Method Article

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

DOI:

10.3791/67367

⸱

September 27th, 2024

 ,  , 
1Pediatrics and Rare Diseases Group, Sanford Research, 2Department of Biochemistry and Molecular Biology, Mayo Clinic, 3Department of Pediatrics, Sanford School of Medicine, University of South Dakota

In This Article

Summary

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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

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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

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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 NDDs2,7,8,9,10,11. 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 conjugates14,15,16. 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, respectively17,18,19,20. 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 analyses22,23. 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 detection12,24. Molecular aspects of DUB activity, such as minimum chain length and linkage specificity25,26,27,28, 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

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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 immunoblot7,32 for ubiquitin and USP27X to analyze the change in mono-ubiquitin presence across selected time points.

Genetic engineering process: transformation, protein expression, cell lysis, affinity purification diagram.
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

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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 incubated with K63-linked di-ubiquitin chains for 1 h. These samples were separated via SDS-PAGE32, and ubiquitin and USP27X proteins were analyzed via immunoblotting. Wild-type USP27X induces di-ubiquitin cleavage, generating mono-ubiquitin (Figure 2B and Supplementary Figure 1). XLID105-associated F313V, Y381H, and S404N USP27X variant proteins did not cleave these chains. Because F313V, Y381H, and S404N variants disrupt USP27X catalytic activity, USP27X functional disruption appears to be the major mechanism underlying XLID1057. Additional quantification and complementary experiments were previously reported7.

Ubiquitin protease domain diagram and gel electrophoresis for USP27X mutations.
Figure 2: Impact of XLID105 variants on USP27X deubiquitylating activity. (A) Diagram of the human USP27X protein structure, showing residue numbers and the location of the XLID105 variants evaluated in the USP domain (purple). (B) GST-tagged wild-type USP27X and XLID105 variants (F313V, Y381H, and S404N) were incubated with K63 di-ubiquitin chains for the indicated times. Immunoblot analysis was performed using anti-GST (USP27X) and ubiquitin antibodies. Please click here to view a larger version of this figure.

Supplementary Figure 1: Uncropped blots of Figure 2B. Please click here to download this File.

Discussion

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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 DUBs that are associated with genetic diseases with poorly understood molecular mechanisms. The optimization of the expression and purification protocol for a specific DUB of interest is crucial. If no specific protocols have been described for a given DUB of interest, the method described here can act as a starting point. When optimizing a protein expression and purification protocol, critical steps include selecting the appropriate parameters for (1) the expression system (e.g., bacteria strains, insect cells, or mammalian cells), (2) the expression vectors, (3) the tag and affinity resin to be used, (4) the growing conditions, (5) the amounts of culture, and (6) the need for further purification (e.g., via size exclusion or ion exchange chromatography). Specific recommendations for these and other aspects of the method have been discussed33.

The in vitro ubiquitin chain cleavage assay is a simple way to measure the deubiquitylating activity of wild-type or mutant DUBs. A time-dependent analysis of ubiquitin cleavage can provide insights into the effects of genetic variation on DUB enzymatic kinetics by allowing the determination of parameters such as the Km and Vmax. This would enable one to distinguish between defects in the DUB active site that impair catalysis (reduced Vmax) and those that impair substrate recognition (increased Km). The versatility of this assay stems from the variety of ubiquitin chains that can be used to provide distinct mechanistic information on a DUB of interest. Ubiquitin chains of different lengths and linkage topologies are commercially available, rendering this assay accessible to non-experts. Specific DUBs have unique properties for enzymatic kinetics, linkage recognition, and cleavage. Critical steps for this assay include (1) defining a working concentration of the DUB, (2) determining the optimal reaction time, (3) choosing the ubiquitin chain linkage type and length, and (4) selecting a detection method.

Ubiquitin chain cleavage assays measure the impact of NDD variants on DUB activity7,11, helping to identify variants that disrupt DUB activity and drive atypical development. This assay can be used in a range of situations where genetic variation drives pathology, including cancer and neurodegeneration. As a substrate-independent assay, prior identification of a specific ubiquitylated protein substrate is not required. However, the assay is limited to measuring changes in ubiquitin chain cleavage activity and cannot assess variants' effects on DUB functions such as protein-protein interactions, subcellular localization, and posttranslational modifications. Identification of the broad signaling pathways where a DUB of interest operates is required to develop specific assays to address how these variants drive discrete NDDs.

Disclosures

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The authors declare no conflicts of interest.

Acknowledgements

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This work was funded by Sanford Research startup funds to FB and the NIH grant R01CA233700 to MJS. The artwork was done by Felipe G. Serrano (www.illustrative-science.com). We thank Dr. Greg Findlay (University of Dundee) for the GST-USP27X plasmid.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Amersham Protran 0.45 NC 200 mm × 200 mm 25/PKCytiva10600041
Ammonium sulfateFisher ScientificAC205872500
AmpicillinFisher ScientificBP1760 25
Anti- Ubiquitin (Mouse monoclonal)BiolegendCat# 646302, RRID:AB_1659269(WB: 1:1000)
Anti-GST (Sheep polyclonal)MRC-PPU Reagents and ServicesCat# S902A Third bleed(WB: 1:1000) https://mrcppureagents.dundee.ac.uk/
Baffled Culture Flasks 2 LFisher Scientific10-042-5N
Bradford ReagentMillipore SigmaB6916-500ML
ChloramphenicolGold BiotechnologyC-105-25
Complete, Protease Inhibitor tabletsMillipore Sigma5056489001
Econo-Column 1.5 × 5 cmBio-Rad7371507
Eppendorf ThermoMixer F1.5 Eppendorf5384000020
ExcelMicrosofthttps://www.microsoft.com/en-us/microsoft-365/excel
GlycerolGenesee Scientific18-205
IllustratorAdobehttps://www.adobe.com/products/illustrator.html
Image StudioLI-COR Bioscienceshttps://www.licor.com/bio/image-studio/
InkscapeInkscapehttps://inkscape.org/
Invitrogen 4-12% NuPAGE 1mm 12 well gelThermo Fisher ScientificNP0322BOX
IPTG (Isopropyl-b-D-Thiogalactopyranoside)Genesee Scientific20-109
IRDye 800CW Donkey anti-Goat IgG Secondary AntibodyLI-COR BiosciencesCat# 926-32214(WB: 1:10000)
IRDye 800CW Donkey anti-Mouse IgG Secondary AntibodyLI-COR BiosciencesCat# 926-32212(WB: 1:10000)
Isotemp Digital Dry BathFisher Scientific88860022
K63 Di-UbiquitinSouth Bay Bio LLCSBB-UP0072
LB AgarGenesee Scientific11-119
LB BrothGenesee Scientific11-118
LysozymeGold BiotechnologyL-040-100
MaxQ 4000 Benchtop Orbital ShakerThermo Fisher ScientificSHKE4000-7
MES-SDS Running BufferBoston Bioproducts IncBP-177
Mini Tube RotatorFisher Scientific88-861-051
NuPage LDS Sample buffer 4xThermo Fisher ScientificNP0007
Odyssey Fc ImagerLI-COR Biosciences43214
PageRuler Plus LadderThermo Fisher Scientific26620
pGEX6P1 human USP27XMRC-PPU Reagents and ServicesDU21193https://mrcppureagents.dundee.ac.uk/
pGEX6P1 human USP27X F313VAddgene225715Koch et at 2024 (PMID: 38182161)
pGEX6P1 human USP27X S404NAddgene225717Koch et at 2024 (PMID: 38182161)
pGEX6P1 human USP27X Y381HAddgene225716Koch et at 2024 (PMID: 38182161)
Pierce Glutathione AgaroseThermo Fisher Scientific16100
PMSF (Phenylmethylsulfonyl fluoride)Gold BiotechnologyP-470-10
Polysorbate 20 (Tween 20)Fisher ScientificAC233360010
Rosetta 2 Competent CellsMillipore Sigma71402-M
SimplyBlue SafeStainThermo Fisher ScientificLC6060
SmartSpec 3000Bio-Rad170-2501
SOC mediumThermo Fisher Scientific15544034
Sodium chlorideGenesee Scientific18-216
Sonifier 250Branson100-132-135
Sorvall RC 6 Plus CentrifugeThermo Fisher Scientific46910
TCEP (Tris-(carboxyethyl) phosphine hydrochloride)Gold BiotechnologyTCEP10
Terrific Broth PowderGenesee Scientific18-225
Tris BaseGenesee Scientific18-146
XCell SureLock Mini-Cell and XCell II Blot Module Thermo Fisher ScientificEI0002

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Neurodevelopmental DisordersEnzymatic Activity AssayUSP27X MutationIn Vitro AssayProtein PurificationSDS PAGEK63 Linked UbiquitinGene Variant Analysis

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