mtarc1 Antibody

What is MTARC1 and why is it important in biomedical research?

MTARC1 (Mitochondrial amidoxime-reducing component 1) is a 337-amino acid protein with a molecular mass of approximately 37.5 kDa that localizes to the mitochondria . It functions as a component that catalyzes the reduction of N-oxygenated molecules, acting as a counterpart to cytochrome P450 and flavin-containing monooxygenases in metabolic pathways .

The protein has gained significant research interest because:

• It is associated with protection from metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD)

• Common missense variants (particularly p.A165T, rs2642438) confer protection against liver steatosis and fibrosis

• It plays a role in hepatocyte lipid metabolism and mitochondrial function

The protein is anchored to the outer mitochondrial membrane in an N-in, C-out orientation through its N-terminal mitochondrial targeting signal .

How are MTARC1 antibodies validated for specificity in research applications?

Proper validation of MTARC1 antibodies is critical for ensuring experimental reliability:

Standard validation methods include:

• Genetic knockout controls: The specificity of anti-MTARC1 antibodies can be validated using MTARC1 knockout cell lines. For example, researchers have demonstrated antibody specificity using HepG2 MTARC1 KO cell lines .

• siRNA knockdown verification: Researchers can confirm antibody specificity by demonstrating reduced signal following MTARC1 siRNA treatment. In mouse models, GalNAc-conjugated siRNA targeting Mtarc1 showed dose-dependent reduction in hepatic Mtarc1 mRNA and protein abundance, providing an excellent control system .

• Overexpression systems: BacMam virus expression systems have been used to express wild-type or mutant MTARC1 variants in cells, allowing for positive control validation .

• Cross-reactivity testing: Testing antibody reactivity across multiple species helps establish specificity. Some commercial antibodies show reactivity with human, mouse, rat, rabbit, bovine, dog, guinea pig, and horse MTARC1 .

When validating an MTARC1 antibody, it's crucial to include these controls and document the specific clone or catalog number used, as antibody performance can vary significantly between suppliers.

What are the optimal experimental conditions for detecting MTARC1 protein expression in liver samples?

Based on published research protocols, the optimal conditions for detecting MTARC1 in liver samples include:

For Western blotting:

• Sample preparation: Liver tissue should be homogenized in RIPA buffer containing protease inhibitors

• Protein loading: 25-50 μg of total protein per lane

• Antibody concentration: Anti-MTARC1 antibodies (such as Sigma HPA028702) are typically used at 1:100 to 1:1000 dilution

• Controls: Include MTARC1 knockout liver samples or siRNA-treated samples as negative controls

For immunohistochemistry/immunofluorescence:

• Fixation: 3.7% formaldehyde fixation for 20 minutes

• Permeabilization: 0.1% Triton X-100 in HBSS with 20 mM HEPES and 3% BSA

• Antibody incubation: Anti-MTARC1 antibody (1:100 dilution) overnight at 4°C

• Co-staining: MitoTracker dyes can be used for co-localization studies (100 nM, 30-minute incubation)

Notably, researchers have observed that detection of the MTARC1 p.A165T variant may require higher antibody concentrations due to its lower steady-state protein levels compared to wild-type .

What methods are available for modulating MTARC1 expression in experimental models?

Several validated approaches have been used to modulate MTARC1 expression in research:

Genetic approaches:

• Global knockout models: Complete genetic deletion of Mtarc1 in mice has been achieved and validated through genotyping and protein expression analysis

• Heterozygous models: Mtarc1+/- mice show intermediate protein expression levels, providing a dose-response model

RNA interference approaches:

• GalNAc-conjugated siRNA: This liver-specific delivery approach achieves 75% knockdown of Mtarc1 RNA in mouse liver with biweekly dosing over 8 weeks

• Validated siRNA sequences: Effective siRNA triggers have been identified with the antisense strand starting at positions 917 and 575 in the full mRNA transcript

Overexpression systems:

• BacMam virus expression: Effective for expressing wild-type or mutant MTARC1 in cell culture

• AAV8-TBG vector system: Used for liver-specific expression of MTARC1 variants in mice

Protein stability modulation:

• Cycloheximide chase: Used to measure MTARC1 protein half-life (wild-type ~11.5h vs. A165T ~3.5h)

• Proteasome inhibition: MG-132 (0.6 μM) can be used to block degradation and assess ubiquitination

Each approach has specific advantages depending on research questions and should be selected based on the experimental timeline and degree of knockdown/expression required.

How do different MTARC1 variants affect protein stability and function?

Research has revealed significant differences in stability and function among MTARC1 variants:

MTARC1 p.A165T variant (rs2642438):

• Associated with protection from MASLD/NAFLD and all-cause cirrhosis

• Shows significantly reduced protein stability with a shorter half-life (3.5h vs. 11.5h for wild-type)

• Exhibits increased ubiquitination and proteasomal degradation

• Maintains mitochondrial localization despite lower abundance

• Shows discrepancy between mRNA (increased) and protein levels (decreased)

MTARC1 p.M187K variant:

• Associated with protection from liver disease

• Does not affect protein stability compared to wild-type

MTARC1 p.R200Ter variant:

• Maintains mitochondrial localization similar to wild-type

MTARC1 p.C273A variant:

• Maintains normal protein expression levels comparable to wild-type

Multiple A165 substitutions:

• A165S, A165N, A165V, A165G, and A165D all show dramatically decreased stability

• Suggests the alanine residue at position 165 is critical for MTARC1 protein stability

These findings indicate that variants affecting MTARC1 stability rather than enzymatic activity may be responsible for the hepatoprotective effects observed in human genetic studies.

What are the recommended protocols for detecting ubiquitination of MTARC1 variants?

The following protocol has been successfully used to detect ubiquitination of MTARC1 variants:

Cell preparation:

• Seed cells (e.g., HepG2 dKO) at 1.84 × 10^7 cells/flask

• Transduce with BacMam virus expressing Flag-tagged MTARC1 (wild-type or variants)

• Incubate for 16 hours in a CO₂ incubator

Treatment conditions:

• Add cycloheximide (20 μM) or DMSO control for 8 hours

• Add proteasome inhibitor MG-132 (0.6 μM) or DMSO control for 2 hours

Immunoprecipitation:

• Harvest and lyse cells

• Perform immunoprecipitation using anti-DYKDDDDK (Flag) magnetic agarose

• Detect ubiquitinated MTARC1 with rabbit anti-ubiquitin antibody

• Detect total immunoprecipitated MTARC1 with anti-MTARC1 antibody

Data analysis:

• Quantify protein bands using ImageJ

• Normalize ubiquitinated MTARC1 to non-ubiquitinated MTARC1

This protocol has revealed that the MTARC1A165T variant exhibits significantly higher ubiquitination compared to wild-type, explaining its reduced protein stability .

How can researchers effectively distinguish between MTARC1 and MTARC2 in experimental models?

Distinguishing between MTARC1 and MTARC2 is critical for specific targeting and analysis:

Antibody selection:

• Use well-characterized antibodies specific to each paralog

• Validate antibody specificity using knockout models of each protein

Gene expression analysis:

• Design PCR primers specific to unique regions of each gene

• Monitor for compensatory expression changes (studies have shown MTARC2 RNA levels are not altered when MTARC1 is knocked down)

Functional discrimination:

• MTARC1 is the main contributor to the metabolic reduction of N-hydroxylated substrates

• In MTARC1 knockout models, plasma concentrations of ¹³C,¹⁵N-labeled hydroxyurea are maintained while formation of ¹³C,¹⁵N-urea is reduced

Knockout models:

• MTARC1 knockout does not affect MTARC2 expression, suggesting minimal compensatory mechanisms

• An inverse relationship exists between MTARC1 abundance and plasma retention of labeled hydroxyurea (R² = 0.89 at mRNA level; R² = 0.79 at protein level)

When designing experiments targeting MTARC1, researchers should always confirm that interventions don't inadvertently affect MTARC2 expression or function to maintain experimental specificity.

What are the optimal methods for subcellular localization studies of MTARC1 variants?

For accurate subcellular localization studies of MTARC1 variants, researchers have successfully employed the following protocols:

Fluorescence microscopy approach:

• Cell preparation:

  • Transduce cells (e.g., HepG2 dKO) in suspension with BacMam viruses expressing different MTARC1 variants (wild-type, A165T, C273A) at MOT 7

  • Plate cells at 1.2 × 10⁴ cells/well in collagen-coated 96-well imaging plates

  • Incubate overnight to allow protein expression

• Mitochondrial co-staining:

  • Add MitoTracker Deep Red (100 nM in culture media) to each well

  • Incubate for 30 minutes in a CO₂ incubator

  • Wash 3× with PBS

• Fixation and immunostaining:

  • Fix cells with 3.7% formaldehyde for 20 minutes

  • Wash 3× with PBS

  • Block/permeabilize with HBSS + 0.1% Triton X + 20 mM HEPES + 3% BSA for 1.5 hours

  • Incubate with anti-MTARC1 antibody (1:100 dilution) overnight at 4°C

Controls and variants:

• Include a MTARC1 construct lacking amino acids 1-50 (mitochondrial targeting signal) as a negative control for mitochondrial localization

• Test both N-terminal and C-terminal tagged variants to ensure tag position doesn't interfere with localization

• Compare wild-type with disease-relevant variants (A165T, M187K, R200Ter, C273A)

This approach has revealed that protective variants like A165T, M187K, and R200Ter all maintain mitochondrial localization despite differences in expression levels .

How do researchers reconcile contradictory findings regarding MTARC1's role in liver disease?

Several studies have reported conflicting results regarding MTARC1's role in liver disease, requiring careful interpretation:

Contradictory findings:

• Protective effect timing: Some studies report that MTARC1 knockout is protective in early to moderate liver disease but not in severe disease. Smagris et al. found no protection in advanced disease models, while other researchers demonstrated protection in moderate models .

• Intervention timing: The therapeutic window appears critical - siRNA intervention at week 16 improved liver fibrosis, but intervention at week 24 showed no benefit, suggesting disease stage-dependent effects .

• Protein abundance in human tissue: One study using mass spectrometry found no difference in protein abundance between A165A and A165T variants in pediatric livers, while another using immunoblotting showed ~50% decrease in A165T protein levels in human liver samples .

Reconciliation approaches:

• Model selection: Use "fit-for-purpose" model selection and temporal profiling of interventions in well-established MASH and liver fibrosis models .

• Disease stage consideration: Stratify analyses by disease severity and duration to account for stage-specific effects.

• Multiple methodologies: Apply both genetic knockout and siRNA approaches to distinguish between developmental and acute effects of MTARC1 loss.

• Complementary techniques: Use both mass spectrometry and immunoblotting for protein quantification, as each has different sensitivity profiles.

The temporal effect of MTARC1 modulation appears to be an essential consideration for reconciling contradictory findings in the literature.

What methodological approaches are most effective for analyzing MTARC1's impact on liver histopathology?

Advanced histopathological methods have proven valuable for detailed analysis of MTARC1's impact on liver disease:

Traditional histopathology:

• Hematoxylin and eosin staining for assessment of steatosis, inflammation, ballooning, and MASLD activity scores

• Collagen staining for fibrosis assessment

AI-powered digital pathology advantages:

• Zonal analysis: Differentiates effects across zones of the liver lobule

  • Zone 1 (portal region): Inflammation assessment

  • Zone 2 (perisinusoidal region): Fibrosis measurement

  • Zone 3 (central vein region): Steatosis characterization

• Lipid droplet characterization:

  • Distinguishes between macrosteatosis and microsteatosis

  • Quantifies total steatosis area

• Collagen fibril properties:

  • Measures length, thickness, and area of collagen fibrils

  • Provides more detailed fibrosis assessment than traditional scoring

• Co-localization analysis:

  • Quantifies fibrosis-steatosis co-localization

  • Measures fibrosis-inflammation co-localization

Results from AI-powered digital pathology revealed that hepatocyte-specific Mtarc1 knockdown resulted in reduced steatosis across all liver zones, decreased zone 2 fibrosis, and reduced fibrosis-steatosis co-localization - insights not detectable through traditional histopathology alone .

How does MTARC1 modulation affect hepatocyte metabolism and lipid profiles?

Recent research has revealed significant metabolic effects of MTARC1 modulation:

Cellular bioenergetics:

• MTARC1 knockdown improves cellular bioenergetics in response to lipotoxic stress

• Decreases mitochondrial superoxide production during lipotoxic stress

Liver lipid metabolism:

• Downregulation of MTARC1 reduces neutral lipid content in primary human hepatocytes homozygous for the wild-type protein (p.A165)

• This reduction is mediated by increased fatty acid utilization through beta-oxidation

• Carriers of the rs2642438 minor allele show higher 3-hydroxybutyrate levels (a by-product of β-oxidation)

Plasma lipid profile changes:
After hepatocyte-specific Mtarc1 knockdown in mice:

• Decreased total and HDL cholesterol

• Altered triacylglycerol and diacylglycerol species (increased)

• Reduced phosphatidylcholine and phosphatidylinositol lipid species

Untargeted lipidomics findings:

• Of 515 lipids quantified, 74 were differentially changed with siMtarc1 treatment:

  • 35 lipids increased in abundance

  • 39 lipids decreased in abundance

• Trend toward increased chain length in samples treated with siMtarc1

These findings suggest MTARC1 plays a crucial role in regulating hepatic lipid metabolism, particularly fatty acid oxidation pathways, which may explain its association with protection from fatty liver disease.

What are the most promising research directions for therapeutic targeting of MTARC1?

Based on current research, several promising therapeutic approaches targeting MTARC1 are emerging:

RNA interference approaches:

• GalNAc-conjugated siRNA shows effective hepatocyte-specific knockdown

• Biweekly administration achieves ~75% knockdown of Mtarc1 RNA in mouse liver

• Demonstrates therapeutic benefit in multiple models of MASH and liver fibrosis

Pharmacological modulation:

• Targeting protein stability: Since the protective A165T variant has reduced stability, compounds accelerating MTARC1 degradation could mimic this effect

• Small molecule inhibitors: Development of specific inhibitors of MTARC1's catalytic activity could provide therapeutic benefit

Biomarker development:

• MTARC1 genetic variants (rs2642438) could help identify patients most likely to benefit from therapy

• Plasma lipidomic profiles altered by MTARC1 modulation could serve as pharmacodynamic biomarkers

Therapeutic windows:

• Intervention timing appears critical - early to moderate disease shows better response to MTARC1 modulation than advanced disease

• Stratification of patients based on disease stage may be necessary for clinical trials

Combined approaches:

• Targeting MTARC1 along with pathways involved in extracellular matrix remodeling and collagen formation

• Combination with agents modulating fatty acid oxidation pathways

These approaches represent promising avenues for translating the hepatoprotective effects observed in carriers of MTARC1 variants into therapeutic interventions for metabolic liver disease.