Grim stability.

Conclusions:

  • Grim is a caspase substrate whose cleavage limits its ubiquitination and proteasomal degradation.
  • Caspase-mediated cleavage of Grim enhances its stability, promoting a feed-forward loop for greater cell death.
  • This mechanism provides target-specific regulation of apoptosis by limiting Grim turnover.

More Related Videos

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model09:00

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

Published on: April 17, 2026

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Related Concept Videos

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 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 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 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...
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.