Related Experiment Video

Updated: Mar 26, 2026

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells
09:15

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

Published on: October 20, 2022

2.9K

Regulation of RIPK3- and RHIM-dependent Necroptosis by the Proteasome

Kenta Moriwaki1, Francis Ka-Ming Chan1

  • 1From the Department of Pathology, Immunology and Microbiology Program, University of Massachusetts Medical School, Worcester, Massachusetts 01605.

The Journal of Biological Chemistry
|January 21, 2016
PubMed

Insights

Proteasome inhibitors like MG132 activate the RIPK3-MLKL necroptosis pathway, independent of caspase inhibition but requiring RIP homotypic interaction motif (RHIM). This reveals the ubiquitin-proteasome system as a novel regulator of necroptosis.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Receptor-interacting protein kinase 3 (RIPK3) is crucial for necroptosis, a programmed cell death pathway.
  • RIPK3 activity is regulated by phosphorylation and activates mixed lineage kinase domain-like (MLKL) to induce necroptosis.
  • The ubiquitin-proteasome system degrades cellular proteins and is implicated in cell death pathways; proteasome inhibitors are explored as anti-cancer agents.

Purpose of the Study:

  • To investigate the role of the ubiquitin-proteasome system in regulating RIPK3-dependent necroptosis.
  • To determine if proteasome inhibitors activate necroptosis and elucidate the underlying molecular mechanisms.
  • To explore RIPK3's function beyond necroptosis, including apoptosis and cytokine signaling.

Main Methods:

  • Treatment of mouse fibroblasts and human leukemia cells with proteasome inhibitors (MG132, bortezomib).
  • Analysis of RIPK3 and MLKL activation, necroptosis induction, and apoptosis.
  • Investigation of the requirement for caspase inhibition and the RIP homotypic interaction motif (RHIM).
  • Mutation of Lys-264 in RIPK3 to assess ubiquitination and its effect on RIPK3 function.

Main Results:

  • Proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in various cell types.
  • Necroptosis induced by proteasome inhibitors does not require caspase inhibition but depends on an intact RHIM.
  • Restricting MLKL recruitment to RIPK3 leads to RIPK3-dependent apoptosis upon proteasome inhibition.
  • Proteasome inhibition causes accumulation of K48-linked ubiquitinated RIPK3, with Lys-264 playing a partial role.

Conclusions:

  • The ubiquitin-proteasome system is a novel regulator of RIPK3-dependent necroptosis.
  • Proteasome inhibitors can trigger necroptosis through RIPK3 activation, offering potential therapeutic strategies.
  • RIPK3's function is context-dependent, mediating necroptosis or apoptosis based on MLKL recruitment and ubiquitination status.
Keywords:
anticancer drugapoptosiscell deathnecrosis (necrotic death)proteasomeubiquitylation (ubiquitination)

More Related Videos

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis08:55

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Published on: August 7, 2018

11.4K
Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System08:15

Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System

Published on: April 11, 2025

1.1K

Related Concept Videos

Necrosis01:16

Necrosis

Necrosis is considered as an “accidental” or unexpected form of cell death that ends in cell lysis. The first noticeable mention of “necrosis” was in 1859 when Rudolf Virchow used this term to describe advanced tissue breakdown in his compilation titled “Cell Pathology”.
Morphological Manifestations of Necrosis
Necrotic cells show different types of morphological appearance depending on the type of tissue and infection. In coagulative necrosis, cells become...
7.2K
Regulation of the Unfolded Protein Response01:31

Regulation of the Unfolded Protein Response

Inositol-requiring kinase one or IRE1 is the most conserved eukaryotic unfolded protein response (UPR) receptor. It is a type I transmembrane protein kinase receptor with a distinctive site-specific RNase activity. As the binding mechanics of the misfolded proteins with the N-terminal domain of IRE-1 are unclear, three binding models — direct, indirect, and allosteric -- are proposed for receptor activation. Nevertheless, it is known that once a misfolded protein associates with IRE1, it...
3.2K
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...
9.2K
Regulated Protein Degradation02:58

Regulated Protein Degradation

3.3K
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...
5.5K
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a...
6.3K