BCAT2 Antibody

What is BCAT2 and what cellular functions does it regulate?

BCAT2 (Branched-Chain Amino Acid Transaminase 2) is a mitochondrial enzyme that catalyzes the first reaction in the catabolism of essential branched-chain amino acids (BCAAs): leucine, isoleucine, and valine. BCAT2 reversibly transaminated BCAAs to form branched-chain α-keto acids (BCKAs), which can then enter the TCA cycle for energy production . In humans, BCAT2 encodes a mitochondrial protein that is ubiquitously expressed in most organs (except hepatocytes) . In addition to its catalytic function, BCAT2 may also function as a transporter of branched-chain alpha-keto acids .

Unlike its cytoplasmic counterpart BCAT1 (primarily expressed in specialized tissues like the brain and ovaries), BCAT2 shows broader tissue distribution and different regulatory mechanisms in various diseases . Recent research indicates that BCAT2 plays crucial roles in cancer metabolism, cellular senescence, and ferroptotic cell death, making it an important target for research in multiple fields .

What applications are BCAT2 antibodies validated for in research protocols?

BCAT2 antibodies have been validated for multiple research applications:

For optimal results, researchers should perform antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) when conducting IHC . Most commercially available BCAT2 antibodies detect a protein between 38-42 kDa, although the calculated molecular weight is 44 kDa .

How can researchers distinguish between BCAT1 and BCAT2 in experimental systems?

Despite having similar substrate specificities, BCAT1 and BCAT2 have different amino acid sequences, subcellular localizations, and tissue expression patterns :

For experimental distinction, researchers should:

• Use antibodies raised against unique epitopes specific to each isoform

• Validate specificity through knockout/knockdown experiments

• Consider subcellular fractionation to separate mitochondrial (BCAT2) from cytoplasmic (BCAT1) components

• Perform parallel immunostaining with both antibodies to confirm distinct localization patterns

What controls should researchers include when working with BCAT2 antibodies?

For rigorous experimental design, researchers should incorporate these controls:

• Positive controls: HepG2 cells, MCF-7 cells, L02 cells, Jurkat cells, or Raji cells are established to express detectable levels of BCAT2 .

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG)

  • BCAT2 knockout/knockdown samples (several publications validate BCAT2 antibody specificity using KD/KO approaches)

• Peptide blocking: For site-specific antibodies (such as K44Ac antibody), perform peptide blocking experiments to confirm specificity. Research shows that K44-acetylated peptide can block K44Ac antibody signal significantly .

• Overexpression validation: Transfection with Flag-BCAT2 constructs can serve as positive controls to validate antibody specificity .

• Tissue controls: Human skin tissue and human placenta tissue have been validated for IHC applications .

How does acetylation regulate BCAT2 function and stability in normal and disease states?

BCAT2 undergoes post-translational modification through acetylation, particularly at lysine 44 (K44), an evolutionarily conserved residue. This modification significantly impacts its stability and function:

• Mechanism of acetylation regulation:

  • BCAT2 is acetylated at K44 by the acetyltransferase CBP (CREB-binding protein)

  • SIRT4 functions as the deacetylase for BCAT2

  • Acetylation leads to BCAT2 degradation through the ubiquitin-proteasome pathway

• Regulation by BCAA availability:

  • BCAA deprivation stimulates BCAT2 acetylation

  • CBP and SIRT4 bind to BCAT2 and control K44 acetylation levels in response to BCAA availability

• Functional consequences:

  • K44 acetylation promotes BCAT2 ubiquitylation and subsequent degradation

  • NAM (nicotinamide, a SIRT inhibitor) treatment increases BCAT2 acetylation and decreases protein levels

  • The K44R mutant (acetylation-resistant) shows increased stability and enhanced BCAA catabolism

• Disease relevance:

  • In pancreatic ductal adenocarcinoma (PDAC), the stable K44R mutant promotes cell proliferation and tumor growth

  • Under BCAA deprivation conditions, stable K44R mutant cells showed increased BCAA uptake

  • Xenograft experiments demonstrated that the K44R mutant induced significantly faster tumor growth than wild-type BCAT2

Researchers studying BCAT2 post-translational modifications should consider using the K44-specific acetylation antibody (K44Ac) for detection, which has been validated through dot blot assays and peptide blocking experiments .

What is the role of BCAT2 in cancer metabolism and how can it be targeted therapeutically?

BCAT2 plays critical roles in cancer metabolism that vary by cancer type:

• Pancreatic Ductal Adenocarcinoma (PDAC):

  • BCAT2 is frequently overexpressed in PDAC

  • BCAT2 knockout reduces cell proliferation and survival both in vitro and in vivo

  • BCAT2 enhances BCAA catabolism, which promotes tumor growth

  • BCAT2 K44 acetylation inhibits pancreatic tumor growth by decreasing BCAA catabolism

• Bladder Cancer:

  • BCAT2 shapes a noninflamed tumor microenvironment (TME)

  • BCAT2 has an inhibitory effect on cytotoxic lymphocyte recruitment

  • BCAT2 expression negatively correlates with secretion of CD8+ T-cell-related chemokines

  • CD8+ T cells show a decreasing tendency around BCAT2+ tumor cells from far to near

• Therapeutic targeting approaches:

  • Combination with immunotherapy: BCAT2 deficiency combined with anti-PD-1 antibody shows synergistic effects in vivo

  • Metabolic vulnerability: CBP-mediated acetylation of BCAT2 could be enhanced to promote BCAT2 degradation

  • Collateral lethality: BCAT2 is required for collateral lethality caused by deletion of PDAC malic enzyme

  • Predictive biomarker: BCAT2 expression may predict efficacy of immunotherapy in multiple cohorts

• Ferroptotic cell death regulation:

  • BCAT2 overexpression protects cancer cells from ferroptotic cell death

  • Treatment with glutaminase inhibitor (DON) abolishes the protective effect of BCAT2 in ferroptotic cancer cell death

  • BCAT2 may represent a potential target for overcoming resistance to ferroptosis inducers

For researchers developing therapeutic strategies, targeting BCAT2 in combination with immune checkpoint blockade represents a promising approach, particularly in cancers with noninflamed TME .

What methodologies are optimal for detecting BCAT2 post-translational modifications?

Detecting BCAT2 post-translational modifications, particularly acetylation, requires specialized techniques:

• Site-specific acetylation antibody approach:

  • Generate site-specific antibodies against acetylated peptides (e.g., K44Ac antibody)

  • Validate antibody specificity through dot blot assays using both acetylated and non-acetylated peptides

  • Perform peptide blocking experiments to confirm specificity

  • Treat cells with deacetylase inhibitors (NAM for sirtuins, TSA for HDACs) to increase acetylation levels

• Mass spectrometry-based detection:

  • Immunoprecipitate BCAT2 using tagged constructs (e.g., Flag-BCAT2) or specific antibodies

  • Digest proteins and analyze by LC-MS/MS to identify acetylated lysine residues

  • BCAT2 has three putative acetylation lysine (K) residues: K44, K321, and K374

• Mutation-based functional studies:

  • Generate lysine-to-arginine (K→R) mutants as deacetylation mimetics

  • Generate lysine-to-glutamine (K→Q) mutants as acetylation mimetics

  • Compare the stability and function of wild-type and mutant proteins to assess the impact of acetylation

• Western blotting for acetylation detection:

  • Immunoprecipitate BCAT2 from cells treated with/without deacetylase inhibitors

  • Probe with pan-specific anti-acetylated lysine antibody

  • Alternatively, use site-specific acetylation antibodies (e.g., K44Ac)

  • Quantify band intensity to determine relative acetylation levels

Researchers should note that BCAT2 acetylation increases ~2.1-fold after treatment with nicotinamide (NAM) and trichostatin A (TSA), with NAM being the more effective inducer of acetylation .

How does BCAT2 expression influence cellular senescence and aging processes?

Recent evidence indicates that BCAT2 plays a crucial role in regulating cellular senescence and aging:

• BCAT2 expression in senescence:

  • Various senescence-inducing stressors and aging itself reduce BCAT2 expression

  • Reduced BCAT2 expression decreases cellular BCAA catabolism

  • BCAT2 knockdown alone can induce cellular senescence in multiple cell types

• Experimental evidence:

  • BCAT2 knockdown by shRNA increases intracellular BCAA levels and decreases the BCKA to BCAA ratio

  • BCAT2 knockdown inhibits cell proliferation, increases SA-β-Gal activity, upregulates p16, p21, and SASP-related gene expression, and decreases lamin B1 expression

  • Exogenous BCAT2 overexpression suppresses cellular senescence in experimental models

• Mechanistic insights:

  • The shortage of BCAA catabolites (rather than accumulation of BCAAs) appears to induce cellular senescence

  • Branched-chain acyl-carnitines (BC-carnitines) can rescue growth arrest in senescent cells more effectively than BCAAs

  • BC-carnitines (isobutyryl-carnitine, isovaleryl-carnitine, and methylbutyryl-carnitine) can be incorporated into mitochondria and interconverted with downstream BCKA metabolites

• Aging-related changes:

  • Normal aging results in decreased Bcat2 expression and BCKA production in mouse adipose tissue

  • Alterations in BCAA metabolism are observed in human aging as well

  • BCAA transamination may be a crucial step in the aging process

Researchers studying aging and senescence should consider BCAT2 as a potential intervention target, as evidence suggests that modulating BCAA metabolism may impact longevity in various experimental models .

What are the optimal experimental conditions for detecting BCAT2 in different tissue and cell types?

Detecting BCAT2 requires optimized protocols depending on the application and sample type:

• Western Blotting (WB):

  • Dilution range: 1:500-1:2000

  • Positive controls: HepG2, MCF-7, L02, Jurkat, Raji cells

  • Sample preparation: Total protein extraction with protease inhibitors

  • Observed molecular weight: 38-42 kDa (calculated: 44 kDa)

  • Buffer system: Standard SDS-PAGE with transfer to PVDF or nitrocellulose membrane

  • Loading control: Mitochondrial proteins (e.g., COX IV) may be more appropriate than cytosolic housekeeping genes

• Immunohistochemistry (IHC):

  • Dilution range: 1:20-1:200

  • Validated tissues: Human skin tissue, human placenta tissue

  • Antigen retrieval: TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0

  • Detection system: Standard ABC or polymer-based systems

  • Counterstaining: Hematoxylin provides optimal contrast

• Immunofluorescence (IF/ICC):

  • Dilution range: 1:50-1:500

  • Validated cells: C2C12 cells

  • Counterstaining: Mitochondrial markers (e.g., MitoTracker) for colocalization studies

  • Fixation: 4% paraformaldehyde preferred over methanol-based fixatives

• Flow Cytometry (FC):

  • Concentration: 0.80 μg per 10^6 cells in a 100 μl suspension

  • Validated cells: HepG2 cells

  • Permeabilization: Required due to mitochondrial localization

  • Controls: Include isotype control to determine background staining

• Special considerations:

  • BCAT2 is primarily localized to mitochondria, requiring permeabilization for intracellular staining

  • For tissue analysis, optimal fixation time depends on tissue type and thickness

  • For dual staining, consider using antibodies raised in different host species to avoid cross-reactivity

  • When studying acetylation, pretreatment with NAM increases detectability of acetylated forms

Researchers should always perform sample-dependent titration to determine optimal conditions for their specific experimental system .

How can researchers effectively study BCAT2 interaction with key regulatory proteins?

Studying BCAT2 protein-protein interactions requires specialized approaches:

• Co-immunoprecipitation (Co-IP):

  • For studying interactions with acetyltransferases (CBP) and deacetylases (SIRT4)

  • For detecting ubiquitylation of BCAT2 following acetylation

  • Cell lysates should be prepared with mild detergents to preserve protein-protein interactions

  • Include proteasome inhibitors (MG132) when studying ubiquitylation

  • Include deacetylase inhibitors (NAM, TSA) when studying acetylation-dependent interactions

• His-BCAT2 pull-down assay:

  • Useful for verifying direct protein interactions

  • Has been successfully used to demonstrate direct interaction between BCAT2 and CBP

  • Requires purified recombinant proteins or tagged proteins expressed in suitable systems

• Proximity ligation assay (PLA):

  • Enables visualization of protein-protein interactions in situ

  • Particularly useful for studying interactions in specific subcellular compartments

  • Can detect transient interactions that might be missed by Co-IP approaches

• FRET/BRET approaches:

  • For studying dynamics of interactions in living cells

  • Requires fusion proteins with appropriate fluorescent/luminescent tags

  • Enables real-time monitoring of interactions in response to treatments

  • Particularly useful for studying the kinetics of BCAT2 interactions with regulatory proteins

• Mass spectrometry-based interactomics:

  • For unbiased discovery of novel BCAT2-interacting proteins

  • Can be combined with quantitative approaches to study how interactions change under different conditions

  • Requires careful optimization of sample preparation to preserve interactions

When studying BCAT2 interactions with CBP and SIRT4, researchers should consider how these interactions are modulated by BCAA availability, as evidence indicates that CBP and SIRT4 control BCAT2 K44 acetylation level in response to BCAA availability .