HERC5 Antibody

What is HERC5 and what are its primary functions?

HERC5 is an interferon (IFN)-induced HECT-type E3 protein ligase that mediates ISGylation, a post-translational modification process involving the conjugation of ISG15 (Interferon-stimulated gene 15) to target proteins . Structurally, HERC5 possesses an N-terminal region containing multiple RCC1 (regulator of chromatin condensation 1) motifs which collectively form an RCC1-like domain (RLD), and a C-terminal HECT domain critical for its ligase activity .

HERC5 plays a central role in mammalian innate immunity by ISGylating viral proteins to disrupt viral replication . For example, HERC5 uses its RLD domain to bind viral proteins such as influenza A virus NS1, hepatitis C virus NS5A, and multiple HIV gag particle precursor proteins . More recently, HERC5 has been found to suppress Mycobacterium tuberculosis infection through ISGylating PTEN, thereby promoting its degradation and facilitating cytokine production for bacterial clearance .

How is HERC5 expression regulated in cells?

HERC5 expression is primarily regulated by type I interferons. In both HeLa and A549 cell lines, while basal levels of HERC5 mRNA can be detected, IFN-β treatment rapidly induces HERC5 mRNA expression . Experimental data shows that within 6 hours of IFN-β stimulation, HERC5 mRNA is significantly upregulated, and by 12 hours, there is approximately a 30-fold increase in HERC5 mRNA expression compared to untreated controls . This elevated expression continues at 24 and 48 hours post-treatment .

The timing of HERC5 induction is noteworthy as it precedes the detection of ISG15 protein conjugates, which only become observable at 24 hours after IFN-β treatment. This sequential timing parallels observations made for other components of the ISGylation machinery, such as Ube1L and UbcH8 .

What are the key structural domains of HERC5 and their functions?

HERC5 contains distinct structural domains that contribute to its specialized functions:

The N-terminal RLD domain of HERC5 is responsible for recognizing and coordinating viral substrates for ISGylation . Mutation studies have shown that a conserved cysteine residue (C994) in the HECT domain is critical for its ligase activity; changing this residue to alanine abolishes the protein's ability to transfer ISG15 to target proteins .

What are the recommended applications for HERC5 antibodies in research?

HERC5 antibodies can be utilized in multiple experimental applications, with each providing distinct information about HERC5 expression, localization, or function:

What controls should be included when validating HERC5 antibody specificity?

Proper validation of HERC5 antibody specificity requires several controls:

• Positive Control: IFN-β treated cells (HeLa or A549) which show robust induction of HERC5 expression compared to untreated cells .

• Negative Control:

  • Cells where HERC5 expression has been knocked down using siRNA or shRNA

  • HERC5-deficient cell lines

  • Primary antibody omission control

• Specificity Control: Tests to ensure the antibody doesn't cross-react with the closest homolog of HERC5, which is HERC6 . This is particularly important as HERC6 shares structural similarities with HERC5.

• Peptide Competition Assay: Pre-incubating the antibody with the immunizing peptide (position F793-G1024 for the antibody described in search result #2) should eliminate specific staining .

In published validation studies, researchers have successfully used siRNA targeting nucleotide sequences 536-554 bp and 715-735 bp of HERC5, achieving approximately 80% reduction in HERC5 mRNA expression after 24 hours of IFN-β treatment . Additionally, a shRNA construct targeting region 1,606-1,639 bp has been effective for stable knockdown in HeLa cells .

How can HERC5 antibodies be used to study ISGylation in the context of viral infections?

To study ISGylation mediated by HERC5 during viral infections, researchers can employ several methodological approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Immunoprecipitate ISG15 conjugates using anti-ISG15 antibodies and probe for specific viral proteins

  • Alternatively, immunoprecipitate specific viral proteins and probe for ISG15 modification

  • Include HERC5 antibodies to verify the presence of HERC5 in the protein complexes

• Comparative analysis of ISGylation patterns:

  • Compare ISGylation profiles between wild-type cells and HERC5-depleted cells during viral infection

  • Use HERC5 antibodies in Western blot analysis alongside anti-ISG15 antibodies to correlate HERC5 expression with ISGylation levels

• Immunofluorescence co-localization studies:

  • Use HERC5 antibodies in conjunction with antibodies against viral proteins to visualize their co-localization

  • Determine whether HERC5 is recruited to viral replication complexes or virion assembly sites

Research has shown that HERC5 uses its RLD domain to bind viral proteins, including influenza A virus NS1, hepatitis C virus NS5A, and HIV gag proteins . When designing experiments to study these interactions, it's important to consider the timing of IFN induction, as HERC5 expression precedes the appearance of ISGylated proteins by approximately 18 hours .

What approaches can be used to identify novel HERC5 substrates?

Identifying novel substrates of HERC5-mediated ISGylation requires comprehensive experimental strategies:

• Proteomics-based approaches:

  • Affinity purification of ISGylated proteins followed by mass spectrometry (MS)

  • Stable isotope labeling with amino acids in cell culture (SILAC) comparing control vs. HERC5-overexpressing cells

  • Proximity-dependent biotin identification (BioID) using HERC5 as the bait protein

• Candidate-based verification:

  • After identifying potential substrates through proteomic screening, validate individual targets using:

    • Co-immunoprecipitation with HERC5 and candidate proteins

    • In vitro ISGylation assays with purified components

    • Site-directed mutagenesis of potential ISGylation sites

Previous research has successfully identified 174 candidate proteins that were covalently conjugated or interacted with ISG15 upon IFN treatment . Of 27 target proteins examined in one study, 24 were confirmed to be conjugated with ISG15, and 3 were found to interact with ISG15 without conjugation . The verification rate suggests that large-scale proteomic approaches can yield highly reliable candidates for HERC5 substrates.

How can HERC5 antibodies be used to study bacterial infections like tuberculosis?

Recent research has revealed that HERC5 plays a role in antimycobacterial immunity, particularly against Mycobacterium tuberculosis . When designing experiments to study this function:

• Infection models:

  • Use macrophage cell lines (human or mouse) infected with M. tuberculosis

  • Compare wild-type cells with HERC5-depleted cells (or HERC6-depleted in mice, which is the functional equivalent)

  • Monitor bacterial clearance, cytokine production, and signaling pathway activation

• Mechanistic studies:

  • Use HERC5 antibodies to immunoprecipitate HERC5 and identify interacting partners during infection

  • Examine ISGylation of PTEN and subsequent degradation using Western blot

  • Monitor activation of the PI3K-AKT signaling pathway, which is regulated by PTEN ISGylation

• In vivo relevance:

  • Analyze HERC5 expression in human tuberculosis patient samples

  • Correlate HERC5 levels with disease progression and immune response metrics

Research has shown that HERC5-mediated ISGylation of PTEN promotes its degradation, alleviating its suppression of the PI3K-AKT signaling pathway and enhancing cytokine production that facilitates clearance of M. tuberculosis .

What are common challenges when detecting HERC5 and how can they be addressed?

Researchers may encounter several challenges when working with HERC5 antibodies:

• Low basal expression levels:

  • Challenge: HERC5 has low basal expression in many cell types, making detection difficult without stimulation

  • Solution: Treat cells with type I interferons (particularly IFN-β) for at least 12-24 hours to induce robust HERC5 expression

• Non-specific binding:

  • Challenge: Some antibodies may cross-react with related HERC family proteins

  • Solution: Validate antibody specificity using HERC5 knockdown controls and peptide competition assays

• Signal variability:

  • Challenge: Variable induction of HERC5 depending on cell type and experimental conditions

  • Solution: Include positive controls (IFN-treated samples) and standardize treatment protocols

• Protein solubility issues:

  • Challenge: Difficulty expressing full-length HERC5 or its HECT domain in bacterial systems for in vitro studies

  • Solution: Consider mammalian expression systems or focus on specific domains separately

Research has shown that detecting endogenous HERC5 can be challenging, and some researchers have had difficulty expressing either the full-length or the HECT domain of HERC5 in bacterial systems . Alternative approaches include using mammalian expression systems or focusing on functional studies through knockdown and overexpression approaches.

How should samples be prepared for optimal HERC5 detection?

Proper sample preparation is crucial for successful detection of HERC5:

For Western blot analysis of ISGylated proteins, it's important to include N-ethylmaleimide (NEM) in the lysis buffer to inhibit deISGylating enzymes that might remove ISG15 modifications during sample preparation . Additionally, when studying the effect of HERC5 on viral infections, timing is critical – samples should be collected at multiple time points to capture the dynamic process of ISGylation.

What experimental approaches can be used to study HERC5 function beyond antibody-based detection?

While antibodies are valuable tools for studying HERC5, complementary approaches provide additional insights:

• Genetic manipulation:

  • siRNA/shRNA-mediated knockdown: Target sequences 536-554 bp, 715-735 bp, or 1,606-1,639 bp of HERC5 have proven effective

  • CRISPR-Cas9 gene editing: Generate HERC5-knockout cell lines for functional studies

  • Overexpression systems: Co-express HERC5 with Ube1L and UbcH8 to reconstitute ISGylation machinery

• Domain-specific functional analysis:

  • Generate constructs expressing specific domains of HERC5 (RLD or HECT domains)

  • Create point mutations in critical residues (e.g., C994A in the HECT domain)

  • Perform domain swapping with other HERC family members

• Transcriptional analysis:

  • Real-time PCR to quantify HERC5 mRNA expression changes

  • RNA-seq to identify global transcriptional changes upon HERC5 manipulation

  • ChIP-seq to identify transcription factors regulating HERC5 expression

Research has demonstrated that HERC5 mRNA expression can be quantified using real-time PCR, and that IFN-β treatment induces approximately 30-fold increments in HERC5 mRNA expression within 12 hours . Additionally, the co-expression of HERC5 with Ube1L and UbcH8 is sufficient to mediate ISG15 conjugation in vivo even in the absence of IFN treatment .

How might HERC5 antibodies be used to explore the role of ISGylation in emerging viral infections?

As new viral threats emerge, HERC5 antibodies will be valuable tools for understanding host-pathogen interactions:

• Comparative analysis across viral families:

  • Examine whether HERC5 targets conserved viral proteins across different virus families

  • Determine if viruses have evolved mechanisms to evade or antagonize HERC5-mediated ISGylation

  • Identify viral proteins that are preferentially ISGylated during infection

• High-throughput screening approaches:

  • Develop cell-based assays using HERC5 antibodies to screen for compounds that enhance HERC5 expression or activity

  • Screen viral protein libraries to identify targets of HERC5-mediated ISGylation

  • Use CRISPR screens to identify host factors that regulate HERC5 function

• Translational applications:

  • Investigate whether HERC5 expression levels correlate with disease severity in viral infections

  • Determine if genetic variants in HERC5 impact susceptibility to viral diseases

  • Explore potential therapeutic approaches targeting the HERC5-ISGylation pathway

Current research has established that HERC5 targets viral proteins from influenza A virus, hepatitis C virus, and HIV . Future studies might expand this knowledge to emerging viral threats and potentially develop therapeutic approaches that enhance this innate immune defense mechanism.

What role might HERC5 play in other infectious diseases beyond viral infections and tuberculosis?

The discovery of HERC5's role in antimycobacterial immunity raises questions about its potential functions in other infectious contexts:

• Bacterial infections:

  • Investigate whether HERC5 mediates resistance to other intracellular bacterial pathogens (e.g., Salmonella, Listeria)

  • Examine if bacterial proteins can be targets of ISGylation

  • Determine if bacteria have evolved mechanisms to manipulate the ISGylation pathway

• Parasitic infections:

  • Study HERC5 expression and function during parasitic infections

  • Examine whether ISGylation affects parasite replication or host response

• Fungal infections:

  • Investigate potential roles of HERC5 in antifungal immunity

  • Determine if fungal infections induce ISGylation and HERC5 expression

The recent finding that HERC5 suppresses M. tuberculosis infection by ISGylating PTEN suggests a broader role for this E3 ligase in antibacterial immunity . Further research may uncover similar mechanisms operating against other classes of pathogens, potentially revealing common principles of ISGylation-mediated host defense.