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

Summary

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Abstract

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

Introduction

Neurodevelopmental disorders (NDDs) arise from diverse etiologies with environmental or genetic determinants that drive atypical nervous system development¹. Next-generation sequencing genetic testing has linked an increasing number of variants in ubiquitin system-related genes with genetic NDDs². The ubiquitin system catalyzes the ligation of the small protein modifier ubiquitin to primarily lysine residues in protein substrates to drive changes in cellular behavior, including localization, stability, protein-protein interactions, or activity³. Ubiquitylation is mediated by E1 activating, E2 conj....

Protocol

The following protocol can be adapted for recombinant proteins using various affinity tags expressed in different strains of competent cells. Depending on the protein being expressed, the culturing and overnight expression conditions may require optimization of the OD₆₀₀ at expression induction, expression time, expression temperature, and IPTG concentration. An overview of the protocol is illustrated in Figure 1. The details of the reagents and the equipment used in this study ar.......

Discussion

This article presents a protocol for the expression and purification of recombinant USP27X DUBs and an in vitro ubiquitin chain cleavage assay to compare the deubiquitylating activity of wild-type USP27X and NDD-associated variant proteins. This assay determined that XLID105-associated variants disrupt USP27X catalytic activity⁷. This mechanistic insight helped us define XLID105 as a USP27X functional disruption disorder.

This protocol can be adapted to other D.......

Materials

Name	Company	Catalog Number	Comments
Amersham Protran 0.45 NC 200 mm × 200 mm 25/PK	Cytiva	10600041
Ammonium sulfate	Fisher Scientific	AC205872500
Ampicillin	Fisher Scientific	BP1760 25
Anti- Ubiquitin (Mouse monoclonal)	Biolegend	Cat# 646302, RRID:AB_1659269	(WB: 1:1000)
Anti-GST (Sheep polyclonal)	MRC-PPU Reagents and Services	Cat# S902A Third bleed	(WB: 1:1000) https://mrcppureagents.dundee.ac.uk/
Baffled Culture Flasks 2 L	Fisher Scientific	10-042-5N
Bradford Reagent	Millipore Sigma	B6916-500ML
Chloramphenicol	Gold Biotechnology	C-105-25
Complete, Protease Inhibitor tablets	Millipore Sigma	5056489001
Econo-Column 1.5 × 5 cm	Bio-Rad	7371507
Eppendorf ThermoMixer F1.5	Eppendorf	5384000020
Excel	Microsoft		https://www.microsoft.com/en-us/microsoft-365/excel
Glycerol	Genesee Scientific	18-205
Illustrator	Adobe		https://www.adobe.com/products/illustrator.html
Image Studio	LI-COR Biosciences		https://www.licor.com/bio/image-studio/
Inkscape	Inkscape		https://inkscape.org/
Invitrogen 4-12% NuPAGE 1mm 12 well gel	Thermo Fisher Scientific	NP0322BOX
IPTG (Isopropyl-b-D-Thiogalactopyranoside)	Genesee Scientific	20-109
IRDye 800CW Donkey anti-Goat IgG Secondary Antibody	LI-COR Biosciences	Cat# 926-32214	(WB: 1:10000)
IRDye 800CW Donkey anti-Mouse IgG Secondary Antibody	LI-COR Biosciences	Cat# 926-32212	(WB: 1:10000)
Isotemp Digital Dry Bath	Fisher Scientific	88860022
K63 Di-Ubiquitin	South Bay Bio LLC	SBB-UP0072
LB Agar	Genesee Scientific	11-119
LB Broth	Genesee Scientific	11-118
Lysozyme	Gold Biotechnology	L-040-100
MaxQ 4000 Benchtop Orbital Shaker	Thermo Fisher Scientific	SHKE4000-7
MES-SDS Running Buffer	Boston Bioproducts Inc	BP-177
Mini Tube Rotator	Fisher Scientific	88-861-051
NuPage LDS Sample buffer 4x	Thermo Fisher Scientific	NP0007
Odyssey Fc Imager	LI-COR Biosciences	43214
PageRuler Plus Ladder	Thermo Fisher Scientific	26620
pGEX6P1 human USP27X	MRC-PPU Reagents and Services	DU21193	https://mrcppureagents.dundee.ac.uk/
pGEX6P1 human USP27X F313V	Addgene	225715	Koch et at 2024 (PMID: 38182161)
pGEX6P1 human USP27X S404N	Addgene	225717	Koch et at 2024 (PMID: 38182161)
pGEX6P1 human USP27X Y381H	Addgene	225716	Koch et at 2024 (PMID: 38182161)
Pierce Glutathione Agarose	Thermo Fisher Scientific	16100
PMSF (Phenylmethylsulfonyl fluoride)	Gold Biotechnology	P-470-10
Polysorbate 20 (Tween 20)	Fisher Scientific	AC233360010
Rosetta 2 Competent Cells	Millipore Sigma	71402-M
SimplyBlue SafeStain	Thermo Fisher Scientific	LC6060
SmartSpec 3000	Bio-Rad	170-2501
SOC medium	Thermo Fisher Scientific	15544034
Sodium chloride	Genesee Scientific	18-216
Sonifier 250	Branson	100-132-135
Sorvall RC 6 Plus Centrifuge	Thermo Fisher Scientific	46910
TCEP (Tris-(carboxyethyl) phosphine hydrochloride)	Gold Biotechnology	TCEP10
Terrific Broth Powder	Genesee Scientific	18-225
Tris Base	Genesee Scientific	18-146
XCell SureLock Mini-Cell and XCell II Blot Module	Thermo Fisher Scientific	EI0002