HMGCS2 Antibody, HRP conjugated

What is HMGCS2 and what is its fundamental biological function?

HMGCS2 is a mitochondrial enzyme that catalyzes the first irreversible step in ketogenesis. The canonical HMGCS2 protein is 508 amino acids in length with a molecular weight of 56.6 kDa and primarily localizes to mitochondria. It functions in identical protein binding and hydroxymethylglutaryl-CoA synthase activity, playing a crucial role in metabolism. HMGCS2 is a member of the HMG-CoA synthase protein family and is also known as hydroxymethylglutaryl-CoA synthase, mitochondrial or 3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial) . While its primary role is in ketone body synthesis during fasting or carbohydrate restriction, recent research has revealed HMGCS2 may have additional functions in cancer progression through non-metabolic pathways .

How does HMGCS2 contribute to the process of ketogenesis?

HMGCS2 catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form HMG-CoA, which represents the first rate-limiting and irreversible step in ketogenesis . This enzymatic reaction is critical during periods of low carbohydrate availability when the body shifts to using fatty acids as an alternative energy source. After HMG-CoA formation, this intermediate can follow two distinct pathways: it can be converted by HMG-CoA reductase (HMGCR) into mevalonate in the cholesterol synthesis pathway, or it can proceed through the ketogenesis pathway to form ketone bodies (acetoacetate, β-hydroxybutyrate, and acetone) . The ketogenic pathway becomes particularly important during fasting states when ketone bodies serve as alternative energy substrates for tissues like the brain.

What are the advantages of using HRP-conjugated antibodies for HMGCS2 detection?

HRP-conjugated antibodies for HMGCS2 detection offer several methodological advantages in research applications. The horseradish peroxidase enzyme provides exceptional signal amplification properties, significantly increasing detection sensitivity in various immunoassays. This is particularly valuable when studying HMGCS2 in tissues with variable expression levels. The enzyme's stability allows for extended storage and consistent performance across experiments. Additionally, HRP-conjugated antibodies enable versatile detection methods including chromogenic, chemiluminescent, and fluorescent visualization depending on the substrate used, facilitating adaptation to different experimental requirements. For immunohistochemical applications, HRP-conjugated detection systems have been successfully used to visualize HMGCS2 in paraffin-embedded human liver tissue sections .

What applications are most suitable for HMGCS2 antibodies?

HMGCS2 antibodies have been validated for multiple research applications. According to manufacturer specifications, antibodies like the rabbit polyclonal HMGCS2 antibody (ab236667) are suitable for immunohistochemistry on paraffin-embedded sections (IHC-P) and immunocytochemistry/immunofluorescence (ICC/IF), with confirmed reactivity to human samples . Researchers have successfully employed HMGCS2 antibodies in Western blot analysis using liver lysates to detect the approximately 56.6 kDa protein . In cancer research, these antibodies have been instrumental in immunoprecipitation experiments to study protein-protein interactions, particularly the association between HMGCS2 and PPARα . When selecting an HMGCS2 antibody, researchers should consider the specific application needs, species reactivity, and whether HRP conjugation is beneficial for their detection method.

How can researchers validate the specificity of HMGCS2 antibodies for experimental use?

Validating HMGCS2 antibody specificity requires a multi-faceted approach. First, researchers should perform Western blot analysis using positive control tissues (liver samples are ideal as they express high levels of HMGCS2) and negative control tissues or cell lines with minimal expression. A specific HMGCS2 antibody should detect a single band at approximately 56.6 kDa . Second, implement genetic validation through HMGCS2 knockdown or knockout models; the antibody signal should correspondingly decrease or disappear in these systems. Third, conduct peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific binding. For immunohistochemical applications, include appropriate tissue controls and compare staining patterns with known HMGCS2 expression profiles. For HRP-conjugated antibodies specifically, researchers should carefully titrate antibody concentrations to optimize signal-to-noise ratios and include isotype controls to identify any non-specific binding attributed to the conjugation process.

What are the optimal experimental conditions for immunohistochemical detection of HMGCS2?

Successful immunohistochemical detection of HMGCS2 requires careful optimization of multiple parameters. Based on validated protocols, researchers should prepare paraffin-embedded tissue sections following standard histological procedures. Antigen retrieval is critical and should be performed using high-pressure treatment in citrate buffer (pH 6.0) to expose epitopes that may be masked during fixation. Effective blocking consists of applying 10% normal goat serum for 30 minutes at room temperature to minimize non-specific binding. The primary antibody (such as ab236667) should be diluted to 1/400 in 1% BSA solution and incubated at 4°C overnight for optimal binding . Detection systems utilizing biotinylated secondary antibodies followed by HRP-conjugates have shown excellent results. The visualization step can employ DAB (3,3'-diaminobenzidine) or other chromogens compatible with HRP. This protocol has been successfully applied to human liver tissue, which serves as an excellent positive control for HMGCS2 expression .

How does HMGCS2 expression correlate with cancer progression and patient prognosis?

HMGCS2 expression exhibits significant correlations with cancer progression and patient outcomes in multiple cancer types. In colorectal cancer (CRC), higher HMGCS2 mRNA expression is strongly associated with advanced TNM staging (P = 0.009), lymph node metastasis (P < 0.001), and disease recurrence (P = 0.002) . Similar patterns appear in oral squamous cell carcinoma (OSCC), where elevated HMGCS2 mRNA correlates with advanced TNM staging (P = 0.029), lymph node metastasis (P = 0.030), and recurrence (P = 0.0014) . These correlations translate to significant survival differences: CRC patients with low HMGCS2 expression (n = 55) demonstrated a 95.1% five-year survival rate compared to only 38.2% for those with high expression (n = 57; P < 0.001) . The table below summarizes these clinical correlations:

Furthermore, comparative analysis of paired samples revealed that HMGCS2 mRNA expression was significantly lower in normal tissue compared to cancerous tissue in both CRC (P = 0.042) and OSCC (P = 0.037) , suggesting its specific upregulation during carcinogenesis.

What is the molecular relationship between HMGCS2, PPARα, and Src in cancer metastasis?

HMGCS2 promotes cancer metastasis through a novel mechanism involving direct interaction with PPARα (peroxisome proliferator-activated receptor alpha) and subsequent activation of Src signaling. Immunoprecipitation-Western analysis has demonstrated that HMGCS2 physically interacts with PPARα, with the HMGCS2/PPARα complex being more abundant in HMGCS2-stable transfectants than in control clones in both SW480 (colorectal cancer) and Cal27 (oral cancer) cell lines . Subcellular fractionation experiments revealed that HMGCS2 localizes to both cytoplasmic and nuclear compartments, with the HMGCS2/PPARα complex particularly enriched in the nuclear fraction .

How can researchers differentiate between ketogenesis-dependent and independent functions of HMGCS2?

Distinguishing between the metabolic (ketogenesis-dependent) and non-metabolic functions of HMGCS2 requires sophisticated experimental approaches. First, researchers should employ domain-specific mutational analysis, creating HMGCS2 constructs with targeted mutations in catalytic domains that abolish ketogenic activity while preserving protein structure for potential protein-protein interactions. The functional consequences of these mutations should be compared with wild-type HMGCS2 in both metabolic assays (measuring ketone body production) and cellular phenotypic assays (such as migration, invasion, and proliferation).

Second, subcellular localization studies are essential, as HMGCS2's ketogenic function occurs in mitochondria, while its interaction with PPARα involves nuclear translocation . Immunofluorescence microscopy and subcellular fractionation can identify non-mitochondrial pools of HMGCS2 that likely mediate ketogenesis-independent functions. Third, metabolic manipulation approaches, such as using inhibitors of other ketogenic enzymes or altering cellular metabolic states, can help determine if HMGCS2's effects on cell phenotypes persist when ketogenesis is disrupted. Finally, protein interaction network analysis through techniques like proximity labeling, co-immunoprecipitation coupled with mass spectrometry, or yeast two-hybrid screening can identify HMGCS2 binding partners beyond the ketogenic pathway, as exemplified by the discovery of the HMGCS2-PPARα interaction .

What are the known pathogenic mutations in HMGCS2 and how do they affect protein function?

Functional analysis has identified several pathogenic variants in the HMGCS2 gene associated with HMGCS2 deficiency, a rare metabolic disorder. Five novel mutations—M235T, V253A, G219E, S392L, and R500C—have been characterized using both eukaryotic and bacterial expression systems to determine their effects on protein function . The M235T, S392L, and R500C mutations resulted in complete loss of enzymatic activity in purified protein assays, indicating these variants produce catalytically inactive enzymes . The V253A variant retained some residual enzymatic activity, suggesting a partial loss of function that may correlate with a milder clinical phenotype . The G219E variant demonstrated protein instability in transient expression experiments, indicating this mutation likely affects protein folding or stability rather than catalytic function directly .

Clinical data from patients with these mutations reveal the consequences of HMGCS2 deficiency, characterized by hypoketotic hypoglycemia and metabolic acidosis:

These findings demonstrate how specific mutations affect HMGCS2 function and contribute to understanding genotype-phenotype correlations in this disorder .

How can researchers establish reliable cellular models for studying HMGCS2 function?

Establishing reliable cellular models for HMGCS2 functional studies requires strategic selection of appropriate systems and genetic manipulation approaches. For overexpression studies, researchers should generate stable cell lines expressing wild-type or mutant HMGCS2 through transfection or viral transduction methods, as demonstrated with SW480 and Cal27 cell lines . These models are valuable for assessing gain-of-function phenotypes and structure-function relationships. For loss-of-function studies, RNA interference (shRNA) or CRISPR-Cas9 technologies can effectively reduce or eliminate HMGCS2 expression, as shown with DLD-1/shHMGCS2 and SAS/shHMGCS2 transfectants .

To study specific mutations, site-directed mutagenesis should be employed to create constructs with disease-associated variants, as described in functional analyses of HMGCS2 mutations . For biochemical and enzymatic studies, bacterial expression systems can be utilized to produce and purify wild-type and mutant HMGCS2 proteins for direct measurement of enzymatic activity . Reporter assay systems incorporating promoter constructs (like the Src promoter) provide valuable tools for studying HMGCS2's role in transcriptional regulation . When selecting cell lines, researchers should consider endogenous HMGCS2 expression levels and the presence of relevant interacting partners such as PPARα to ensure physiological relevance of their models.

What are the best practices for quantifying HMGCS2 expression levels in patient samples?

Accurate quantification of HMGCS2 expression in patient samples requires rigorous methodological approaches. For mRNA analysis, real-time quantitative RT-PCR represents the gold standard, as successfully employed in studies of colorectal cancer (n = 112) and oral squamous cell carcinoma (n = 140) . Researchers should collect paired tumor and adjacent normal tissue samples whenever possible to enable direct comparative analysis. Careful selection of stable reference genes for normalization is essential to ensure reliable quantification across diverse tissue samples.

For protein-level analysis, immunohistochemistry with optimized protocols (as detailed in section 2.2) provides spatial information about HMGCS2 expression patterns. Standardized scoring systems should be implemented, with multiple independent observers to ensure reproducibility. For more precise quantification, Western blot analysis with densitometry or enzyme-linked immunosorbent assays (ELISAs) may be employed if suitable antibodies are available.

Statistical analysis should include determination of appropriate cutoff values for categorizing high versus low expression; receiver operating characteristic (ROC) curve analysis represents a robust approach for establishing these thresholds . For prognostic evaluation, Kaplan-Meier survival analyses should be performed to assess correlations between HMGCS2 expression and patient outcomes, with multivariate analyses to determine independence from established prognostic factors . This comprehensive approach enables meaningful correlation of HMGCS2 expression with clinical parameters and patient outcomes.