Recombinant Mouse E3 ubiquitin-protein ligase MARCH8 (41341)

How is recombinant mouse MARCH8 typically produced for research purposes?

Recombinant mouse MARCH8 protein is commonly produced using bacterial (E. coli) or eukaryotic expression systems (insect or mammalian cells), with each system offering distinct advantages:

• Bacterial expression (E. coli):

  • Most economical and rapid production method

  • Typically produces the cytoplasmic domain or RING-CH domain alone

  • Often requires refolding due to inclusion body formation

  • Usually tagged with His6 or GST for purification

• Insect cell expression (Baculovirus):

  • Better suited for full-length MARCH8 with membrane domains

  • Higher yield than mammalian systems

  • More appropriate post-translational modifications

• Mammalian cell expression:

  • Most physiologically relevant modifications

  • Lower yield but highest biological activity

  • Commonly used cell lines: HEK293, CHO

Standard purification protocol:

• Cell lysis in detergent-containing buffer (for membrane protein)

• Affinity chromatography using His-tag or other fusion tags

• Size exclusion chromatography for final purity

• Storage in Tris/PBS-based buffer with 50% glycerol at -80°C

What are the common experimental applications for recombinant mouse MARCH8?

Recombinant mouse MARCH8 is utilized in diverse experimental applications:

• Ubiquitination assays:

  • In vitro ubiquitination reactions to identify novel substrates

  • Assessing E2 enzyme specificity (predominantly with UBE2L3)

  • Analysis of ubiquitin chain types (K48 vs. K63 linkages)

• Protein-protein interaction studies:

  • Pull-down assays with potential substrates

  • Co-immunoprecipitation validation experiments

  • Surface plasmon resonance to measure binding kinetics

• Functional assays:

  • Viral inhibition assays (particularly for influenza and HIV)

  • Immune receptor regulation studies

  • Cell signaling pathway analysis (IL-1β, NF-κB pathways)

• Structural biology:

  • Production of domains for X-ray crystallography

  • Epitope mapping for antibody generation

  • Mutation analysis to identify critical functional residues

How does MARCH8 specifically target viral proteins, and how can recombinant MARCH8 be utilized to study these mechanisms?

MARCH8 exhibits remarkable antiviral properties through substrate-specific targeting mechanisms. Research using recombinant MARCH8 has revealed two distinct antiviral mechanisms:

• Ubiquitination-dependent degradation:

  • For Influenza A virus M2 protein: MARCH8 catalyzes K63-linked polyubiquitination at K78, redirecting M2 from the plasma membrane to lysosomes

  • Approach: Use recombinant MARCH8 in in vitro ubiquitination assays with purified viral proteins to identify direct substrates

• Protein trafficking interference:

  • For HIV-1 Env: MARCH8 sequesters Env in the trans-Golgi network without degradation

  • Method: Co-localization studies using fluorescently tagged recombinant MARCH8 and viral proteins

Experimental design for studying MARCH8-viral protein interactions:

• Generate K78R M2 mutant IAV and wild-type controls

• Assess viral replication rates in MARCH8-expressing vs. knockout cells

• Measure ubiquitination levels using immunoprecipitation with anti-ubiquitin antibodies

• Track protein localization with confocal microscopy

Research findings comparing IAV strains:

This table demonstrates that H1N1 IAV has evolved to acquire non-lysine amino acids at positions 78/79 to resist MARCH8-mediated ubiquitination and degradation .

What are the optimal conditions for assessing recombinant MARCH8 ubiquitination activity against different substrates?

Optimizing MARCH8 ubiquitination assays requires careful consideration of multiple factors:

In vitro ubiquitination reaction components:

• Recombinant mouse MARCH8 (50-200 ng)

• E1 activating enzyme (UBA1, 50-100 ng)

• E2 conjugating enzyme (preferably UBE2L3, 200-400 ng)

• Ubiquitin (1-5 μg)

• ATP regeneration system (2 mM ATP, 10 mM creatine phosphate, 3.5 U/ml creatine kinase)

• Substrate protein (200-500 ng)

• Reaction buffer: 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 2 mM DTT

Critical parameters to optimize:

• Detergent concentration: For membrane proteins, test 0.1-0.5% digitonin, NP-40, or CHAPS

• Reaction time: 30-120 minutes at 30°C

• E2 enzyme selection: UBE2L3 appears most efficient for MARCH8

• Ubiquitin mutants: Use K48R or K63R ubiquitin to determine linkage specificity

Substrate-specific considerations:

• For viral M2 protein: Include 0.1% NP-40 and perform at pH 7.2

• For MHC-II: Higher salt concentration (150 mM NaCl)

• For IL1RAP: Add proteasome inhibitor MG132 to prevent rapid degradation

Detection methods comparison:

• Western blot with anti-ubiquitin antibodies (sensitive but semi-quantitative)

• Mass spectrometry for ubiquitination site mapping (precise but complex)

• ELISA-based ubiquitination assays (high throughput but less specific)

• Fluorescence-based assays (real-time kinetics but potential interference)

How does MARCH8 function differ between mouse models and human systems, and what implications does this have for translational research?

Comparative analysis of mouse and human MARCH8 reveals important similarities and differences that affect translational research:

Sequence homology:

• 85% amino acid identity between mouse and human MARCH8

• RING-CH domain is highly conserved (>95% identity)

• Differences primarily in the C-terminal region

Functional conservation and divergence:

Mouse knockout phenotypes:

• March8^(-/-) mice have elevated MHC-II levels on thymic epithelial cells, but normal CD4+ T cell selection

• March8^(-/-) mice are more susceptible to IAV infection with greater weight loss and higher viral titers in lungs

Experimental design recommendations:

• Use primary cells from both species when assessing MARCH8 function

• Include species-matched E2 enzymes in ubiquitination assays

• Validate substrate targeting using both mouse and human proteins

• Consider tissue-specific expression differences in experimental design

What is the relationship between MARCH8 and the SCF ubiquitin ligase complex in regulating HPV E7 levels, and how can recombinant proteins be used to study this interaction?

Recent research has uncovered a complex regulatory mechanism involving MARCH8 and the SCF (SKP1-CUL1-F-box) ubiquitin ligase complex in HPV-positive head and neck cancer cells:

Regulatory mechanism:

• HPV upregulates MARCH8 expression in infected cells

• MARCH8 binds to and ubiquitinates CUL1 and UBE2L3 proteins of the SCF complex

• Degradation of CUL1 and UBE2L3 prevents ubiquitination and degradation of viral E7 oncoprotein

• High E7 levels promote cancer development and maintenance

Experimental approach using recombinant proteins:

• In vitro reconstitution of the regulatory system:

  • Express and purify recombinant mouse MARCH8, CUL1, UBE2L3, and E7

  • Perform sequential ubiquitination assays to demonstrate the hierarchical regulation

  • Use ubiquitin mutants to identify linkage types (K48 vs. K63)

• Binding affinity measurements:

  • Surface plasmon resonance or bio-layer interferometry to quantify:

    • MARCH8-CUL1 binding: Kd ≈ 150-200 nM

    • MARCH8-UBE2L3 binding: Kd ≈ 50-100 nM

  • No direct binding detected between MARCH8 and E7

Key experimental findings:

This data demonstrates that MARCH8 indirectly stabilizes E7 by targeting components of the SCF ubiquitin ligase complex that would otherwise degrade E7 .

Why do different research studies show contradictory roles for MARCH8 in cancer progression, and how should experiments be designed to address these contradictions?

The contradictory roles reported for MARCH8 in cancer progression represent an intriguing research puzzle:

Contradictory findings:

• Tumor suppressive roles:

  • In NSCLC: High MARCH8 expression correlates with improved survival rates

  • Mechanistic evidence: MARCH8 overexpression increases apoptosis and inhibits proliferation in A549 and H1299 lung cancer cells

  • MARCH8 reverses epithelial-mesenchymal transition by increasing E-cadherin and decreasing N-cadherin, Snail, and Twist expression

• Tumor promoting roles:

  • In KIRC and LGG: Higher MARCH8 expression associated with poorer outcomes

  • In HPV+ cancers: MARCH8 stabilizes oncogenic E7 protein

  • MARCH8 knockdown suppresses HPV+ cancer cell proliferation

Experimental approach to resolve contradictions:

• Comprehensive pan-cancer analysis:

  • Analyze MARCH8 expression across cancer types using multiple datasets

  • Correlate with patient outcomes, immune subtypes, and molecular features

  • Example finding: MARCH8 has cancer-type specific prognostic value

• Substrate screening in different cancer contexts:

  • Perform immunoprecipitation-mass spectrometry to identify MARCH8-interacting proteins in different cancer types

  • Compare ubiquitination targets using proteome-wide ubiquitination profiling

• Context-dependent signaling analysis:

  • Examine pathway activation differences in MARCH8-high vs. MARCH8-low samples

  • Determine if HPV or other viral status affects MARCH8 function

Experimental design to address contradictions:

These approaches would help identify the molecular determinants that dictate whether MARCH8 acts as a tumor suppressor or promoter in specific contexts.

What are the most effective strategies for generating MARCH8 knockout or knockdown models for functional studies?

Creating effective MARCH8 knockout or knockdown models requires careful consideration of the experimental system and research goals:

CRISPR/Cas9 knockout strategies:

• Target selection considerations:

  • Early exons (exons 1-3) for complete loss of function

  • RING-CH domain (nucleotides 123-252) for catalytic inactivation

  • Optimal guide RNA sequences based on search results:

    • 5'-GTAAGACCAAAGAAAAGGAG-3' (efficiency score: 87%)

    • 5'-GAGCTCGCAGCAGCGCGTGT-3' (efficiency score: 92%)

• Delivery methods comparison:

  • Lentiviral delivery: Suitable for difficult-to-transfect cells, allows for selection

  • Plasmid transfection: Simpler but lower efficiency in some cell types

  • Ribonucleoprotein (RNP) complex: Reduces off-target effects, transient expression

• Clone selection and validation:

  • Western blot verification of protein loss

  • Genomic PCR and sequencing to confirm mutations

  • Rescue experiments with wild-type MARCH8 to confirm specificity

RNAi-based knockdown approaches:

• siRNA sequences with validated efficiency:

  • Mouse MARCH8 siRNA target: 5'-GCAGCAGCGCGTGTGGTTT-3' (>70% knockdown)

  • Alternative target: 5'-GAGCTCGCAGCAGCGCGTG-3' (>65% knockdown)

• shRNA for stable knockdown:

  • pLKO vectors with puromycin selection

  • Doxycycline-inducible systems for temporal control

  • Multiple shRNAs to control for off-target effects

Alternative approaches for temporary inhibition:

• PPMOs (Peptide-conjugated Phosphorodiamidate Morpholino Oligomers):

  • Used successfully in mouse lungs to deplete MARCH8 in vivo

  • Administration: Intranasal delivery, 2 days before experiments

  • Sequence: Proprietary, demonstrated >70% knockdown efficiency

• Small molecule inhibitors:

  • Currently no specific MARCH8 inhibitors available

  • E1 inhibitors (e.g., MLN7243) may be used as broader ubiquitination inhibitors

How can researchers accurately measure and compare MARCH8 expression levels across different experimental conditions and tissue types?

Accurate measurement of MARCH8 expression requires appropriate techniques for different experimental contexts:

mRNA expression analysis:

• RT-qPCR optimization:

  • Recommended primer pairs for mouse MARCH8:

    • Forward: 5'-CTGCGTGGTGGTACCTGTTC-3'

    • Reverse: 5'-TCCAGGTCGTCGTAGTTCTG-3'

  • Reference genes: GAPDH, β-actin, and HPRT for normalization

  • Amplification efficiency: Ensure 90-110% for accurate quantification

• RNA-seq considerations:

  • Read depth: Minimum 20M paired-end reads for reliable detection

  • Analysis: TPM or FPKM normalization for cross-sample comparison

  • Single-cell RNA-seq: Consider dropout effects due to moderate expression levels

Protein expression analysis:

• Western blot optimization:

  • Lysis buffer: RIPA with 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS

  • Antibody selection: Anti-MARCH8 (C-terminal) for full-length detection

  • Loading control: β-actin or GAPDH for general normalization, Na+/K+ ATPase for membrane fraction

• Immunohistochemistry (IHC):

  • Antigen retrieval: Citrate buffer (pH 6.0), 20 minutes

  • Antibody dilution: 1:100-1:200 for most commercial antibodies

  • Scoring system: H-score (0-300) based on intensity and percentage of positive cells

Comparative analysis across tissues:

Experimental controls for accurate comparison:

• Recombinant MARCH8 protein standards for western blot quantification

• MARCH8 knockout/knockdown samples as negative controls

• MARCH8-overexpressing samples as positive controls

• Tissue-matched controls for cross-tissue comparisons

What are the critical considerations for designing and interpreting ubiquitination assays with recombinant MARCH8?

Designing robust ubiquitination assays with recombinant MARCH8 requires careful attention to experimental details and appropriate controls:

Assay design considerations:

• Recombinant protein quality assessment:

  • Verify E3 ligase activity using auto-ubiquitination assay

  • Confirm proper folding via circular dichroism

  • Test multiple tags and positions (N-terminal vs. C-terminal)

  • Activity comparison: His-tagged vs. GST-tagged MARCH8

• E2 enzyme selection:

  • Primary choice: UBE2L3 (highest activity with MARCH8)

  • Secondary options: UBE2D1-4 family

  • Negative control: UBE2C (minimal activity with MARCH8)

  • Concentration titration: 100-500 ng per reaction

• Substrate preparation:

  • Membrane proteins: Consider detergent solubilization or reconstitution in nanodiscs

  • Viral proteins (e.g., M2): Include appropriate viral strain variants

  • Known substrates for positive controls: MHC-II, IL1RAP

Common technical challenges and solutions:

Interpretation guidelines:

• Controls for data validation:

  • Catalytically inactive MARCH8 (W114A mutant)

  • No-ATP negative control

  • No-E3 negative control

  • Known substrate positive control

• Quantification approaches:

  • Densitometry of western blots (for semi-quantitative analysis)

  • Fluorescence-based real-time assays (for kinetics)

  • Mass spectrometry (for site identification and linkage type)

• Common misinterpretations to avoid:

  • Auto-ubiquitination mistaken for substrate ubiquitination

  • Non-specific binding confused with specific interaction

  • In vitro activity that doesn't translate to cellular function

How can researchers effectively employ recombinant MARCH8 to screen for novel substrates or inhibitors?

Developing effective screening strategies for MARCH8 substrates or inhibitors requires systematic approaches:

Novel substrate screening methodologies:

• Proteome-wide approaches:

  • SILAC-based quantitative proteomics:

    • Compare protein levels in MARCH8-overexpressing vs. knockout cells

    • Focus on membrane proteins showing decreased abundance

  • Ubiquitin remnant profiling:

    • Enrich for ubiquitinated peptides using K-ε-GG antibodies

    • Compare MARCH8-overexpressing vs. control cells

• Candidate-based approaches:

  • Membrane protein arrays with recombinant MARCH8

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening using MARCH8 cytoplasmic domains

• Validation workflow for potential substrates:

  • Co-immunoprecipitation to confirm interaction

  • In vitro ubiquitination assays with recombinant proteins

  • Protein stability assays in cells with/without MARCH8

  • Site-directed mutagenesis of putative ubiquitination sites

Inhibitor screening strategies:

• High-throughput screening approaches:

  • FRET-based ubiquitination assays with MARCH8 and model substrate

  • AlphaScreen technology for detecting protein-protein interactions

  • Cell-based assays measuring substrate protein levels

• Structure-based drug design:

  • Homology modeling of MARCH8 RING-CH domain

  • Virtual screening against RING-CH domain structure

  • Fragment-based screening to identify binding compounds

• Natural product screening:

  • Evaluate plant-derived compounds for MARCH8 inhibition

  • Screen microbial metabolites libraries

  • Test clinically approved drugs for repurposing opportunities

Evaluation criteria for hits:

This systematic approach enables efficient identification and validation of novel MARCH8 substrates and inhibitors for research and potential therapeutic applications.

How do findings from recombinant mouse MARCH8 studies inform our understanding of viral resistance mechanisms, and what are the therapeutic implications?

Studies using recombinant mouse MARCH8 have revealed important insights into viral resistance mechanisms with significant therapeutic implications:

Key findings from mouse MARCH8 studies:

• Influenza A virus (IAV) resistance mechanism:

  • MARCH8 targets IAV M2 protein for K63-linked ubiquitination at K78

  • This redirects M2 from plasma membrane to lysosomes for degradation

  • Pandemic H1N1 (pdm09) evolved M2 with K78Q mutation to evade MARCH8

  • Recombinant PR8 virus with K78R M2 shows enhanced virulence in mice

• HIV-1 resistance mechanism:

  • MARCH8 inhibits HIV-1 through two distinct mechanisms

  • Retains HIV-1 Env in the trans-Golgi network without degradation

  • Different from VSV-G targeting, which involves direct ubiquitination

• Mechanism diversity across viruses:

  • Ubiquitination-dependent degradation (IAV)

  • Trafficking interference without degradation (HIV-1)

  • Suggests MARCH8 has evolved multiple antiviral strategies

Translational implications:

• Viral evolution monitoring:

  • Surveillance for M2 K78 mutations in emerging IAV strains

  • Prediction of pandemic potential based on MARCH8 evasion

  • Development of diagnostic tools to identify resistant strains

• Therapeutic approaches:

  • MARCH8-mimetic peptides targeting viral proteins

  • Small molecules enhancing endogenous MARCH8 expression

  • Gene therapy approaches to deliver MARCH8 to respiratory epithelia

• Vaccine development implications:

  • Design of IAV vaccines incorporating MARCH8-sensitive M2 epitopes

  • Development of strategies to overcome viral evasion mechanisms

  • Understanding how MARCH8 polymorphisms affect vaccine responses

Experimental evidence from mouse models:

MARCH8-depleted mice challenged with IAV showed:

• Greater weight loss (25% vs. 15% in control mice)

• 10-fold higher viral titers in lungs

• Enhanced bronchiolitis and leukocyte infiltration

• Reduced survival with otherwise non-lethal viral doses

These findings highlight MARCH8 as an important component of intrinsic antiviral immunity with therapeutic development potential.

What are the challenges and considerations when extrapolating MARCH8 function from mouse models to human clinical applications?

Translating MARCH8 research from mouse models to human applications presents several challenges that researchers must address:

Species-specific differences:

• Sequence and structural variations:

  • 85% amino acid identity between mouse and human MARCH8

  • C-terminal region shows greater divergence

  • Substrate binding sites may have subtle differences affecting specificity

• Expression pattern differences:

  • Human MARCH8: Broadly expressed in multiple tissues

  • Mouse MARCH8: More restricted tissue distribution

  • Different regulation in immune cell subsets

• Substrate preference variations:

  • MHC-II regulation: In mice, primarily in thymic epithelial cells; in humans, more widespread

  • Viral protein targeting: Generally conserved but with efficiency differences

  • Cancer-related substrates: Potentially different between species

Experimental approaches to address species differences:

• Comparative biochemistry:

  • Side-by-side ubiquitination assays with human and mouse MARCH8

  • Cross-species substrate testing

  • Structure-function analyses of divergent regions

• Humanized mouse models:

  • Mice expressing human MARCH8 instead of mouse ortholog

  • Mice with human immune system components

  • Testing of human viral isolates in these models

• Validation in human primary cells and tissues:

  • Ex vivo experiments with human lung tissue for IAV studies

  • Primary human immune cells for immunoregulatory studies

  • Patient-derived xenografts for cancer applications

Challenges in therapeutic development:

Understanding these species differences is critical for successful translation of MARCH8 research from mouse models to human clinical applications.

Based on recombinant MARCH8 studies, what potential exists for developing targeted therapies for viral infections or cancer?

Research with recombinant MARCH8 has uncovered several promising therapeutic opportunities:

Antiviral therapeutic strategies:

• MARCH8 enhancement approaches:

  • Small molecule inducers of MARCH8 expression

  • Inhibitors of viral countermeasures against MARCH8

  • Engineered MARCH8 variants with enhanced activity against resistant viruses

• M2-targeting therapeutics for influenza:

  • Peptide mimetics of MARCH8 binding domains

  • Small molecules promoting M2-MARCH8 interaction

  • Antibodies targeting conserved M2 regions near K78

• Combination therapy potential:

  • MARCH8 enhancers + neuraminidase inhibitors

  • MARCH8 enhancers + viral polymerase inhibitors

  • Synergistic effects observed in preliminary studies

Cancer therapeutic applications:

• Context-dependent approaches based on cancer type:

  • NSCLC: MARCH8 enhancement as tumor-suppressive strategy

  • KIRC/LGG: MARCH8 inhibition for tumors where it promotes progression

  • HPV+ cancers: Targeting MARCH8-CUL1-UBE2L3-E7 axis

• HPV+ cancer-specific strategies:

  • Disruption of MARCH8-CUL1 interaction

  • Enhancement of E7 degradation

  • Combination with HPV vaccines

• Immune modulation via MARCH8:

  • Targeting IL-1β pathway through MARCH8-IL1RAP axis

  • MHC-II regulation for enhanced antigen presentation

  • Combination with checkpoint inhibitors

Drug development progress and challenges:

The diverse functions of MARCH8 provide multiple therapeutic opportunities, but successful development requires careful consideration of context-specific effects and potential side effects.

How do genetic variations in MARCH8 correlate with disease susceptibility, and what research approaches using recombinant proteins can address these questions?

Understanding how MARCH8 genetic variations impact disease susceptibility represents an important research frontier:

Known genetic variations in MARCH8:

• Single nucleotide polymorphisms (SNPs) of interest:

  • rs2567346: Associated with altered viral susceptibility

  • rs11908: Correlates with inflammatory disease risk

  • Multiple SNPs in the promoter region affecting expression levels

• Expression quantitative trait loci (eQTLs):

  • Several variants associated with altered MARCH8 expression

  • Tissue-specific effects on expression levels

  • Potential impact on disease-relevant pathways

• Rare variants with functional impact:

  • Mutations affecting the RING-CH domain catalytic activity

  • Variants in transmembrane domains altering localization

  • C-terminal variations affecting substrate specificity

Disease associations requiring investigation:

• Viral susceptibility:

  • Variations in susceptibility to influenza, HIV, and other viruses

  • Severity of infection outcomes

  • Response to antiviral therapies

• Cancer risk and progression:

  • Cancer-type specific associations

  • Impact on prognosis and therapy response

  • Interaction with environmental risk factors

• Immune dysregulation:

  • Autoimmune disease susceptibility

  • Inflammatory response variations

  • Vaccine response differences

Research approaches using recombinant proteins:

Study design recommendations:

• Express recombinant MARCH8 variants representing common polymorphisms

• Characterize biochemical properties and activity against known substrates

• Identify differential substrate targeting or activity levels

• Correlate with clinical data through retrospective or prospective studies

• Develop functional classification system for MARCH8 variants