ACOT13 Human

What is ACOT13 and what is its basic biochemical function?

ACOT13 (Acyl-CoA thioesterase 13) is a member of the thioesterase superfamily that catalyzes the hydrolysis of Coenzyme A esters to free fatty acids plus CoA. The reaction proceeds as: CoA ester + H₂O → free acid + coenzyme A. In humans, ACOT13 co-localizes with microtubules and is essential for sustained cell proliferation . The protein forms a homotetramer and functions primarily as a medium- and long-chain acyl-CoA thioesterase . Multiple transcript variants encoding different isoforms have been identified for the human ACOT13 gene .

What experimental approaches should be used to characterize ACOT13's subcellular localization?

Accurate determination of ACOT13's subcellular localization requires a multi-method approach:

• Protease protection assays: Expose isolated mitochondria to proteases (trypsin or proteinase K) with/without membrane-disrupting agents (Triton X-100 or digitonin). ACOT13 follows the digestion pattern of matrix proteins like PDH, being digested only when Triton X-100 is present .

• Differential centrifugation: Separate cellular fractions through high-speed centrifugation, then perform immunoblot analysis on total homogenate, supernatant, and mitochondrial pellet fractions .

• Quantitative enrichment analysis: Calculate the ratio of ACOT13 in mitochondrial fractions versus total homogenate to determine compartmental distribution. Studies show that while some ACOT13 may be present in cytoplasm, the majority localizes to the mitochondrial matrix .

How does ACOT13 target to mitochondria despite low mitochondrial targeting sequence (MTS) prediction scores?

ACOT13 presents an interesting case of mitochondrial targeting despite relatively low MTS prediction scores:

• Mouse ACOT13 has MTS probability of ~30% (TargetP) or 39% (MitoProt II)

• Human ACOT13 isoform 1 has higher MTS scores (~52% TargetP; 63% MitoProt II)

• Human ACOT13 isoform 2 has lower scores (~12% TargetP; 50% MitoProt II)

This highlights limitations in current MTS prediction algorithms, which show significant false negative rates (60%, 56%, and 38% for TargetP, MitoFates, and MitoProt II respectively) when using a 65% probability cutoff . Many confirmed mitochondrial matrix proteins in MitoCarta2.0 have similarly low MTS predictions, including ribosomal proteins MRPS22, MRPL42, and MRPL48 . Searches for internal targeting sequences by sequential N-terminal shortening have not revealed alternative targeting motifs with probability >20% .

How can researchers experimentally distinguish matrix versus membrane localization of ACOT13?

To definitively determine ACOT13's submitochondrial localization, researchers should use:

• Protease protection assays with differential membrane disruption:

  • Tom20 (outer membrane marker): digested by protease alone

  • Tim23 (inner membrane marker): requires digitonin for digestion

  • PDH (matrix marker): only digested with Triton X-100

  • ACOT13: follows PDH pattern in liver, kidney, and heart mitochondria

• Quantitative distribution analysis comparing ACOT13 with established markers of different submitochondrial compartments across tissue types .

How does ACOT13 expression vary across different tissues and what is the significance?

ACOT13 shows tissue-specific expression patterns that differ from other mitochondrial acyl-CoA thioesterases:

• ACOT13 is expressed in multiple tissues including liver, heart, kidney, and skeletal muscle .

• Unlike ACOT9, which shows higher expression in glycolytic muscle (white gastrocnemius) compared to oxidative muscles, ACOT13 expression is similar across different muscle fiber types (glycolytic and oxidative) .

• When analyzing skeletal muscle, it's important to distinguish between soleus (highly oxidative), white quadriceps (glycolytic), and red gastrocnemius (mixed oxidative) .

The distinct tissue expression pattern suggests ACOT13 may have tissue-specific functions rather than being functionally redundant with other mitochondrial ACOTs .

What is the relationship between ACOT13 and other mitochondrial thioesterases?

ACOT13 is one of multiple acyl-CoA thioesterases found in mitochondria:

• At least five ACOTs localize to the mitochondrial matrix: ACOT2, ACOT7, ACOT9, ACOT13, and ACOT15 .

• These enzymes show distinct tissue expression patterns, substrate preferences, and regulatory properties, suggesting they occupy different functional niches rather than being redundant .

• To determine the quantitative contribution of specific ACOTs to total thioesterase activity, researchers have developed approaches using tissue-specific knockout models (e.g., ACOT2 depletion models) .

• When studying ACOT13, researchers should consider the entire complement of ACOTs in the tissue of interest to understand potential compensatory mechanisms .

What antibodies and validation methods are recommended for ACOT13 detection?

For reliable ACOT13 detection, researchers should:

• Use validated antibodies such as HPA019881 (Atlas Antibodies), which has been tested in human kidney and skeletal muscle tissues with corresponding RNA-seq validation .

• Confirm antibody specificity using ACOT13 knockout or knockdown controls .

• Include appropriate controls when performing Western blotting:

  • Positive controls from tissues with known high ACOT13 expression

  • Loading controls appropriate for mitochondrial proteins (e.g., VDAC, Complex I subunits)

  • Size verification using recombinant ACOT13 protein

• For immunohistochemistry, verify staining patterns correlate with mRNA expression data and include negative controls (no primary antibody, isotype controls) .

How should researchers measure ACOT13 enzymatic activity?

To accurately measure ACOT13 activity:

• Isolate highly purified mitochondria through differential centrifugation and density gradient purification to minimize contamination with cytosolic thioesterases .

• Use spectrophotometric assays coupling CoA release to a detectable reaction (e.g., with DTNB).

• Test multiple acyl-CoA substrates of varying chain lengths to determine substrate specificity.

• Include controls for non-enzymatic hydrolysis and background activity.

• When comparing activities across tissues, normalize to markers of mitochondrial content (e.g., citrate synthase activity).

• To distinguish ACOT13 activity from other thioesterases, use recombinant enzyme assays or ACOT13-depleted samples as controls .

How does ACOT13 contribute to fatty acid metabolism and energy homeostasis?

ACOT13 plays important roles in regulating intracellular levels of acyl-CoAs, which affects multiple aspects of cellular metabolism:

• By hydrolyzing acyl-CoA esters, ACOT13 influences substrate availability for β-oxidation and other metabolic pathways.

• Acyl-CoAs function as allosteric regulators of key metabolic enzymes including acetyl-CoA carboxylase, hexokinase IV, and citrate condensing enzyme; ACOT13 can thus indirectly regulate these pathways .

• Long-chain acyl-CoAs also regulate ATP-sensitive potassium channels and Calcium ATPases, potentially affecting insulin secretion and calcium homeostasis .

• Research indicates ACOT13 (THEM2) plays a role in adaptive thermogenesis in mice, suggesting involvement in energy expenditure regulation .

What is the relationship between ACOT13 and PC-TP/StarD2?

A significant research question concerns the interaction between ACOT13 and PC-TP (Phosphatidylcholine Transfer Protein, also known as StarD2):

• Researchers have investigated whether StarD2 co-localizes with ACOT13 in the mitochondrial matrix using protease protection assays .

• This potential interaction may coordinate functions in lipid metabolism and transport.

• To study this relationship, researchers should employ:

  • Co-immunoprecipitation studies

  • Subcellular co-localization analysis

  • Functional assays to determine whether the proteins influence each other's activities

How can researchers distinguish ACOT13-specific functions from other mitochondrial thioesterases?

To differentiate ACOT13's specific roles from other ACOTs:

• Develop tissue-specific conditional knockout models to avoid developmental compensation seen in global knockouts.

• Perform substrate specificity profiling using comprehensive acyl-CoA panels to identify unique activity signatures.

• Use proteomics to determine relative abundance of each ACOT in specific tissues and submitochondrial compartments.

• Generate catalytically inactive ACOT13 mutants to separate structural from enzymatic functions.

• Create mathematical models incorporating kinetic parameters of all relevant ACOTs to predict their relative contributions under different physiological conditions .

What emerging research directions are important for ACOT13?

Key emerging research areas include:

• Investigation of ACOT13's role in microtubule association and cell proliferation in humans .

• Further characterization of ACOT13's role in adaptive thermogenesis and metabolic disease models .

• Exploration of potential post-translational modifications and allosteric regulators affecting ACOT13 activity.

• Analysis of ACOT13 expression and function in pathological states involving altered lipid metabolism.

• Systematic comparison of human ACOT13 isoforms and their potentially distinct functions and subcellular distributions .

What are common pitfalls in ACOT13 research and how can they be avoided?

Researchers should be aware of these common challenges:

• Mitochondrial targeting sequence prediction: Standard algorithms show high false negative rates; experimental localization studies are essential rather than relying solely on prediction tools .

• Antibody cross-reactivity: Careful validation with knockout controls is crucial as ACOT family members share sequence similarity.

• Sample preparation: Preserve ACOT13 activity by avoiding freeze-thaw cycles and maintaining appropriate buffer conditions.

• Activity assays: Control for non-enzymatic hydrolysis of acyl-CoA substrates and background activity from other thioesterases.

• Functional redundancy: Design experiments to account for potential compensation by other ACOTs when ACOT13 is depleted .

What experimental design considerations are crucial for ACOT13 functional studies?

When designing ACOT13 experiments:

• Include multiple tissues in comparative studies, as ACOT13's function may be tissue-specific.

• When using skeletal muscle, distinguish between glycolytic and oxidative fiber types .

• Consider metabolic state (fed, fasted) and environmental conditions (temperature) that may affect ACOT13 expression and activity.

• Include appropriate timepoints to capture both acute and chronic adaptations.

• When manipulating ACOT13 levels, measure expression of other ACOTs to identify compensatory changes.

• For thermogenesis studies, include thermoneutral controls and measure multiple parameters beyond gene expression (e.g., metabolic rate, core temperature) .