HERC4 Antibody

What is HERC4 and what cellular functions does it perform?

HERC4 (HECT domain and RCC1-like domain-containing protein 4) functions as an E3 ubiquitin-protein ligase that plays crucial roles in protein trafficking and cellular structure distribution. It's integral to the ubiquitin-proteasome system, accepting ubiquitin from E2 ubiquitin-conjugating enzymes via thioester bonds before transferring it to target substrates . HERC4 is notably required for proper spermatozoon maturation, fertility, and the removal of cytoplasmic droplets from spermatozoa . Research has also established its functional relationship with tumor suppressor p53 and other E3 ligases that regulate proteolytic balance within cells . Recent studies have identified its important role in regulating MafA protein stability through K63-linked polyubiquitination and interaction with GSK3β, affecting transcriptional activity in multiple myeloma pathways .

What are the different isoforms of HERC4 and their significance in experimental design?

Human HERC4 exists in at least 6 isoforms produced through alternative splicing . When designing experiments, researchers must consider which isoforms are relevant to their study system. The calculated molecular weights of these isoforms include 12kDa, 13kDa, 106kDa, 109kDa, 117kDa, and 118kDa forms, though Western blot typically detects the 118-119kDa species . This diversity necessitates careful antibody selection to ensure recognition of relevant isoforms.

For accurate experimental design, researchers should determine which isoforms are expressed in their model system before selecting appropriate antibodies.

What are the optimal conditions for Western blot detection of HERC4?

For successful Western blot detection of HERC4, consider the following optimized protocol:

• Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors.

• Gel percentage: Use 6-8% SDS-PAGE gels to properly resolve the 119kDa HERC4 protein.

• Transfer conditions: Transfer proteins to PVDF membrane at 30V overnight at 4°C for high molecular weight proteins.

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute HERC4 antibody 1:500 to 1:2000 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG at 1:5000 dilution.

• Detection: Develop using ECL technique with 30-second exposure for optimal signal .

Expected results: A distinct band should be visible at approximately 119kDa . Multiple bands may indicate detection of different isoforms or post-translationally modified variants of HERC4.

How can I design experiments to study HERC4's ubiquitin ligase activity?

To investigate HERC4's E3 ubiquitin ligase activity, consider this experimental approach:

• Co-immunoprecipitation assays: Transfect cells with tagged HERC4 and potential substrate proteins (e.g., MafA). Immunoprecipitate with anti-tag antibody and immunoblot for ubiquitinated species.

• Ubiquitination assays: Co-transfect cells with HERC4, substrate, and tagged ubiquitin (WT-Ub or mutants like K63R-Ub to determine linkage specificity).

• Controls:

  • Include HERC4 ΔHECT domain mutant (enzymatically inactive form) as negative control

  • Include wild-type HERC4 as positive control

  • Use substrate-specific siRNA to verify specificity

• Linkage analysis: Use antibodies specific for different ubiquitin linkages (K48, K63, K11, K29) to determine ubiquitination patterns .

Research demonstrates that HERC4 primarily induces K63-linked polyubiquitination of substrates like MafA, with minor K48-linked ubiquitination . When K63 is mutated (K63R-Ub), HERC4-mediated ubiquitination of MafA is significantly reduced, confirming specificity of linkage .

What is the recommended protocol for immunoprecipitation using HERC4 antibodies?

For successful immunoprecipitation of HERC4 and its interacting partners:

• Cell lysis: Prepare whole cell lysate (1mg protein) in IP lysis buffer containing protease inhibitors.

• Pre-clearing: Incubate lysate with protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Antibody binding: Add HERC4 antibody at 3μg/mg of lysate and incubate overnight at 4°C with gentle rotation .

• Bead capture: Add protein A/G beads and incubate for 2-4 hours at 4°C.

• Washing: Wash beads 4-5 times with IP wash buffer.

• Elution: Elute bound proteins by boiling in SDS sample buffer.

• Analysis: Load 20% of IP for Western blot detection .

Always include an IgG control IP to identify non-specific interactions . For detecting interactions with specific partners like MafA or GSK3β, use appropriate antibodies in subsequent Western blots.

How does HERC4 regulate MafA stability and function in cellular systems?

HERC4 regulates MafA through a dual mechanism involving both ubiquitination and phosphorylation:

• Stabilization via K63-linked ubiquitination:

  • HERC4 increases MafA protein stability by catalyzing K63-linked polyubiquitination at lysine 33 (K33)

  • This stabilization occurs in a HECT domain-dependent manner, as HERC4 ΔHECT fails to increase MafA levels

  • Cycloheximide chase assays confirm HERC4 extends MafA half-life

• Regulation via GSK3β interaction:

  • HERC4 directly interacts with GSK3β, inhibiting its ability to phosphorylate MafA

  • This inhibition suppresses MafA transcriptional activity

  • The inhibition of MafA phosphorylation by HERC4 leads to reduced STAT3 activation and decreased expression of STAT3 target genes (Bcl-2, Mcl-1, CCND2)

This dual regulation makes HERC4 a potential therapeutic target in multiple myeloma, where MafA overexpression contributes to disease progression.

What binding domains of HERC4 are critical for substrate interaction?

HERC4 contains multiple functional domains that mediate its interactions with substrates:

• HECT domain: Located at the C-terminus, this ~350 amino acid domain is essential for ubiquitin ligase activity, catalyzing thioester formation with ubiquitin before substrate transfer .

• RCC1-like domain (RLD): Functions as a guanine nucleotide exchange factor for small G proteins .

• MafA binding region: Research has identified amino acids 376-728 as the critical fragment for MafA interaction . Truncation studies showed that only HERC4 constructs containing this region could bind MafA.

These domain-specific interactions should be considered when designing experiments to study HERC4 function or develop inhibitors.

Why might I observe multiple bands when using HERC4 antibodies in Western blot?

Multiple bands in HERC4 Western blots can occur for several reasons:

• Detection of different isoforms: HERC4 has six known isoforms with molecular weights ranging from 12kDa to 118kDa . Commercial antibodies may detect multiple isoforms depending on the epitope.

• Post-translational modifications: HERC4 undergoes modifications that alter its migration pattern. These may include:

  • Ubiquitination (self-ubiquitination is common for E3 ligases)

  • Phosphorylation

  • Other modifications affecting protein mobility

• Degradation products: Sample preparation issues or endogenous proteases can generate fragmented HERC4.

• Non-specific binding: Some antibodies may cross-react with other HERC family members.

Troubleshooting approach:

• Validate bands using HERC4 knockout or knockdown samples

• Use different antibodies targeting distinct epitopes

• Include phosphatase treatment to eliminate modification-based mobility shifts

• Optimize sample preparation with fresh protease inhibitors

• For expected results, refer to product data showing a predominant band at 119kDa

How can I determine if HERC4 is functioning as expected in my experimental system?

To verify HERC4 functionality in your system:

• Expression verification: Confirm HERC4 protein expression by Western blot using validated antibodies .

• Ubiquitination activity assay:

  • Overexpress HERC4 alongside known substrates (e.g., MafA)

  • Compare wild-type HERC4 with HERC4 ΔHECT (enzymatically dead version)

  • Examine substrate stability via cycloheximide chase assays

  • Expected result: Wild-type HERC4 should increase MafA stability while HERC4 ΔHECT should not

• Interaction validation:

  • Perform co-immunoprecipitation of HERC4 with known binding partners (MafA, GSK3β)

  • Expected result: HERC4 should co-precipitate with these proteins

• Functional readouts:

  • Measure STAT3 phosphorylation and target gene expression

  • Expected result: HERC4 overexpression should decrease STAT3 phosphorylation and downregulate Bcl-2, Mcl-1, and CCND2 expression in MM cells

These approaches provide multiple lines of evidence for proper HERC4 function.

How can HERC4 be targeted in therapeutic approaches for multiple myeloma?

Research indicates several promising strategies for targeting HERC4 in multiple myeloma therapy:

• GSK3β inhibition approach:

  • Lithium chloride (LiCl), a known GSK3β inhibitor, upregulates HERC4 expression in MM cells in a concentration-dependent manner

  • This upregulation correlates with increased MafA protein levels

  • Combining LiCl with dexamethasone (Dex) shows enhanced anti-myeloma activity compared to either agent alone

• Experimental validation:

  • In vitro: Combined LiCl and Dex treatment showed synergistic effects on MM cell viability

  • In vivo: Human myeloma xenografts in nude mice showed significantly reduced tumor growth with combination therapy compared to single agents

  • Mechanism: Combined treatment significantly upregulated both HERC4 and MafA in myeloma xenografts

• Therapeutic implications:

  • Targeting the HERC4/GSK3β/MafA axis represents a novel treatment modality for MM

  • Upregulation of HERC4 may be beneficial in MM contexts where it is downregulated during disease progression

This approach leverages HERC4's natural regulatory function to inhibit pathological MafA-driven transcriptional programs in MM.

What are the challenges in studying HERC4's substrate specificity?

Investigating HERC4's substrate specificity presents several methodological challenges:

• Differential substrate recognition:

  • HERC4 shows specific binding preferences even among related proteins

  • Example: HERC4 interacts with MafA and c-Maf but not with the related MafB protein

  • This requires careful validation of each potential substrate

• Domain-specific interactions:

  • Different HERC4 domains mediate distinct substrate interactions

  • The aa 376-728 fragment is critical for MafA binding

  • Mapping these interactions requires extensive truncation and mutagenesis studies

• Linkage-specific ubiquitination:

  • HERC4 preferentially catalyzes K63-linked polyubiquitination on MafA, with minor K48-linked activity

  • This pattern may vary for different substrates

  • Requires specialized ubiquitin mutants (K63R-Ub, K48R-Ub, etc.) to determine linkage type

• Context-dependent activity:

  • HERC4 activity may vary across cell types and physiological conditions

  • Example: HERC4 is gradually downregulated during myelomagenesis

  • This necessitates studying HERC4 in multiple relevant cellular contexts

These challenges highlight the need for comprehensive approaches when investigating HERC4 substrate specificity in your research system.