Related Experiment Video

Updated: May 29, 2025

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

10.6K

Deubiquitinase processing of a non-natural linkage of ubiquitinated-PTEN

Reina Iwase1, Isabella Jaen Maisonet2, Kwangwoon Lee1

  • 1Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, United States; Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, United States.

Bioorganic Chemistry
|February 7, 2025
PubMed

Insights

Researchers explored how deubiquitinases (enzymes that remove ubiquitin) process monoubiquitinated PTEN using a novel aminoAla-Cys linkage. Several USP family deubiquitinases effectively processed this linkage, aiding deubiquitinase activity studies.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • PTEN, a crucial tumor suppressor, is regulated by ubiquitination.
  • Deubiquitinases (DUBs) remove ubiquitin, modulating protein function.
  • Studying DUB activity requires well-defined ubiquitinated substrates.

Purpose of the Study:

  • To evaluate the processing of monoubiquitinated PTEN by various deubiquitinases.
  • To assess the utility of a semisynthetic strategy employing an aminoAla-Cys linkage for PTEN ubiquitination.
  • To determine which deubiquitinases can hydrolyze the aminoAla-Cys linked ubiquitin on PTEN.

Main Methods:

  • Developed a semisynthetic strategy to site-specifically attach monoubiquitin to PTEN via an aminoAla-Cys linkage.
  • Tested the ability of several known deubiquitinases (USP10, USP11, USP15, BAP1, OTUD3) to process the modified PTEN.
  • Utilized biochemical assays to monitor the deubiquitination of PTEN.

Main Results:

  • The aminoAla-Cys linked monoubiquitination of PTEN was successfully achieved.
  • Deubiquitinases USP10, USP11, and USP15 effectively processed the aminoAla-Cys linked monoubiquitinated PTEN.
  • Deubiquitinases BAP1 and OTUD3 did not process the substrate under the tested conditions.

Conclusions:

  • Ubiquitin linked via the aminoAla-Cys functionality is recognized and hydrolyzed by specific USP family deubiquitinases.
  • This semisynthetic approach provides a versatile tool for studying deubiquitinase activity and substrate specificity.
  • Enables systematic evaluation of deubiquitinase engagement with monoubiquitinated protein substrates.
Keywords:
DeubiquitinasePTENProtein SemisynthesisUbiquitin

More Related Videos

Purification of Ubiquitinated p53 Proteins from Mammalian Cells10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

2.0K
Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay07:05

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Published on: September 27, 2024

393

Related Concept Videos

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...
803
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...
7.1K
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....
6.8K
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...
680
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...
3.5K
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...
4.4K