HERC5 Antibody, FITC conjugated

What is HERC5 and why is it significant in research?

HERC5 (HECT domain and RCC1-like domain containing E3 ubiquitin protein ligase 5) is an interferon-induced protein with significant roles in innate immunity. It contains an amino-terminal Regulator of Chromosome Condensation 1 (RCC1)-like domain and a carboxyl-terminal Homologous to the E6-AP Carboxyl Terminus (HECT) domain . HERC5 functions primarily as the main cellular E3 ligase that conjugates the IFN-induced protein ISG15 to target proteins (ISGylation), which is a post-translational modification similar to ubiquitination .

Research significance:

• Key player in antiviral responses, particularly against HIV-1 and other viruses

• Functions in ISG15 conjugation pathway, critical for innate immunity

• Shows evolutionary signatures of positive selection in the RCC1-like domain

• Implicated in cancer development, particularly oral squamous cell carcinoma (OSCC)

What does FITC conjugation mean for HERC5 antibodies and how does it work?

FITC conjugation involves the chemical labeling of antibodies with Fluorescein Isothiocyanate (FITC), a fluorescent dye widely used in biological research. This process creates a covalent interaction between the isothiocyanate group of FITC and the primary amines located on lysine residues of the antibody, establishing a stable thiourea bond .

The conjugation process:

• The reaction typically occurs under mild conditions (pH 8.0-9.5)

• The isothiocyanate group of FITC reacts with primary amines on lysine residues

• A stable thiourea bond forms between FITC and the antibody

• The resulting conjugate maintains antibody specificity while gaining fluorescent properties

The FITC-conjugated HERC5 antibody exhibits the following spectral characteristics:

• Absorption maximum: 495 nm (blue spectrum)

• Emission maximum: 519 nm (green spectrum)

• Observable as green fluorescence under appropriate microscopy conditions

How should HERC5 antibody, FITC conjugated be stored and handled to maintain optimal activity?

For optimal stability and performance of FITC-conjugated HERC5 antibodies:

Storage conditions:

• Temperature: Store at 2-8°C for short-term (1 month) or aliquot and store at -20°C for long-term

• Buffer components: Typically preserved in 0.03% Proclin 300, 50% Glycerol, 0.01M PBS at pH 7.4

• Light exposure: Protect from light to prevent photobleaching of the FITC fluorophore

• Avoid freeze-thaw cycles: Minimize repeated freezing and thawing (aliquot before freezing)

Handling recommendations:

• Centrifuge briefly before opening to ensure all liquid is at the bottom of the vial

• Use low-protein binding tubes and pipette tips

• When diluting, use buffers at pH 7.2-7.6 (optimal for FITC fluorescence)

• For maximum fluorescence stability, include antifade agents in mounting media

• Avoid azide-containing preservatives when using in applications involving peroxidase activity

What are the most effective applications for HERC5 antibody, FITC conjugated in research settings?

The FITC-conjugated HERC5 antibody is suitable for multiple research applications with particular effectiveness in:

• Immunofluorescence microscopy:

  • Direct detection of HERC5 in fixed cells and tissues

  • Colocalization studies with other proteins in the ISGylation pathway

  • Tracking HERC5 redistribution following interferon stimulation

• Flow cytometry:

  • Quantitative analysis of HERC5 expression across cell populations

  • Cell sorting based on HERC5 expression levels

  • Monitoring HERC5 induction following interferon treatment

• Confocal microscopy:

  • High-resolution imaging of HERC5 subcellular localization

  • Live-cell imaging for HERC5 trafficking studies

  • 3D reconstruction of HERC5 distribution patterns

• Immunohistochemistry (IHC):

  • Detection of HERC5 in paraffin-embedded tissue sections

  • Analysis of HERC5 expression in disease states (especially cancer tissues)

The reactivity of commercially available HERC5 antibodies has been confirmed for human samples, with some antibodies showing cross-reactivity with other species including bovine, horse, rabbit, and monkey proteins .

How should I design control experiments when using HERC5 antibody, FITC conjugated?

A robust experimental design using FITC-conjugated HERC5 antibody should include these controls:

Negative controls:

• Isotype control: Use a FITC-conjugated non-specific antibody of the same isotype (e.g., rabbit IgG for polyclonal HERC5 antibodies)

• Blocking peptide competition: Pre-incubate the antibody with excess immunizing peptide (if available)

• Untreated cells: Compare HERC5 expression in cells without interferon stimulation

• HERC5 knockdown: Use cells expressing HERC5-specific shRNA

Positive controls:

• Interferon-stimulated cells: HERC5 is strongly induced by type I interferons

• Overexpression system: Cells transfected with HERC5 expression plasmids

• Tissues known to express HERC5: Certain immune cells or interferon-treated cells

Specificity controls:

• Secondary-only staining: When using indirect immunofluorescence protocols

• Autofluorescence control: Unstained samples to assess background

• Cross-reactivity test: Test for reactivity with related HERC family proteins

For quantitative experiments, include calibration controls with known fluorescence intensities to enable standardization across experiments.

What are the optimal protocols for immunofluorescence detection of HERC5 using FITC-conjugated antibodies?

Optimized Immunofluorescence Protocol for HERC5 Detection:

Sample preparation:

• Grow cells on glass coverslips or prepare tissue sections (5 μm)

• Fix with 4% paraformaldehyde (10 min, room temperature)

• Permeabilize with 0.1% Triton X-100 in PBS (5 min)

• Block with 1% bovine serum albumin (BSA) in PBS (30-60 min)

Staining procedure:

• Dilute FITC-conjugated HERC5 antibody to optimal concentration (typically 1:100-1:200)

• Incubate samples with diluted antibody (1-2 hours at room temperature or overnight at 4°C)

• Wash 3× with PBS (5 min each)

• Counterstain nuclei with DAPI (1 μg/mL, 5 min)

• Wash 2× with PBS

• Mount with antifade mounting medium

Optimization considerations:

• Fixation method: Test both PFA and methanol fixation (certain epitopes may be sensitive)

• Antibody concentration: Perform titration experiments (1:50 to 1:500)

• Incubation time and temperature: Adjust based on signal strength

• Signal amplification: For weak signals, consider TSA (tyramide signal amplification)

Image acquisition parameters:

• Excitation wavelength: 488 nm laser or FITC filter set

• Emission filter: 515-540 nm bandpass

• Exposure time: Adjust to prevent photobleaching while maximizing signal

• Z-stack: Consider for complete 3D visualization

For dual or triple staining with other antibodies, select complementary fluorophores with minimal spectral overlap (e.g., TRITC, Cy5) and include appropriate compensation controls.

How can I study the relationship between HERC5-mediated ISGylation and viral restriction?

To investigate the relationship between HERC5-mediated ISGylation and viral restriction:

Experimental Approach:

• ISGylation analysis:

  • Transfect cells with plasmids encoding FLAG-tagged HERC5 (wild-type), HERC5-C994A (catalytically inactive mutant), and HA-tagged ISG15

  • Analyze ISGylation patterns by immunoprecipitation and Western blotting

  • Visualize ISGylated proteins using anti-HA or anti-ISG15 antibodies

• Viral restriction assays:

  • Express HERC5 (wild-type or C994A mutant) in target cells

  • Challenge with virus (e.g., HIV-1)

  • Measure viral replication through various methods:

    • Infectious virus release assay

    • Intracellular viral protein expression by Western blot

    • Viral RNA levels by qPCR

• Structure-function analysis:

  • Generate domain deletion mutants (e.g., HERC5-ΔRLD)

  • Test their ability to inhibit viral replication

  • Compare with wild-type HERC5 and catalytically inactive mutant (C994A)

• Protein interaction studies:

  • Perform co-immunoprecipitation to detect HERC5 interaction with viral proteins

  • Use FLAG-tagged HERC5 and viral protein-specific antibodies

  • Compare binding of wild-type HERC5 and catalytic mutants

Research findings show that HERC5 restricts HIV-1 assembly by two distinct mechanisms:

• ISG15 conjugation to viral proteins (requiring E3 ligase activity)

• Inhibition of nuclear export of Rev/RRE-dependent RNA (independent of E3 ligase activity, requiring RCC1-like domain)

What methodologies can be employed to identify novel targets of HERC5-mediated ISGylation?

To identify novel targets of HERC5-mediated ISGylation, consider these advanced approaches:

Proteomics Workflows:

• Affinity purification-mass spectrometry (AP-MS):

  • Express HA-tagged ISG15, FLAG-tagged HERC5, and the E1/E2 enzymes (Ube1L/UbcH8)

  • Purify ISGylated proteins using anti-HA immunoprecipitation

  • Identify targets by LC-MS/MS analysis

  • Compare HERC5 wild-type vs. C994A mutant conditions

• SILAC (Stable Isotope Labeling with Amino acids in Cell culture):

  • Grow cells in heavy or light isotope-labeled media

  • Express HERC5 in heavy-labeled cells and control vector in light-labeled cells

  • Purify ISGylated proteins and analyze by MS

  • Quantify relative abundance based on heavy/light peptide ratios

• Proximity-based labeling:

  • Generate HERC5-BioID or HERC5-APEX fusion proteins

  • Identify proteins in close proximity to HERC5 during ISGylation

  • Validate candidates using co-immunoprecipitation

  • Confirm ISGylation using in vitro assays

• Site-specific ISGylation mapping:

  • Use diGly remnant antibodies to enrich ISGylated peptides

  • Identify specific lysine residues modified by ISG15

  • Compare modification patterns with and without HERC5 expression

Validation Methods:

• In vitro ISGylation assays:

  • Purify recombinant proteins of interest

  • Incubate with E1 (Ube1L), E2 (UbcH8), E3 (HERC5) and ISG15

  • Detect ISGylation by Western blot

  • Compare wild-type target vs. lysine mutants

• Cell-based validation:

  • Generate lysine-to-arginine mutants of candidate proteins

  • Co-express with HERC5 and ISG15

  • Assess ISGylation by immunoprecipitation/Western blot

  • Evaluate functional consequences of ISGylation

Recent studies using these approaches have identified cGAS as a novel target of HERC5-mediated ISGylation, with modification occurring at 4 specific lysine residues .

How can I analyze the impact of HERC5 expression on cancer progression?

To investigate HERC5's role in cancer progression (particularly in oral squamous cell carcinoma):

Comprehensive Experimental Strategy:

• Expression analysis in patient samples:

  • Perform immunohistochemistry using anti-HERC5 antibodies on tissue microarrays

  • Compare HERC5 expression between tumor and adjacent normal tissues

  • Correlate expression levels with clinical parameters and survival data

• Functional studies in cancer cell lines:

  • Generate stable HERC5 overexpression models:

    • Use vectors like pCDNA3.1-CMV/eGFP-HERC5

    • Select stable clones using G418 selection (150 mg/ml)

    • Confirm expression by qPCR and Western blot

  • Create HERC5 knockdown models:

    • Design specific shRNAs targeting HERC5 mRNA

    • Use vectors like pGCsi-H1/Neo/GFP

    • Validate knockdown efficiency by qPCR and Western blot

• Phenotypic assays:

  • Proliferation assays (MTT, colony formation)

  • Migration and invasion assays (transwell, wound healing)

  • Apoptosis and cell cycle analysis (flow cytometry)

  • Drug resistance studies (e.g., cisplatin sensitivity)

• In vivo tumor models:

  • Subcutaneous xenografts with HERC5-modified cancer cells

  • Metastasis assays (tail vein injection)

  • Drug treatment studies (e.g., cisplatin, 3 mg/kg twice weekly)

  • Analysis by fluorescence imaging and histology

• Molecular mechanism investigation:

  • Identify binding partners by co-immunoprecipitation

  • Study ISGylation of cancer-related proteins

  • Analyze effects on specific signaling pathways

  • Investigate the relationship between HERC5 and SNAI1 expression

Research data indicates that HERC5 promotes OSCC progression by catalyzing UGDH ISGylation, which in turn promotes SNAI1 expression, suggesting HERC5 may serve as a potential therapeutic target in OSCC .

How can I optimize Western blot protocols using HERC5 antibodies?

Optimized Western Blot Protocol for HERC5 Detection:

Sample preparation:

• Lyse cells in RIPA buffer containing protease inhibitors

• Determine protein concentration (Bradford or BCA assay)

• Mix 20-50 μg protein with Laemmli buffer containing DTT

• Heat at 95°C for 5 minutes

Gel electrophoresis considerations:

• Use 8% SDS-PAGE gel (HERC5 is a large protein, ~118 kDa)

• Run at 100V until samples enter resolving gel, then increase to 150V

• Include molecular weight markers covering 75-150 kDa range

Transfer parameters:

• Use PVDF membrane (pre-activated with methanol)

• Transfer at 100V for 90 minutes in cold transfer buffer containing 20% methanol

• For larger HERC5 isoforms, consider overnight transfer at 30V, 4°C

Immunoblotting optimization:

• Block with 5% non-fat milk in TBST (1 hour, room temperature)

• Dilute primary HERC5 antibody 1:500-1:1000 in blocking buffer

• Incubate overnight at 4°C with gentle rocking

• Wash 3× with TBST (10 minutes each)

• Incubate with HRP-conjugated secondary antibody (1:5000, 1 hour)

• Wash 3× with TBST (10 minutes each)

• Develop using enhanced chemiluminescence (ECL) substrate

Troubleshooting common issues:

• High background: Increase blocking time, reduce antibody concentration

• Weak signal: Increase protein amount, extend primary antibody incubation

• Multiple bands: Validate with positive controls, consider using blocking peptide

• No signal: Confirm HERC5 expression (induced by type I IFNs)

Special considerations:

• Use IFN-β treated cells (1000 U/ml, 24h) as positive control

• Include HERC5 knockdown samples as negative control

• For detecting ISGylated proteins, use non-reducing conditions

• Consider using gradient gels (4-15%) to better resolve HERC5 and its ISGylated targets

What factors should I consider when analyzing HERC5 expression in different experimental conditions?

When analyzing HERC5 expression across experimental conditions:

Critical Factors to Consider:

• Baseline expression levels:

  • HERC5 is minimally expressed in most cell types under basal conditions

  • Expression varies by cell type (higher in immune cells)

  • Confirm baseline expression before starting experiments

• Induction parameters:

  • Type I interferons (IFN-α/β) strongly induce HERC5 expression

  • Optimal concentration: 1000 U/ml IFN-β

  • Time course: Expression typically peaks at 24-48 hours post-induction

  • Consider dose-response and time-course analyses

• Expression analysis methods:

  • qRT-PCR: For mRNA quantification, use properly validated primers

  • Western blot: For protein analysis, include positive controls

  • Immunofluorescence: For localization studies

  • Flow cytometry: For single-cell quantification

• Statistical considerations:

  • Perform at least three independent biological replicates

  • Use appropriate statistical tests (t-test for two conditions, ANOVA for multiple)

  • Account for variation in IFN responsiveness between experiments

• Controls and references:

  • Include untreated controls in each experiment

  • Use known IFN-stimulated genes (e.g., MX1, OAS1) as reference controls

  • Consider using HERC5 knockdown or knockout models as negative controls

• Confounding factors:

  • Cell density affects IFN responsiveness

  • Serum components may contain cytokines affecting baseline expression

  • Mycoplasma contamination can trigger innate immune responses

  • Viral infection status of cultured cells

Published data shows that when using shRNA targeting HERC5, an average 3.2-fold reduction in HERC5 RNA levels can be achieved compared to control cells expressing scrambled shRNA, as determined by qPCR .

What strategies can I use to evaluate the specificity of HERC5 antibodies?

To ensure HERC5 antibody specificity for reliable experimental results:

Comprehensive Validation Strategy:

• Genetic approach validation:

  • Compare staining in HERC5 knockdown/knockout vs. control cells

  • Analyze cells transfected with HERC5-specific shRNA vs. scrambled shRNA

  • Confirm specificity using cells overexpressing HERC5

• Biochemical validation:

  • Pre-absorb antibody with immunizing peptide (if available)

  • Perform Western blot to confirm single band of expected size (~118 kDa)

  • Compare staining pattern with multiple HERC5 antibodies targeting different epitopes

• Expression pattern validation:

  • Verify IFN-inducibility (HERC5 should increase after type I IFN treatment)

  • Compare with published expression patterns

  • Ensure correct subcellular localization (primarily cytoplasmic)

• Cross-reactivity assessment:

  • Test reactivity with recombinant HERC family proteins (HERC1-6)

  • Evaluate species cross-reactivity (commercially available antibodies may react with human, monkey, gorilla, and other species)

  • Check for non-specific binding to other proteins

• Application-specific controls:

  • For immunofluorescence: Include secondary-only and isotype controls

  • For flow cytometry: Use fluorescence-minus-one (FMO) controls

  • For Western blot: Include molecular weight markers and positive controls

A systematic validation approach should include multiple methodologies to confirm antibody specificity before proceeding with major experiments.

How should I interpret conflicting results between HERC5 mRNA expression and protein detection?

When facing discrepancies between HERC5 mRNA and protein data:

Systematic Analysis Approach:

• Technical considerations:

  • RNA detection methods (qPCR, RNA-seq) - check primer specificity and efficiency

  • Protein detection methods (Western blot, IHC) - validate antibody specificity

  • Sample preparation differences - protein extraction methods may affect results

  • Different sensitivities of detection methods

• Biological explanations:

  • Post-transcriptional regulation:

    • miRNA-mediated repression of HERC5 translation

    • RNA stability factors affecting HERC5 mRNA half-life

  • Post-translational regulation:

    • Protein degradation rates (HERC5 may be subject to proteasomal degradation)

    • Protein modifications affecting antibody recognition

  • Temporal dynamics:

    • Time lag between mRNA induction and protein accumulation

    • Different half-lives of mRNA vs. protein

• Experimental validation:

  • Time-course analysis of both mRNA and protein after IFN stimulation

  • Protein stability assays (cycloheximide chase)

  • mRNA stability assays (actinomycin D treatment)

  • Analysis of polysome-associated HERC5 mRNA (translation efficiency)

  • Use of proteasome inhibitors to assess degradation

• Reconciliation strategies:

  • Consider both measurements in context of biological question

  • Determine which measure correlates better with functional outcomes

  • Use additional methods to validate results (e.g., reporter assays)

  • Isolate cellular compartments to check for protein localization issues

Research data demonstrates that HERC5 shRNA-expressing cells exhibited substantially more intracellular HIV-1 Gag protein than control cells, despite only achieving a 3.2-fold reduction in HERC5 RNA levels , highlighting the importance of assessing both mRNA and protein levels.

How can I design experiments to distinguish between the E3 ligase-dependent and independent functions of HERC5?

To differentiate between HERC5's E3 ligase-dependent and independent functions:

Strategic Experimental Design:

• Genetic approach using HERC5 mutants:

  • Wild-type HERC5: Full functionality

  • HERC5-C994A: Catalytically inactive mutant (disrupts E3 ligase activity)

  • HERC5-ΔRLD: RCC1-like domain deletion mutant

  • Express these constructs in relevant cell models

• Functional readouts:

  • ISGylation assays:

    • Co-express constructs with ISG15, Ube1L, and UbcH8

    • Analyze by Western blot for ISG15 conjugates

    • Expected result: WT HERC5 promotes ISGylation; C994A mutant does not

  • HIV-1 restriction assays:

    • Co-transfect with HIV-1 proviral constructs

    • Measure virus production and infectivity

    • Expected result: Both WT and C994A restrict HIV-1 (RLD-dependent mechanism)

    • ΔRLD mutant should show impaired restriction

  • Nuclear export inhibition:

    • Assess Rev/RRE-dependent RNA export

    • Analyze by cellular fractionation and RNA quantification

    • Expected result: WT and C994A inhibit export; ΔRLD mutant does not

• Biochemical approach:

  • Co-immunoprecipitation studies:

    • Test interaction with known binding partners

    • Compare binding profiles of WT vs. mutant HERC5

    • Identify domain-specific interactions

  • Subcellular localization:

    • Fluorescence microscopy of tagged HERC5 variants

    • Co-localization with cellular markers

    • Track redistribution after stimulation

• Combined approaches for pathway analysis:

  • ISGylome analysis with each HERC5 variant

  • Transcriptome analysis to identify differentially regulated genes

  • Interactome studies using proximity labeling

Research findings demonstrate that HERC5-C994A (lacking E3 ligase activity) can still inhibit HIV-1 particle production by affecting Rev-dependent RNA export, while HERC5-ΔRLD fails to inhibit HIV-1 release, providing evidence for domain-specific functions .

What are the key considerations for investigating HERC5-mediated ISGylation in primary cells vs. cell lines?

When transitioning HERC5 research from cell lines to primary cells:

Critical Experimental Considerations:

• Baseline expression differences:

  • Primary immune cells (especially macrophages) may have higher basal HERC5 expression

  • Primary cells often show more heterogeneous expression patterns

  • Cell type-specific expression should be characterized before experiments

• IFN responsiveness variations:

  • Primary cells generally have intact IFN signaling pathways

  • Many immortalized cell lines have attenuated IFN responses

  • Dose-response relationship may differ significantly

  • Time course of induction may vary between primary cells and cell lines

• Transfection/transduction challenges:

  • Primary cells are typically more difficult to transfect than cell lines

  • Use optimized protocols (nucleofection, viral transduction)

  • Lower efficiency may require sorting/selection strategies

  • Consider potential activation of innate immune pathways by transfection

• Physiological relevance:

  • Primary macrophages show relevant HIV-1 restriction phenotypes

  • Effects observed in primary cells may better reflect in vivo situations

  • Context-dependent interactions may be preserved in primary cells

  • Primary cells from different donors introduce genetic variation

• Technical adaptations:

  • Western blot: May need larger cell numbers for primary cells

  • Immunofluorescence: Account for autofluorescence in primary cells

  • RNA analysis: Consider reference gene selection carefully

  • Flow cytometry: Include lineage markers for mixed populations

• Validation strategies:

  • Confirm key findings in primary cells from multiple donors

  • Use matched pairs of primary cells and cell lines when possible

  • Control for activation state of primary cells

  • Consider tissue-specific effects

Research data shows that HERC5 knockdown in primary human macrophages from two different donors resulted in substantially more intracellular HIV-1 Gag protein compared to control cells, similar to findings in 293T cells, demonstrating consistency of HERC5 function across cell types .

What strategies can I employ to optimize FITC-conjugated antibody performance in multicolor flow cytometry?

For optimal performance of FITC-conjugated HERC5 antibodies in multicolor flow cytometry:

Advanced Technical Optimization:

• Panel design considerations:

  • FITC characteristics:

    • Excitation maximum: 495 nm (optimal for 488 nm laser)

    • Emission maximum: 519 nm (green channel)

    • Brightness: Moderate (brighter than Alexa Fluor 488)

    • Sensitivity to photobleaching: High

  • Complementary fluorophores:

    • Avoid spectral overlap: PE-Cy5, APC, APC-Cy7 work well with FITC

    • Problematic combinations: PE shows significant spillover into FITC

    • For high-parameter panels: Consider brighter alternatives for dim antigens

• Sample preparation optimization:

  • Fixation impact:

    • Paraformaldehyde (1-4%) preserves FITC fluorescence well

    • Methanol fixation may reduce FITC signal intensity

    • Optimize fixation time to maintain epitope accessibility

  • Permeabilization for intracellular HERC5:

    • Saponin (0.1-0.5%): Gentle, reversible, good for FITC

    • Triton X-100 (0.1%): More stringent, may increase background

    • Commercial kits (e.g., BD Cytofix/Cytoperm): Standardized protocols

• Signal optimization techniques:

  • Antibody titration:

    • Create a titration series (1:50 to 1:1000)

    • Calculate signal-to-noise ratio for each dilution

    • Select optimal concentration with highest signal-to-noise

  • Staining conditions:

    • Temperature: 4°C reduces internalization but slows binding kinetics

    • Time: 30-60 minutes is typically optimal for most applications

    • Buffer: Include protein (0.5-1% BSA) to reduce non-specific binding

• Compensation and controls:

  • Single-color controls:

    • Use cells rather than beads for most accurate compensation

    • Ensure positive population has similar brightness to experimental samples

  • Critical controls:

    • Fluorescence-minus-one (FMO): Essential for gating FITC+ populations

    • Isotype control: Helps identify non-specific binding

    • Biological controls: IFN-stimulated vs. unstimulated cells

• Instrument settings:

  • Voltage optimization:

    • Set FITC PMT voltage to place negative population at 10^2-10^3

    • Ensure positive signal falls within detector linear range

  • Data analysis considerations:

    • Use biexponential display for FITC channel

    • Consider dimension reduction techniques for complex datasets

How can I accurately quantify the degree of ISGylation mediated by HERC5 in different experimental systems?

To accurately quantify HERC5-mediated ISGylation:

Quantitative Analysis Methods:

• Western blot-based quantification:

  • Semi-quantitative approach:

    • Detect ISGylated proteins using anti-ISG15 antibody

    • Quantify the intensity of high-molecular-weight smear

    • Normalize to loading control (β-actin, GAPDH)

    • Compare wild-type HERC5 vs. C994A mutant conditions

  • Target-specific ISGylation:

    • Immunoprecipitate protein of interest

    • Probe for ISG15 modification

    • Quantify ratio of modified to unmodified protein

• Mass spectrometry-based quantification:

  • Global ISGylome analysis:

    • Enrich ISGylated proteins using anti-ISG15 immunoprecipitation

    • Perform LC-MS/MS analysis

    • Use label-free quantification or SILAC for relative abundance

    • Compare conditions with and without HERC5

  • Site-specific quantification:

    • Identify ISG15-modified peptides using diGly remnant enrichment

    • Determine stoichiometry of modification at specific lysine residues

    • Compare modification rates between HERC5 variants

• Cellular imaging-based quantification:

  • Microscopy approach:

    • Co-express fluorescently tagged ISG15 and HERC5

    • Quantify colocalization and intensity

    • Measure changes after stimulation or inhibition

  • Flow cytometry approach:

    • Use intracellular staining for ISG15

    • Quantify mean fluorescence intensity

    • Compare population distributions

• Reporter systems:

  • FRET-based sensors:

    • Design substrate-ISG15-fluorophore constructs

    • Measure FRET efficiency changes upon ISGylation

    • Real-time monitoring in living cells

  • Luciferase complementation:

    • Split luciferase reporters to monitor protein ISGylation

    • Quantify luminescence as readout of modification

• Functional readouts:

  • Measure antiviral activity:

    • Use HIV-1 restriction as functional readout

    • Quantify infectious virus production

    • Compare wild-type vs. enzymatically inactive HERC5

Research data indicates that HERC5 co-expression with Ube1L and UbcH8 induces ISG15 conjugation in vivo independent of IFN stimulation, while a targeted substitution of Cys-994 to Ala in the HECT domain completely abrogates this E3 ligase activity .

What are the methodological challenges in studying the evolutionary aspects of HERC5 function?

Investigating the evolutionary aspects of HERC5 function presents several methodological challenges:

Evolutionary Analysis Methodology:

• Sequence acquisition and alignment challenges:

  • Database limitations:

    • Incomplete genome annotations in non-model organisms

    • Misannotation of HERC family members

    • Limited information on ortholog relationships

  • Alignment difficulties:

    • HERC5 is a large protein (~1024 amino acids)

    • Domain architecture varies across species

    • RCC1-like domain contains repeat structures challenging for alignment

• Selection analysis considerations:

  • Appropriate methods selection:

    • Site-specific methods (PAML, SLAC, FEL)

    • Branch-site methods for lineage-specific selection

    • Alignment quality critically affects results

  • Statistical challenges:

    • False positives in detection of positive selection

    • Power limitations with small datasets

    • Multiple testing correction needed for robust results

• Functional validation strategies:

  • Cross-species activity testing:

    • Express HERC5 orthologs in human cells

    • Measure ISGylation activity and antiviral function

    • Assess species-specific substrate preferences

  • Chimeric protein approach:

    • Create domain swaps between human and non-human primate HERC5

    • Map functional differences to specific domains/regions

    • Focus on regions under positive selection

• Structural biology integration:

  • Homology modeling limitations:

    • Few structures available for HECT E3 ligases

    • Uncertainty in modeling positively selected sites

  • Structure-function relationships:

    • Map positively selected sites onto structural models

    • Predict impact on substrate binding or catalytic activity

    • Experimental validation of predictions

• Evolutionary context considerations:

  • Virus-host co-evolution:

    • Temporal matching of HERC5 evolution with viral challenges

    • Correlation with primate lentivirus emergence

    • Integration with viral antagonist evolution

  • Gene family evolution:

    • HERC5 arose from gene duplication events

    • Functional specialization within HERC family

    • Comparison with other ISGylation machinery components

Research has identified a region in the RCC1-like domain of HERC5 that exhibits an exceptionally high probability of having evolved under positive selection, and this region is required for HERC5-mediated inhibition of nuclear export, suggesting viral selective pressure has shaped HERC5 evolution .

What are the emerging roles of HERC5 beyond viral restriction and ISGylation?

Beyond its established roles in viral restriction and ISGylation, HERC5 is emerging as a multifunctional protein with diverse biological activities:

Emerging HERC5 Functions:

• Cancer biology:

  • Tumor progression:

    • HERC5 promotes oral squamous cell carcinoma (OSCC) development

    • Mechanism involves UGDH ISGylation, which promotes SNAI1 expression

    • High HERC5 expression correlates with poor outcomes in certain cancers

  • Therapeutic implications:

    • Potential target for cancer therapy

    • Role in regulating response to chemotherapeutic agents

    • Involvement in epithelial-mesenchymal transition

• Innate immune signaling:

  • cGAS-STING pathway regulation:

    • HERC5 interacts robustly with cGAS

    • Catalyzes multi-site ISGylation of cGAS

    • Modification occurs at 4 specific lysine residues

    • ISGylation is indispensable in DNA-induced antiviral responses

  • Cytokine regulation:

    • Potential role in modulating interferon production

    • Impact on inflammatory cytokine expression

    • Cross-talk with NF-κB signaling

• Cellular stress responses:

  • Protein quality control:

    • Interface between ISGylation and ubiquitination systems

    • Potential role in targeting misfolded proteins

    • Stress granule regulation

  • Metabolic regulation:

    • Emerging connections to cellular metabolism

    • ISGylation of metabolic enzymes

    • Potential role in metabolic reprogramming during immune responses

• Post-translational modification crosstalk:

  • Phosphorylation-ISGylation interplay:

    • ISGylation affects tyrosine phosphorylation of UGDH at Tyr473

    • Complex interplay between different modification systems

    • Hierarchical modification patterns

  • Integration with other UBL systems:

    • Competition or cooperation with ubiquitination

    • Sequential or combinatorial modifications

    • Differential outcomes of various modifications

These emerging areas represent promising frontiers for HERC5 research, expanding our understanding beyond its canonical functions in antiviral immunity.

How can advanced imaging techniques be applied to study HERC5 localization and dynamics?

Advanced imaging approaches for investigating HERC5 localization and dynamics:

Cutting-Edge Imaging Strategies:

• Super-resolution microscopy:

  • STED (Stimulated Emission Depletion):

    • Resolution: ~50-70 nm

    • Advantage: Compatible with standard fluorophores including FITC

    • Application: Precise localization of HERC5 relative to cellular structures

  • PALM/STORM:

    • Resolution: ~20-30 nm

    • Requirement: Photoconvertible fluorophores

    • Application: Single-molecule mapping of HERC5 distribution

  • SIM (Structured Illumination Microscopy):

    • Resolution: ~100-120 nm

    • Advantage: Compatible with live cell imaging

    • Application: Dynamic redistribution of HERC5 after stimulation

• Live-cell imaging approaches:

  • FRAP (Fluorescence Recovery After Photobleaching):

    • Create FITC-HERC5 fusion protein or use anti-HERC5-FITC antibody fragment

    • Photobleach region of interest and measure recovery kinetics

    • Determine mobile vs. immobile HERC5 fractions

  • Single-particle tracking:

    • Label HERC5 with quantum dots or other bright, stable markers

    • Track individual molecules in living cells

    • Analyze diffusion characteristics and interaction kinetics

  • FRET biosensors:

    • Design biosensors reporting on HERC5-substrate interactions

    • Monitor ISGylation events in real time

    • Map cellular locations of active ISGylation

• Correlative microscopy:

  • CLEM (Correlative Light and Electron Microscopy):

    • Combine fluorescence imaging of HERC5 with ultrastructural context

    • Immunogold labeling for EM visualization

    • Precise localization at specific cellular structures

  • Multiplexed imaging:

    • Cyclic immunofluorescence or mass cytometry imaging

    • Visualize HERC5 together with multiple markers

    • Create spatial maps of protein interaction networks

• Dynamic analysis techniques:

  • Lattice light-sheet microscopy:

    • Gentle illumination for long-term imaging

    • 3D visualization of HERC5 dynamics

    • Capture rapid trafficking events

  • Optogenetic approaches:

    • Control HERC5 localization using light-inducible systems

    • Observe functional consequences of relocalization

    • Test compartment-specific activities

• Computational analysis:

  • Deep learning for image analysis:

    • Automated detection of HERC5 puncta

    • Track dynamic events over time

    • Correlate with cellular landmarks

Research shows that HERC5 shows a primarily cytoplasmic localization, and imaging approaches could reveal whether cytosolic cGAS undergoes ISGylation while nuclear cGAS does not, as suggested by biochemical studies .

What are the potential therapeutic applications targeting HERC5 pathways in disease?

Therapeutic strategies targeting HERC5 pathways represent an emerging frontier with applications in several disease contexts:

Therapeutic Targeting Strategies:

• Viral infection interventions:

  • Enhancement approaches:

    • Small molecules to increase HERC5 expression or activity

    • Gene therapy to deliver HERC5 to susceptible cells

    • Targeting the RCC1-like domain function to inhibit HIV-1 replication

    • Potential application in HIV-1 and other viral infections

  • Delivery considerations:

    • Tissue-specific targeting to enhance antiviral state

    • Temporal control to avoid chronic activation

    • Combination with other antivirals for synergistic effects

• Cancer therapy applications:

  • Inhibition strategies:

    • Small molecule inhibitors of HERC5 E3 ligase activity

    • Blocking HERC5-substrate interactions

    • Preventing UGDH ISGylation to impair SNAI1-driven tumor progression

    • Particular relevance for oral squamous cell carcinoma

  • Combination approaches:

    • Sensitization to cisplatin and other chemotherapeutics

    • Integration with immune checkpoint inhibitors

    • Targeting cancer stem cell populations

• Immunomodulatory applications:

  • Autoimmunity regulation:

    • Modulation of cGAS-STING pathway activation

    • Control of excessive type I interferon responses

    • Targeting ISGylation to fine-tune innate immunity

  • Inflammatory disease treatment:

    • Regulation of inflammatory cytokine production

    • Adjustment of macrophage activation states

    • Potential applications in chronic inflammatory conditions

• Diagnostic and prognostic applications:

  • Biomarker development:

    • HERC5 expression levels as disease indicators

    • ISGylation patterns as diagnostic signatures

    • Potential for liquid biopsy applications

  • Patient stratification:

    • Prediction of therapy responses

    • Identification of high-risk cancer patients

    • Personalized treatment selection

• Drug development considerations:

  • Target validation:

    • Genetic models (knockout, knockdown)

    • Domain-specific inhibition

    • Specificity within HERC family

  • Screening platforms:

    • High-throughput ISGylation assays

    • Cell-based phenotypic screens

    • Structure-based drug design

Research data indicates that HERC5 promotes cisplatin resistance in OSCC xenografts, suggesting that HERC5 inhibition could sensitize tumors to chemotherapy . Additionally, the role of HERC5 in potentiating cGAS-mediated innate immune responses suggests therapeutic potential in conditions with dysregulated DNA sensing .