Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins.

EMBO Reports
Marie E MorrowCynthia Wolberger

Abstract

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

References

May 1, 2004·The Journal of Biological Chemistry·Suzanne ElsasserDaniel Finley
Dec 2, 2004·Acta Crystallographica. Section D, Biological Crystallography·Paul Emsley, Kevin Cowtan
Dec 6, 2005·Cell·Sebastian M B NijmanRené Bernards
Apr 12, 2006·The Journal of Cell Biology·Nico P DantumaJacques Neefjes
May 20, 2008·Nature Cell Biology·Alwin KöhlerEd Hurt
Jun 6, 2009·Annual Review of Biochemistry·Francisca E Reyes-TurcuKeith D Wilkinson
Jul 25, 2009·Nature Reviews. Molecular Cell Biology·David KomanderSylvie Urbé
Feb 4, 2010·Acta Crystallographica. Section D, Biological Crystallography·Paul D AdamsPeter H Zwart
Aug 21, 2010·Nature·Shinichiro NakadaDaniel Durocher
Apr 5, 2011·Acta Crystallographica. Section D, Biological Crystallography·Martyn D WinnKeith S Wilson
Oct 15, 2011·Oncogene·J M FraileC López-Otín
Feb 14, 2012·Molecular Cell·Yu-Chi JuangDaniel Durocher
Feb 24, 2012·Journal of Cell Science·Michael J ClagueSylvie Urbé
Mar 1, 2012·Nature·Reuven WienerCynthia Wolberger
Aug 1, 2013·Physiological Reviews·Michael J ClagueSylvie Urbé
Nov 12, 2013·Cell Reports·Miklós BékésTony T Huang
Nov 19, 2014·Nature Communications·Marcello ClericiTitia K Sixma
Apr 25, 2015·Trends in Cell Biology·Michael J ClagueSylvie Urbé
May 7, 2015·The EMBO Journal·Yu ZhaoNima Mosammaparast
Feb 26, 2016·Science·Michael T MorganCynthia Wolberger
Oct 21, 2016·Nature·Tycho E T MevissenDavid Komander
Sep 19, 2017·Molecular Cell·Tiago BaptistaLászló Tora
Sep 21, 2017·Nature Reviews. Molecular Cell Biology·Michael Rape
Sep 30, 2017·Nature Reviews. Drug Discovery·Jeanine A HarriganStephen P Jackson
Dec 14, 2017·Nature Communications·Zhengkui ZhangLong Zhang
Mar 27, 2018·Molecular Cell·Peter HaahrNiels Mailand

❮ Previous
Next ❯

Citations

Apr 2, 2020·Molecular and Cellular Biology·Justin H LayerUtpal P Davé
Aug 28, 2020·Frontiers in Cell and Developmental Biology·Gabrielle J VallesNicholas W Ashton
Feb 9, 2019·Nature Reviews. Molecular Cell Biology·Michael J ClagueDavid Komander
Nov 26, 2020·Cell Chemical Biology·Gabriëlle B A van TilburgTitia K Sixma
Oct 27, 2021·The EMBO Journal·Zikou LiuJohn Silke

❮ Previous
Next ❯

Methods Mentioned

BETA
ubiquitination
cleavage
isothermal titration calorimetry
protein folding
gel filtration
gel
Fluorescence
transfection

Software Mentioned

Coot
Aimless
Microcal Origin
MEM
PyMOL
XDS
PHENIX

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.