Related Experiment Video

Updated: Jun 13, 2026

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
07:57

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Published on: January 20, 2023

RET is a heat shock protein 90 (HSP90) client protein and is knocked down upon HSP90 pharmacological block

Luigi Alfano1, Teresa Guida, Livia Provitera

  • 1Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universitá Federico II, 80131 Naples, Italy.

The Journal of Clinical Endocrinology and Metabolism
|May 7, 2010
PubMed
Abstract

Insights

Heat shock protein 90 (HSP90) inhibition by 17-AAG causes RET degradation via the proteasome. This finding is crucial for understanding RET-dependent cancers like medullary thyroid carcinoma (MTC).

Area of Science:

  • Oncology
  • Molecular Biology
  • Biochemistry

Background:

  • Mutations in the RET receptor tyrosine kinase are linked to multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinoma (MTC).
  • Heat shock protein (HSP) 90 is essential for the stability and proper folding of various kinases, including RET.
  • 17-allyl-amino-17-demethoxygeldanamycin (17-AAG) is a specific inhibitor of HSP90.

Purpose of the Study:

  • To determine if RET protein stability is dependent on HSP90.
  • To elucidate the molecular mechanisms by which HSP90 inhibition leads to RET degradation.

Main Methods:

  • Investigated the effects of 17-AAG in RAT1 fibroblasts expressing MEN2-associated RET mutants.
  • Utilized human MTC-derived cell lines for experimental analysis.
  • Assessed protein degradation pathways, including the 26S proteasome and E3 ligase interactions.

Main Results:

  • 17-AAG induced 26S proteasome-dependent degradation of both wild-type RET and MEN2 RET mutants.
  • HSP90 inhibition disrupted the HSP90/RET interaction, stabilized RET binding to HSP70, and promoted RET ubiquitination and proteasomal degradation.
  • 17-AAG effectively blocked RET downstream signaling and RET-dependent transcriptional activation, leading to cell cycle arrest in MTC cells.

Conclusions:

  • RET and its MEN2-associated mutants require HSP90 for their stability.
  • HSP90 blockade by 17-AAG triggers the degradation of RET, offering a potential therapeutic strategy.

More Related Videos

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay06:51

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Published on: July 21, 2021

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding11:07

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

Published on: September 21, 2011

Related Concept Videos

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...
Molecular Chaperones and Protein Folding03:00

Molecular Chaperones and Protein Folding

The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
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...
The Unfolded Protein Response01:37

The Unfolded Protein Response

The ER is the hub of protein synthesis in a cell. It has robust systems to quality control protein folding and also for degradation of terminally misfolded proteins. Under normal conditions, a small proportion of misfolded proteins that cannot be salvaged need to be transported to the cytoplasm by the ER-associated degradation or ERAD pathways. However, if the ERAD cannot handle the misfolded proteins, the cell activates the unfolded protein response or UPR to adjust the protein folding...
Bacterial Protein Maturation01:26

Bacterial Protein Maturation

Bacterial protein maturation is a tightly regulated process that ensures newly synthesized polypeptides achieve correct functional conformations. This maturation involves a series of modifications, folding events, and quality control steps, often assisted by specialized chaperone proteins.N-Terminal ModificationsThe maturation of bacterial polypeptides begins cotranslationally as the polypeptide exits the ribosome. The first amino acid, N-formylmethionine (fMet), is typically modified at the...
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...