ACOT8 Human

What is ACOT8 and what is its cellular localization?

ACOT8 is an enzyme encoded by the ACOT8 gene in humans. It functions as a peroxisomal thioesterase involved primarily in fatty acid oxidation rather than formation . Unlike some Type-I ACOTs with restricted expression patterns, ACOT8 demonstrates a broad tissue expression range in both mice and humans .

Methodologically, ACOT8's peroxisomal localization can be confirmed through:

• Immunofluorescence microscopy with peroxisomal markers

• Subcellular fractionation followed by Western blotting

• Identification of C-terminal peroxisome targeting sequences in the protein structure

ACOT8 belongs to the Type II thioesterases, which are members of the α/β-hydrolase protein family that includes various lipases and esterases . This structural classification provides insight into its evolutionary relationships with other metabolic enzymes.

What enzymatic activities does ACOT8 catalyze?

ACOT8 exhibits remarkably broad substrate specificity compared to other thioesterases. It hydrolyzes fatty acyl-CoAs to generate free fatty acids and CoA, with activity toward:

• Medium-to-long-chain acyl-CoAs

• Methyl-branched acyl-CoAs (including pristanoyl-CoA)

• Intermediates of β-oxidation

• Bile acid-CoAs

The enzyme's activity is notably inhibited by CoA, suggesting feedback regulation mechanisms . When overexpressed, ACOT8 can induce peroxisomal proliferation in both murine and human cell lines, indicating a potential role in organelle biogenesis or maintenance .

For experimental assessment of ACOT8 activity, researchers typically employ:

• Spectrophotometric assays tracking CoA release

• High-performance liquid chromatography (HPLC) for substrate/product quantification

• Radiolabeled substrate assays with thin-layer chromatography separation

How is ACOT8 expression regulated?

ACOT8 expression is dynamically regulated by metabolic conditions. Studies have demonstrated that:

• Fasting conditions upregulate ACOT8 expression

• PPARα (Peroxisome proliferator-activated receptor alpha) activation increases ACOT8 levels

• Multiple transcript variants encoding different isoforms exist for the ACOT8 gene

To investigate ACOT8 regulation, researchers commonly employ:

• Quantitative real-time PCR for mRNA quantification

• Western blot analysis for protein expression levels

• Luciferase reporter assays with the ACOT8 promoter

• Chromatin immunoprecipitation to identify transcription factor binding sites

These methods collectively provide insights into how ACOT8 expression adapts to changing metabolic demands across different tissues and conditions.

What is the molecular basis of ACOT8 interaction with HIV-1 Nef?

The interaction between ACOT8 and HIV-1 Nef represents a fascinating intersection of viral pathogenesis and cellular metabolism. Molecular characterization studies have revealed:

• High charge complementarity exists between Nef and ACOT8 surfaces

• ACOT8 regions Arg 45-Phe 55 and Arg 86-Pro 93 are critical for Nef association

• Lysine 91 plays a pivotal role, as K91S mutation completely abrogates the interaction with Nef

• When associated with ACOT8, Nef may be protected from degradation

• Nef increases the thioesterase activity of ACOT8

This interaction mediates Nef-induced down-regulation of CD4 in T-cells, potentially contributing to viral immune evasion strategies . Methodologically, this interaction has been studied through:

• Co-immunoprecipitation assays

• Immunofluorescence analyses

• In silico structural modeling

• Site-directed mutagenesis of key residues

• Functional assays measuring CD4 downregulation

Understanding this interaction may provide insights into novel therapeutic approaches targeting HIV-1 infection.

How does ACOT8 contribute to cancer progression and patient prognosis?

Recent investigations have established ACOT8 as a potential biomarker in multiple cancer types. Comprehensive analyses reveal:

• Significant upregulation of ACOT8 mRNA in breast cancer (BC), cervical squamous cell carcinoma (CESC), esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and uterine corpus endometrial carcinoma (UCEC)

• Elevated ACOT8 expression associates with unfavorable prognosis in LIHC, uveal melanoma (UVM), mesothelioma (MESO), BC, and prostate adenocarcinoma (PRAD)

• In breast cancer specifically, increased ACOT8 levels correlate with poorer prognosis in luminal A subtype

For investigating ACOT8's role in cancer, researchers employ:

• Differential expression analysis across tumor vs. normal tissues

• Kaplan-Meier survival analysis stratified by ACOT8 expression levels

• Correlation studies with established clinicopathological parameters

• Functional studies manipulating ACOT8 expression in cancer cell lines

What is the relationship between ACOT8 and immune cell infiltration in the tumor microenvironment?

The relationship between ACOT8 and tumor immunology represents an emerging research frontier. Studies have investigated:

• Correlations between ACOT8 expression and immune checkpoint molecules

• Associations with specific immune cell biomarkers

• Impact on the abundance of immune cell populations within the tumor microenvironment

This relationship can be explored through:

• Bioinformatic analysis using the TIMER algorithm and database

• Gene expression correlation studies with immune cell markers via GEPIA

• Flow cytometry assessment of immune populations in experimental models

• Immunohistochemistry of tumor tissues with dual staining for ACOT8 and immune markers

Understanding how ACOT8 influences the tumor immune landscape could inform immunotherapeutic approaches for cancers with high ACOT8 expression.

What experimental models are most appropriate for studying ACOT8 function?

Investigating ACOT8 requires careful selection of experimental models that recapitulate its physiological roles:

In vitro models:

• Cell lines with endogenous ACOT8 expression (hepatocytes, breast cancer lines)

• CRISPR/Cas9-mediated ACOT8 knockout cell lines

• Stable or inducible ACOT8 overexpression systems

• Primary cells from tissues with high ACOT8 expression

In vivo models:

• Acot8 knockout mouse models

• Tissue-specific conditional knockout models

• Transgenic ACOT8 overexpression mice

• Patient-derived xenografts for cancer studies

When designing experiments, researchers should consider ACOT8's broad tissue expression pattern and substrate specificity. Tissue-specific conditional models may be particularly valuable for distinguishing ACOT8's role in different physiological contexts.

How can researchers effectively study ACOT8's impact on lipid metabolism?

Given ACOT8's role in fatty acid metabolism, several complementary approaches can elucidate its functional impact:

• Metabolic flux analysis: Using isotope-labeled fatty acids to track metabolic fate

• Lipidomics profiling: Mass spectrometry-based quantification of lipid species alterations

• Fatty acid oxidation assays: Measuring oxygen consumption rates or radiolabeled CO₂ production

• Peroxisome function assessment: Analyzing catalase activity, peroxisome numbers, and morphology

• Gene expression analysis: Examining changes in related metabolic enzymes following ACOT8 manipulation

These approaches should ideally combine genetic manipulation (overexpression, knockdown, or mutation) with metabolic phenotyping to establish causality in observed effects.

What databases and bioinformatic tools are most valuable for ACOT8 research?

Several specialized databases and tools have proven particularly useful for ACOT8 research:

• TIMER database (https://cistrome.shinyapps.io/timer/): For analyzing immune cell infiltration in relation to ACOT8 expression

• GEPIA database (http://gepia.cancer-pku.cn/): For gene expression profiling and survival analysis using TCGA and GTEx data

• KM plotter (https://kmplot.com/analysis/): For survival analysis based on ACOT8 expression levels

• LinkedOmics (http://www.linkedomics.org/): For investigating ACOT8 interactions with signaling pathways

• UCSC Genome Browser: For examining ACOT8 gene structure and regulatory elements

These resources enable comprehensive analyses of ACOT8's expression patterns, clinical correlations, and functional relationships across diverse biological contexts.

How does ACOT8 expression correlate with clinicopathological features in breast cancer?

Detailed analyses have revealed significant associations between ACOT8 expression and various clinicopathological parameters in breast cancer:

• Cancer staging: ACOT8 expression increases with advancing cancer stages

• Lymph node status: Significant correlation with lymph node metastasis

• Molecular subtypes: Particularly relevant as a prognostic marker in luminal A breast cancer

• Patient demographics: Associations with race, age, and menstrual status

The UALCAN database analysis demonstrates progressive increases in ACOT8 expression from normal tissue through successive cancer stages, with stage 4 showing the highest levels . This correlation with disease progression supports ACOT8's potential utility as both a diagnostic and prognostic marker.

To study these associations, researchers employ:

• Immunohistochemistry of patient tissue samples

• Analysis of public gene expression datasets with linked clinical data

• Multivariate statistical approaches to control for confounding factors

• Survival analysis stratified by ACOT8 expression and clinicopathological features

What mechanisms might explain ACOT8's association with poor prognosis in cancer?

While the precise mechanisms remain under investigation, several potential pathways may explain ACOT8's association with aggressive cancer phenotypes:

• Altered lipid metabolism: Changes in fatty acid availability affecting cancer cell membrane composition, signaling pathways, or energy production

• Peroxisome dysfunction: Disruption of normal peroxisomal processes controlling reactive oxygen species or specialized lipid synthesis

• Immune modulation: Correlations with immune checkpoint molecules suggesting potential impact on anti-tumor immunity

• Interaction with oncogenic pathways: Potential cross-talk with established cancer signaling networks

Researchers investigating these mechanisms typically employ:

• Pathway enrichment analysis of genes correlated with ACOT8 expression

• Functional studies examining cancer hallmarks (proliferation, migration, invasion) following ACOT8 manipulation

• Metabolomic profiling to identify altered metabolic signatures

• Proteomic analyses to identify novel ACOT8 interaction partners in cancer contexts

How might ACOT8 serve as a therapeutic target in disease contexts?

Given ACOT8's implications in cancer progression and viral pathogenesis, several therapeutic strategies could be explored:

For cancer:

• Small molecule inhibitors targeting ACOT8 enzymatic activity

• Antisense oligonucleotides or siRNA approaches to reduce ACOT8 expression

• Combination therapies targeting ACOT8 alongside standard chemotherapeutics

• Immunotherapeutic approaches if ACOT8 modulates tumor immune microenvironments

For viral infections (particularly HIV):

• Peptide-based or small molecule disruptors of the ACOT8-Nef interaction

• Compounds that maintain ACOT8-Nef binding but block functional consequences

• Targeted alteration of ACOT8 expression or activity in HIV-infected cells

Development of such approaches requires:

• High-throughput screening platforms

• Structure-based drug design utilizing ACOT8 structural models

• Medicinal chemistry optimization of lead compounds

• Robust preclinical models for efficacy and toxicity assessment