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 conjugating, and E3 ligase enzymes⁴ and is reversible by the activity of deubiquitylating enzymes (DUBs) that catalyze the cleavage and removal of ubiquitin from protein substrates⁵. Ubiquitin can be ligated to the substrate as a monomer (monoubiquitylation) or polymeric chains (polyubiquitylation) that are formed on any of the seven lysine residues (K6, K11, K27, K29, K33, K48, K63) or the M1 residue of ubiquitin. These different ubiquitin chain topologies and their combinations create a cellular code that is key for signal transduction⁶.

DUBs such as USP27X, USP7, USP9X, USP48, STAMBP, OTUD4, OTUD6B, and OTUD5 have been associated with NDDs^{2,7,8,9,10,11}. For most NDDs, the molecular mechanisms that drive pathogenesis remain experimentally undefined. Some of the DUBs driving recently described disorders are poorly understood and lack known cellular readouts that can be used to assess the impact of genetic variation on protein function. In vitro, ubiquitin chain cleavage assays overcome this limitation as substrate-independent DUB activity readouts that can measure the impact of gene variants on enzymatic activity¹².

In vitro ubiquitin cleavage assays have been used since the 1980s. These assays using radiolabeled substrates allowed for the discovery of the first DUBs, including isopeptidase, identified for its capacity to deubiquitylate Histone H2A¹³, and ubiquitin carboxyl-terminal hydrolase (UCH), identified by its ability to hydrolyze ubiquitin from a variety of chemical conjugates^14,15,16. Further, radiolabeled polyubiquitylated full-length proteins or peptides were used to identify isopeptidase T and several UCHs and ubiquitin-specific proteases (UBPs) from erythrocytes and skeletal muscle, respectively^17,18,19,20. Ubiquitin chains of a defined length and linkage type (K48-linked tetra-ubiquitin) were first used to measure the DUB activity of Isopeptidase T²¹. Since then, this assay has become the gold standard to measure DUB activity in mutational analyses^22,23. The refinement of this assay currently allows visualization of ubiquitin cleavage via electrophoresis and conventional gel stains such as Coomassie blue, SYPRO orange, ruby, and silver stain or fluorescent or immunoblotting-based detection^12,24. Molecular aspects of DUB activity, such as minimum chain length and linkage specificity^25,26,27,28, can be clarified by using ubiquitin chains of different lengths (e.g., di-, tri-, tetra-ubiquitin) and linkages (K6, K11, K27, K29, K33, K48, K63, linear) in functional assays. NDD-associated variants can drive DUB activity defects that are ubiquitin chain linkage type specific¹¹.

A di-ubiquitin cleavage assay using purified recombinant DUB proteins can directly measure the impact of NDD variants on DUB activity. USP27X, which is mutated in the NDD X-linked intellectual disability disorder 105 (XLID105)^7,28 models the process presented here. This approach allows for the determination of how DUB activity is disrupted by gene variants in existing and unknown DUB-associated NDDs.

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 are listed in the Table of Materials.

1. Transformation of competent Rosetta 2 E. coli cells with the recombinant GST-USP27X expression plasmid

NOTE: To maintain the sterility of the bacterial culture, perform steps where media containers are open under a Bunsen burner flame. To allow optimal oxygen transfer, perform bacterial culture shaking in a benchtop temperature-controlled shaker with an orbit of 19 mm to 50 mm and a speed of 200 rpm²⁹.

1. Thaw 20 µL of chemically competent Rosetta 2 E. coli cells on ice immediately before use. Add 1-10 ng of the pGEX6P1-USP27X expression plasmid (encoding for N-terminal Glutathione-S-transferase (GST)³⁰ -tagged USP27X and containing Ampicillin resistance) and incubate 5 min on ice.
2. Heat shock for 30 s at 42 °C in a dry bath. Incubate on ice for 2 min.
3. Add 80 µL of room temperature (RT) SOC medium. Incubate for 60 min at 37 °C with 200 rpm rotation in a 19 mm orbit benchtop temperature-controlled shaker.
4. Plate 50 µL of culture on an LB agar plate supplemented with 25 µg/L Chloramphenicol and 50 µg/L Ampicillin. Incubate for 20 h at 37 °C lid-side down in a temperature-controlled incubator.

2. Overnight bacterial expression of recombinant protein from expression plasmid

NOTES: To maintain the sterility of the bacterial culture, perform steps where media containers are open under a Bunsen burner flame. To allow optimal oxygen transfer, perform bacterial culture shaking in a benchtop temperature-controlled shaker with an orbit of 19 mm to 50 mm and a speed of 200 rpm²⁹. Measure culture OD₆₀₀ using a spectrophotometer.

1. Prepare 1 L of Terrific Broth (TB) medium by adding 47.6 g of TB Powder to 1 L of ultrapure water with 0.4% glycerol in a 2 L baffled culture flask. Autoclave medium for 30 min and cool to RT.
2. Pick a single colony of transformed bacteria and add to 10 mL of sterile LB medium supplemented with 25 µg/L Chloramphenicol and 50 µg/L Ampicillin. Incubate for 20 h at 37 °C with 200 rpm rotation in a 19 mm orbit benchtop temperature-controlled shaker.
3. Add 10 mL overnight culture to 1 L of TB medium supplemented with 25 µg/L Chloramphenicol and 50 µg/L Ampicillin (1:100 ratio of starting culture to expression culture). Incubate at 37 °C with 200 rpm rotation in a 19 mm orbit benchtop temperature-controlled shaker until the culture OD₆₀₀ is between 0.5-0.6.
4. Add 50 µM of IPTG to the culture to induce expression, cool to 16 °C, and incubate for 20 h at 16 °C with 200 rpm rotation.
5. Pellet cells from expression culture by centrifugation for 20 min at ≥3,000 x g and 4 °C. Store the pellet at -80 °C for at least 1 h.
   NOTE: At this point, the experiment can be paused and restarted later (preferably the same week). The pellet can be stored for the long term at -80 °C.

3. Protein purification by gravity-flow affinity column

NOTE: The resin, binding, wash, elution, and storage buffers appropriate for each purification will depend on the recombinant protein being purified. Collect samples from the cell pellet, supernatant, flow-through, wash fractions, and elution fractions in SDS-PAGE buffer. Perform SDS-PAGE and Coomassie staining for the samples to evaluate the success of the purification. Perform purification at 4 °C and handle fractions on ice. Cleavage of protein tags can be performed on or off the column using the appropriate protease to target the relevant protease-specific cleavage site.

1. Secure the empty gravity flow column in the retort stand and fill it with glutathione agarose resin. Use 2-3 mL of resin to purify a cell pellet collected from 1 L of expression culture.
2. Wash the column with one resin-bed volume of 20% ethanol. Wash column 3 times with one resin-bed volume of MS500 buffer (20 mM of Tris pH 7.5, 500 mM of NaCl, 0.5 mM of TCEP). Stop the column so the resin remains covered with buffer to prevent drying while preparing the cell pellet.
3. Thaw the cell pellet at 4 °C. Add 30 mL of MS500 buffer (20 mM of Tris pH 7.5, 500 mM of NaCl, 0.5 mM of TCEP, 60 mg of lysozyme, and one protease inhibitor tablet) to the thawed pellet. Lyse for 30 min at 4 °C with gentle end-over-end rotation.
4. Sonicate lysed cells in either a 50 mL centrifuge tube or a metal beaker on ice until the lysate flows freely when dispensed from a pipette tip. Centrifuge for 30 min at 4 °C and ≥20,000 x g to clear the supernatant.
   NOTE: Determine sonicator settings empirically. Set the sonicator such that 120 s total time is enough to reduce the viscous lysate to a free-flowing and transparent liquid.
5. Decant the supernatant into a beaker and load cleared lysate onto the column. Run lysate through the column by gravity flow while collecting the flow through. Load the column with the collected flow through and run it through the column.
6. Wash the column with at least two resin-bed volumes of MS500 wash buffer (20 mM of Tris pH 7.5, 500 mM of NaCl, 0.5 mM of TCEP) 5 times. Collect the wash flow through in 5 mL fractions. Add 1 µL of each fraction to 100 µL of Bradford reagent to visually check for protein presence. Wash until unbound protein is no longer present in the last wash, adding additional wash steps if necessary.
7. Recover protein by running MS500 elution buffer (MS500 buffer supplemented with 10 mM of glutathione and 10 mM of NaOH) through the column, collecting 5 mL elution fractions. Check for protein presence by adding 1 µL of eluate to 100 µL of Bradford reagent. Stop collecting fractions when the Bradford reagent no longer indicates proteins are present.
8. Perform buffer exchange by precipitation and centrifugation. Precipitate protein by adding 2 volumes of 4 M of ammonium sulfate to the eluate, gently inverting until cloudy, and then centrifuging for 30 min at 4 °C and ≥20,000 x g and again for another 5 min. Remove the supernatant after each centrifugation without disturbing the protein pellet.
9. Re-dissolve and store protein in MS500 supplemented with 25% glycerol. Store protein pellets or protein in storage buffer at -80 °C.

4. In vitro ubiquitin chain cleavage assay

NOTE: Select ubiquitin chain length and linkage types based on DUB specificity described in previous reports³¹ or determined empirically. If necessary, this protocol could be used to test the activity of the wild-type DUB of interest on a panel of commercially available ubiquitin chains of defined length and linkage type. A di-ubiquitin chain amount of 375-750 ng and a DUB concentration of 1-2 µM can be used as starting points for the assay²⁷.

1. Prepare 10x DUB activation buffer (500 mM of Tris-HCl pH 7.5, 500 mM of NaCl, and 100 mM of TCEP).
2. For each time point for each DUB, prepare 10 µL total of 2 µM of purified GST-USP27X in 1x DUB activation buffer (DUB mix). Prepare master mixes and split them into time points.
3. Incubate the DUB mix for 10 min at RT.
4. Add 7 µL of SDS-PAGE loading buffer to time 0 before adding ubiquitin chains to prevent the deubiquitylation reaction from starting.
5. To each time point for each DUB, add 375 ng of K-63 di-ubiquitin chains diluted in 10 µL 1x DUB activation buffer. The total volume is 20 µL per reaction.
6. Incubate the tubes at 30 °C, stopping each time point with 7 µL of SDS-PAGE loading buffer.
7. Perform SDS-PAGE with a 4%-12% gradient gel and immunoblot^7,32 for ubiquitin and USP27X to analyze the change in mono-ubiquitin presence across selected time points.

Figure 1: Schematic of the study design. (A) Transformation of competent E. coli cells with recombinant protein expression plasmid. (B) Overnight bacterial expression of recombinant deubiquitylase protein. (C) Protein purification of recombinant deubiquitylase using a gravity-flow affinity column. (D) In vitro ubiquitin chain cleavage assay to evaluate deubiquitylating activity. Please click here to view a larger version of this figure.

Results

To determine the impact of XLID105-associated variants on USP27X catalytic activity, GST-tagged wild-type USP27X and the XLID105-associated variant F313V, Y381H, and S404N USP27X proteins were purified from bacteria. These variants are located within the USP catalytic domain of USP27X (Figure 2A). Because USP27X was previously reported to cleave K63 ubiquitin chains³¹, wild-type USP27X and the XLID105-associated variant F313V, Y381H, and S404N USP27X proteins were inc...

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