OTUD5 Antibody

Frequently Asked Questions: OTUD5 Antibody Research Applications

Advanced Research Questions

How can contradictory findings about OTUD5’s tumor-suppressive vs. oncogenic roles be resolved?

OTUD5 exhibits context-dependent functions:

• Pro-Tumor Role: In hepatocellular carcinoma, OTUD5 stabilizes TRIM25, suppressing PML expression and accelerating proliferation (Fig. 1E-H, ).

• Anti-Tumor Role: In breast cancer, OTUD5 deubiquitinates ARID1A, enhancing chromatin accessibility at tumor suppressor loci .

Methodological Recommendations:

• Subtype-Specific Models: Use CRISPR-Cas9 knock-in mutations (e.g., OTUD5/C224S catalytic mutant) in isogenic cell lines from different tissues.

• Ubiquitome Profiling: Perform mass spectrometry to identify OTUD5 substrates in a cancer-specific manner (MUDPIT-MS, ).

• Single-Cell RNA-seq: Resolve OTUD5’s differential expression across tumor microenvironments .

Table 2: OTUD5 Functional Dichotomy in Cancer

What advanced techniques are required to study OTUD5’s linkage-specific deubiquitination in chromatin regulation?

OTUD5 preferentially cleaves K48/K63 ubiquitin chains on chromatin remodelers like ARID1A and HCF1 . Critical methods:

• Linkage-Specific Ubiquitin Probes: Use K48- or K63-diUb fluorescein conjugates in in vitro deubiquitination assays with recombinant OTUD5 .

• Chromatin Fractionation: Isolate chromatin-bound OTUD5 via subcellular fractionation and detect co-localization with ARID1A by proximity ligation assay .

• CUT&Tag Sequencing: Map OTUD5-bound genomic regions and correlate with H3K27ac/H3K4me1 marks in neuroectodermal cells .

How do disease-associated OTUD5 mutations (e.g., Gly494Ser) impact experimental approaches for studying LINKED syndrome?

The LINKED syndrome mutation p.Gly494Ser disrupts OTUD5’s ability to cleave K48 ubiquitin chains from chromatin regulators . Key strategies:

• Patient-Derived iPSCs: Differentiate induced pluripotent stem cells into neuroectoderm to assess enhancer accessibility (ATAC-seq) and HDAC2 stability .

• X-Inactivation Skewing: Analyze female carriers via RNA-seq and methylation-sensitive restriction digest to quantify mutant vs. wild-type allele expression (Fig. S1E, ).

• Mouse Knock-In Models: Introduce Otud5 p.Gly494Ser via CRISPR homology-directed repair and track embryonic lethality (E12.5) .

What computational tools can predict OTUD5-substrate interactions to guide experimental validation?

• AlphaFold Multimer: Predict OTUD5’s OTU domain binding to TRIM25’s SPRY domain (Fig. 2D, ).

• UbPred: Identify potential ubiquitination sites on substrates like TRIM25 (Lys568/Lys571) .

• STRING-DB: Prioritize OTUD5 interactors (e.g., TRIM25, PML) based on functional enrichment in cancer pathways .