Recombinant Mouse E3 ubiquitin-protein ligase SHPRH (Shprh), partial

What is the molecular structure and function of E3 ubiquitin-protein ligase SHPRH?

SHPRH is a complex multidomain protein belonging to the RAD5/RAD16-related subgroup of SNF2 family proteins. Its primary function involves DNA repair through its E3 ubiquitin ligase activity, particularly in post-replication repair pathways. The protein contains several functional domains:

• SNF2-family ATPase domain: Responsible for ATP hydrolysis

• RING finger domain: Mediates E3 ubiquitin ligase activity

• PHD (Plant Homeodomain) finger: Involved in chromatin binding

• Linker histone-like domain: Facilitates nucleosome interactions

These domains work together to enable SHPRH's dual functionality in DNA repair and chromatin regulation. Unlike typical SNF2-family enzymes, SHPRH's ATPase activity does not translate into conventional nucleosome remodeling under standard assay conditions, suggesting specialized functions related to ubiquitination rather than chromatin restructuring .

How does SHPRH interact with nucleosomes and chromatin components?

SHPRH demonstrates distinctive binding preferences for chromatin components. Electrophoretic mobility shift assays (EMSAs) have revealed that:

• SHPRH binds equally well to double-stranded DNA and nucleosome core particles

• SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA

• Binding to nucleosomes with extranucleosomal DNA (147-bp core + 80-bp protruding DNA) occurs at significantly lower SHPRH concentrations compared to free DNA or core particles alone

This binding behavior resembles that of ISWI- and CHD-family chromatin remodeling factors, which also preferentially bind to nucleosomes with extranucleosomal DNA. The enhanced binding to these structures suggests that SHPRH may primarily function at nucleosomes during DNA repair processes or transcriptional regulation where partially unwrapped nucleosomes are present .

What is the relationship between SHPRH and circ-SHPRH in disease contexts?

Circular RNA derived from the SHPRH gene (circ-SHPRH) has emerged as a potential biomarker and tumor suppressor distinct from the SHPRH protein itself. Key findings include:

Mechanistically, circ-SHPRH acts as a miRNA sponge, regulating downstream genes and signaling pathways that affect cancer cell proliferation, invasion, and apoptosis. This suggests separate but potentially complementary roles for SHPRH protein and circ-SHPRH in maintaining genomic stability and preventing tumorigenesis .

What are the optimal conditions for assessing SHPRH's ATPase activity?

To accurately measure SHPRH's ATPase activity, researchers should implement the following methodological approach:

Reaction Components:

• Purified recombinant SHPRH (50-100 nM)

• ATP (0.1-1 mM)

• Nucleosomes with extranucleosomal DNA (optimal substrate based on binding studies)

• Magnesium ions (2-5 mM) as cofactor

• Buffer system (typically Tris-HCl pH 7.5-8.0, 50-100 mM NaCl, 1 mM DTT)

Measurement Methods:

• Colorimetric phosphate detection (e.g., malachite green assay)

• Coupled enzyme assay using pyruvate kinase and lactate dehydrogenase

• Radioactive ATP hydrolysis assay with [γ-³²P]ATP

Critical Controls:

• DNA-only reactions

• Nucleosome core particles without extranucleosomal DNA

• No-ATP controls to establish baseline

• Time course measurements to determine linear range

Research has demonstrated that nucleosomes strongly stimulate SHPRH's ATPase activity compared to free DNA, with nucleosomes containing extranucleosomal DNA providing maximal stimulation. Unlike conventional chromatin remodeling enzymes, this ATPase activity does not result in canonical nucleosome repositioning, suggesting a specialized function likely related to facilitating SHPRH's E3 ligase activity .

How can the E3 ubiquitin ligase activity of SHPRH be effectively measured?

Assessment of SHPRH's E3 ubiquitin ligase activity requires careful experimental design:

Reaction Setup:

• Purified recombinant SHPRH

• Ubiquitin (wild-type or tagged versions)

• E1 activating enzyme (typically UBA1)

• E2 conjugating enzymes (research indicates SHPRH works with at least 7 different E2s)

• ATP regeneration system

• Target substrate (nucleosomes, PCNA, or other candidate proteins)

Recommended E2 Enzymes:
The following table outlines key E2 enzymes shown to function with SHPRH:

Detection Methods:

• Western blotting with anti-ubiquitin antibodies

• Mass spectrometry for ubiquitination site and chain type identification

• Fluorescently tagged ubiquitin for in-gel detection

Mass spectrometry analyses have revealed that SHPRH working with UBE2D1 can catalyze the formation of diverse polyubiquitin chains, including:

• Branched polyubiquitin linkages

• Linkages associated with DNA repair factor recruitment

• Linkages involved in proteasomal degradation (K48-linked)

This diversity of chain types indicates that SHPRH may have multiple functions in the DNA damage response pathway .

What techniques can be used to study SHPRH's binding to different nucleosomal substrates?

To characterize SHPRH's interaction with nucleosomes, researchers should consider:

Nucleosome Preparation:

• Reconstitute nucleosomes with defined DNA sequences:

  • 147 bp DNA for core particles

  • 227 bp DNA (147 bp + 80 bp extension) for nucleosomes with extranucleosomal DNA

• Consider incorporating modified histones to assess their effect on binding

Binding Assays:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Titrate increasing SHPRH concentrations with fixed nucleosome concentration

  • Run on native polyacrylamide gels

  • Detect shifts through fluorescence or staining

• Fluorescence Anisotropy:

  • Use fluorescently labeled nucleosomes

  • Monitor changes in anisotropy upon SHPRH binding

  • Calculate binding constants

• Pulldown Assays:

  • Immobilize tagged SHPRH or nucleosomes

  • Assess binding partners and stability

Data Analysis:

• Plot binding curves to determine dissociation constants (Kd)

• Compare binding affinities between different nucleosome constructs

• Analyze binding stoichiometry

Research has demonstrated that SHPRH binding to nucleosomes containing extranucleosomal DNA forms more stable complexes than with either free DNA or nucleosome core particles alone. Quantification of EMSA experiments showed that binding to nucleosomes with extranucleosomal DNA occurred at much lower SHPRH concentrations, indicating higher affinity for these substrates compared to free DNA or core particles .

How does SHPRH's ATPase activity coordinate with its E3 ligase function?

Understanding the interplay between SHPRH's ATPase and E3 ligase activities represents a frontier in current research. Available evidence suggests a complex relationship that can be investigated through:

Domain-Function Analysis:

• Generate SHPRH variants with mutations in:

  • ATPase domain (Walker A/B motifs)

  • RING finger domain

  • PHD finger domain

• Assess each variant for ATPase activity, E3 ligase activity, and nucleosome binding

• Determine interdependence between domains

Reaction Coupling Studies:

• Compare sequential versus simultaneous ATPase and ubiquitination reactions

• Analyze reaction products using mass spectrometry

• Determine if ATP hydrolysis enhances ubiquitination specificity or efficiency

Structural Analysis:

• Perform structural studies of SHPRH in different nucleotide-bound states

• Identify conformational changes that might coordinate the two activities

• Map interaction surfaces for E2 enzymes and nucleosome binding

Current research indicates that while nucleosomes stimulate SHPRH's ATPase activity, this doesn't translate into conventional nucleosome remodeling. Instead, ATP hydrolysis may facilitate conformational changes in SHPRH that enhance its ubiquitination activity. The finding that SHPRH can self-ubiquitinate within functional protein domains suggests a potential autoregulatory mechanism that could coordinate these activities .

What determines the specificity of SHPRH for different E2 enzymes and substrates?

SHPRH demonstrates versatility in its ability to function with multiple E2-conjugating enzymes and ubiquitinate various substrates. Understanding the determinants of this specificity requires:

E2 Enzyme Specificity:

• Perform comprehensive screening with diverse E2 enzyme panels

• Identify structural features that determine E2-SHPRH compatibility

• Characterize ubiquitination patterns resulting from different E2-SHPRH pairs

Substrate Recognition:

• Compare ubiquitination efficiency on different substrates:

  • Free histones vs. nucleosomes

  • Nucleosomes with different DNA lengths

  • Nucleosomes with various histone modifications

• Identify substrate recognition motifs or surfaces

• Determine how nucleosome binding affects target specificity

Mass Spectrometry Analysis:
Analyze ubiquitination sites and chain types formed with different E2-SHPRH combinations:

Research has shown that SHPRH can recruit E2 enzymes such as UBE2D1 to nucleosomes and form stable complexes. This recruitment ability may contribute to SHPRH's specificity in chromatin contexts. Mass spectrometry analyses have indicated that SHPRH with UBE2D1 can ubiquitinate nucleosomes with broad specificity and generate diverse polyubiquitin chains .

How does SHPRH contribute to DNA repair pathways beyond PCNA ubiquitination?

While SHPRH's role in polyubiquitinating PCNA during DNA damage response is established, emerging evidence suggests broader functions:

Chromatin-level Repair Mechanisms:

• Investigate SHPRH recruitment to different types of DNA damage:

  • UV-induced damage

  • Double-strand breaks

  • Interstrand crosslinks

• Characterize changes in chromatin accessibility at damage sites

• Identify additional substrates at damage sites

Nucleosome Ubiquitination:

• Map ubiquitination sites on histones catalyzed by SHPRH

• Determine whether these modifications:

  • Recruit specific repair factors

  • Affect chromatin structure

  • Promote nucleosome removal or exchange

Non-ubiquitination Functions:

• Assess potential roles in:

  • Chromatin remodeling during repair

  • Stabilizing repair intermediates

  • Regulating access to damaged DNA

Research has proposed that beyond PCNA ubiquitination, SHPRH may promote DNA repair or transcriptional regulation through chromatin ubiquitination. Mass spectrometry analyses have revealed that SHPRH can catalyze formation of polyubiquitin linkages associated with the recruitment of DNA repair factors, suggesting a broader role in coordinating repair processes at the chromatin level .

How should mass spectrometry data be analyzed to identify SHPRH-mediated ubiquitination patterns?

Mass spectrometry has become essential for characterizing SHPRH's ubiquitination targets and patterns. Researchers should implement the following analytical approach:

Sample Preparation:

• Perform in vitro ubiquitination reactions with SHPRH, E2 enzymes, and nucleosomal substrates

• Digest samples with trypsin (creates signature peptides with diglycine remnants on ubiquitinated lysines)

• Consider enrichment for ubiquitinated peptides using anti-K-ε-GG antibodies

Data Analysis Workflow:

• Ubiquitination Site Identification:

  • Search for +114.0429 Da mass shift on lysine residues

  • Apply appropriate false discovery rate thresholds

  • Validate with manual spectrum inspection

• Chain Linkage Analysis:

  • Identify signature diglycine-modified peptides from ubiquitin itself

  • Quantify relative abundance of each linkage type

  • Determine branch points by identifying peptides with multiple modifications

Interpretation Framework:

Research has shown that SHPRH working with UBE2D1 can generate polyubiquitin chains with various linkages, including those associated with DNA repair factor recruitment and those involved in proteasomal degradation. This diversity suggests that SHPRH may have multiple functions in the cellular response to DNA damage .

What bioinformatic approaches can identify potential SHPRH binding sites in the genome?

Identifying SHPRH genomic binding sites requires integrative bioinformatic approaches:

Sequence-Based Analysis:

• Identify consensus sequences from known binding sites

• Apply motif discovery algorithms to ChIP-seq or similar data

• Generate position weight matrices for genome-wide prediction

Structural Feature Recognition:

• Characterize DNA structural properties of known binding regions:

  • DNA shape parameters

  • Intrinsic curvature

  • Flexibility and deformability

• Apply machine learning to identify similar regions genome-wide

Chromatin Context Integration:

• Correlate potential binding sites with:

  • Nucleosome positioning data

  • Histone modification patterns

  • DNA accessibility profiles

• Prioritize sites with favorable chromatin contexts

Validation Experiments:

• Perform ChIP-qPCR on predicted high-confidence sites

• Test binding affinity with in vitro assays

• Assess functional relevance through genetic perturbation

Research has shown that SHPRH preferentially binds nucleosomes with extranucleosomal DNA, suggesting that binding sites in vivo may be located at nucleosome boundaries or regions with partial nucleosome unwrapping. This preference should be incorporated into bioinformatic models for predicting SHPRH binding sites with higher accuracy .

How can researchers reconcile seemingly contradictory data about SHPRH and circ-SHPRH functions?

Literature on SHPRH and circ-SHPRH shows some variation across studies and cancer types. To address these apparent contradictions:

Systematic Data Evaluation:

• Categorize findings by:

  • Experimental system (cell lines, tissues, animal models)

  • Analytical methods

  • Cancer type and stage

  • Molecular context (mutations, pathway alterations)

• Identify sources of variability:

  • Technical versus biological variation

  • Context-dependent effects

  • Temporal dynamics

Integrated Analysis Approach:

Cross-validation Strategy:

• Test hypotheses across multiple model systems

• Employ orthogonal experimental approaches

• Perform tissue-specific analyses

How might SHPRH be targeted therapeutically in cancer treatment?

SHPRH's roles in DNA repair and cancer suppression suggest potential therapeutic strategies:

Targeting Approaches:

• Small Molecule Modulators:

  • ATP-competitive inhibitors targeting the ATPase domain

  • Allosteric modulators affecting E3 ligase activity

  • Protein-protein interaction disruptors targeting E2-SHPRH interfaces

• Stabilization Strategies:

  • Compounds that prevent SHPRH degradation

  • Agents that enhance SHPRH expression or activity

  • Drugs targeting negative regulators of SHPRH

Cancer-Specific Considerations:

Research has shown that axitinib, a tyrosine receptor kinase inhibitor used for renal cell carcinoma, appears to act partly by stabilizing SHPRH, which in turn increases ubiquitination and degradation of β-catenin, a central coactivator of oncogenic Wnt-responsive genes. This mechanism suggests that modulating SHPRH activity could be therapeutically beneficial in cancers with hyperactive Wnt signaling .

What are the challenges in developing SHPRH as a therapeutic target?

Despite promising potential, several challenges exist in targeting SHPRH therapeutically:

Biological Challenges:

• Dual Enzymatic Functions:

  • Distinguishing between ATPase and E3 ligase activities

  • Selectively targeting one function while preserving the other

  • Understanding pathway-specific roles

• Context-Dependent Activities:

  • Tissue-specific functions

  • Cancer-type variations

  • Genetic background effects

Technical Challenges:

• Structural Complexity:

  • Large multi-domain protein difficult to express

  • Complex conformational dynamics

  • Multiple interaction surfaces

• Selectivity Issues:

  • Similarity to other SNF2 family members

  • E3 ligase domain homology with other RING proteins

  • Potential off-target effects

Strategic Approaches:

• Conduct comprehensive structural characterization:

  • Apply AI-driven conformational ensemble generation

  • Identify unique binding pockets

  • Develop highly selective modulators

• Exploit cancer-specific vulnerabilities:

  • Synthetic lethal approaches

  • Tumor-specific expression patterns

  • Combination therapies

Advanced AI algorithms have been applied to predict alternative functional states of SHPRH, including large-scale conformational changes, to provide a robust foundation for structure-based drug design. These approaches may help overcome the challenges associated with targeting this complex protein .

How can circ-SHPRH be developed as a biomarker for cancer diagnosis and prognosis?

Circ-SHPRH shows promise as a cancer biomarker based on consistent downregulation across multiple cancer types:

Biomarker Development Pathway:

• Analytical Validation:

  • Standardize detection methods (qRT-PCR, RNA-seq)

  • Establish reference ranges in healthy tissues

  • Determine assay sensitivity and specificity

• Clinical Validation:

  • Correlate with disease stage and progression

  • Assess prognostic value in prospective studies

  • Compare with existing biomarkers

• Clinical Utility:

  • Develop minimally invasive testing (liquid biopsy)

  • Establish decision algorithms

  • Implement in therapeutic selection

Performance Characteristics: