ABHD14B Human

What is ABHD14B and what enzyme family does it belong to?

ABHD14B is a member of the metabolic serine hydrolase family, one of the largest functional enzyme classes in mammals. This family comprises 1-2% of the total human proteome and uses a conserved nucleophilic serine residue in the active site to perform diverse hydrolytic reactions . Specifically, ABHD14B belongs to the AB hydrolase superfamily . The serine hydrolase family in humans includes over 250 members, with approximately 40% remaining unannotated in terms of their endogenous substrates and biological pathways .

What are the alternative names for ABHD14B?

ABHD14B is known by several alternative names in the literature:

• CCG1-interacting factor B (CIB)

• CCG1/TAF II250-interacting factor B

• Abhydrolase domain-containing protein 14B

• Putative protein-lysine deacylase ABHD14B

The name CIB derives from its discovery as an interacting protein partner of the histone acetyl-transferase (HAT) domain of the largest subunit for the TFIID transcription factor CCG1/TAF II250 through a yeast two-hybrid screen .

What is the basic function of ABHD14B?

ABHD14B functions as an atypical protein-lysine deacetylase (KDAC) that catalyzes the deacetylation of lysine residues using CoA as a substrate. This reaction generates acetyl-CoA and regenerates the free amine of protein-lysine residues . The enzyme has also demonstrated hydrolase activity towards various surrogate p-nitrophenyl (pNp) substrates, such as pNp-butyrate, pNp-acetate, and pNp-octanoate in vitro, with a strong preference for pNp-acetate . Additionally, ABHD14B may play a role in transcriptional regulation through its interaction with the transcription factor TFIID .

What is the protein structure of ABHD14B?

The three-dimensional structure of human ABHD14B was determined over a decade ago (PDB: 1IMJ) . The structure revealed that human ABHD14B possesses a nucleophilic serine residue (S111) as part of a non-canonical SxS motif (where x = any amino acid) . The human ABHD14B full-length protein consists of 210 amino acids with a molecular weight of approximately 22.451 kDa . The protein sequence of recombinant human ABHD14B includes the canonical AB hydrolase fold that characterizes this superfamily, with the conserved catalytic serine residue positioned to perform nucleophilic attacks on substrate molecules .

Where is ABHD14B expressed in human tissues?

Research has developed selective ABHD14B antibodies that have been used to map the cellular and tissue distribution of ABHD14B . When studying ABHD14B expression, researchers validate antibody specificity by testing tissues known to express ABHD14B positively and negatively . While the search results don't provide specific details about ABHD14B's tissue distribution pattern in humans, the availability of selective antibodies enables researchers to characterize its expression across different tissues and cell types. For mouse ABHD14B, the gene is located on chromosome 9, specifically at 9 F1 .

What is the mechanism of ABHD14B's lysine deacetylase activity?

ABHD14B employs a unique deacetylation mechanism distinct from traditional HDACs and sirtuins. It transfers an acetyl group from a post-translationally modified protein acetyl-lysine residue to a molecule of CoA to produce acetyl-CoA, while regenerating the free amine of protein lysine residues . This mechanism relies on the conserved nucleophilic serine residue (S111) in the active site, which is part of a non-canonical SxS motif .

The reaction likely proceeds through the following steps:

• The nucleophilic serine (S111) attacks the carbonyl carbon of the acetyl group on the acetylated lysine

• A tetrahedral intermediate forms

• The intermediate breaks down, transferring the acetyl group to CoA

• The free lysine residue is regenerated

This mechanism expands the repertoire of known activities within the metabolic serine hydrolase family and adds another enzyme family capable of deacetylating protein lysine residues, alongside the well-studied sirtuins and histone deacetylase (HDAC) enzymes .

How does ABHD14B differ from other known deacetylases like HDACs and Sirtuins?

ABHD14B represents a novel class of lysine deacetylases distinct from the two well-established families: HDACs and sirtuins:

These fundamental differences suggest that ABHD14B may have distinct biological functions and regulatory mechanisms compared to traditional deacetylases, potentially targeting different substrate proteins or operating in different cellular contexts .

What experimental approaches are used to validate ABHD14B's enzymatic activity?

Several complementary experimental approaches have been employed to validate ABHD14B's enzymatic activity:

• Recombinant protein expression and purification: The abhd14b gene was synthesized as a codon-optimized construct for expression in E. coli and cloned into the pET-30b(+) vector. The protein was then expressed in BL21(DE3) E. coli and purified for subsequent biochemical analysis .

• Site-directed mutagenesis: The S111A ABHD14B mutant was generated using standard Quik-Change site-directed mutagenesis protocols to confirm the role of the S111 residue in the enzyme's activity .

• Activity-based protein profiling (ABPP): ABHD14B was treated with the fluorophosphonate-rhodamine (FP-Rh) activity probe to assess its enzymatic activity. Protein titration experiments were conducted with varying concentrations of ABHD14B (both wild-type and S111A mutant) while keeping FP-Rh constant. Activity probe titration experiments were also performed with constant ABHD14B concentrations and varying FP-Rh concentrations .

• Surrogate substrate assays: ABHD14B's hydrolase activity was tested using various p-nitrophenyl (pNp) substrates, showing a strong preference for pNp-acetate .

• Cellular studies: ABHD14B was knocked down in a mammalian cell line to study its function in a cellular context .

• Structural modeling: Substrates were modeled into the enzyme active site to identify potential binding interactions and substrate preferences .

These multiple lines of evidence collectively support ABHD14B's function as a lysine deacetylase with a unique mechanism involving CoA as a co-substrate.

What are the potential biological pathways regulated by ABHD14B?

Based on current research, several potential biological pathways might be regulated by ABHD14B:

• Transcriptional regulation: ABHD14B was identified as a protein interactor of the histone acetyl-transferase (HAT) domain of the general transcription factor TFIID, suggesting a role in transcriptional regulation . It may activate transcription through modulating protein acetylation status .

• Metabolic regulation: As a lysine deacetylase that generates acetyl-CoA, ABHD14B might play a role in cellular metabolism by influencing acetyl-CoA levels, which is a central metabolite in numerous metabolic pathways including the TCA cycle and fatty acid synthesis .

• Protein acetylation dynamics: By removing acetyl groups from lysine residues, ABHD14B likely influences protein function, stability, or interactions that are regulated by acetylation/deacetylation cycles, potentially affecting various cellular processes .

• Developmental processes: While not specifically mentioned for ABHD14B, the related enzyme ABHD14A has been implicated in embryonic development of the cerebellum , suggesting that ABHD14B might also have developmental roles.

Researchers have mapped "prospective metabolic pathways that this enzyme might biologically regulate" , although specific pathways are not detailed in the provided information. Understanding these pathways represents an important direction for future research.

How can researchers generate and validate specific antibodies against ABHD14B?

Based on reported methodologies and standard practices, researchers can generate and validate specific antibodies against ABHD14B using the following approach:

• Antigen preparation:

  • Express and purify recombinant human ABHD14B protein as described in the literature

  • Alternatively, design synthetic peptides based on unique regions of ABHD14B that don't share homology with other ABHD family members

• Antibody production:

  • Immunize animals (rabbits, mice, or rats) with the purified ABHD14B protein or peptide

  • For monoclonal antibodies, harvest B cells from immunized animals and fuse them with myeloma cells to create hybridomas

  • For polyclonal antibodies, collect serum from immunized animals

• Antibody validation protocols:

  • Specificity testing: Test the antibodies on tissues known to express ABHD14B positively and negatively

  • Western blot: Confirm the antibody detects a protein of the expected molecular weight (~22.45 kDa)

  • Knockdown controls: Test the antibody in samples where ABHD14B has been knocked down to confirm specificity

  • Cross-reactivity testing: Ensure the antibody doesn't detect other ABHD family members

The development of "a much-needed, exquisitely selective ABHD14B antibody" has been reported , enabling the mapping of ABHD14B's cellular and tissue distribution. Commercial sources like Boster Biologics offer validated ABHD14B antibodies with publications and validation images , which may serve as valuable resources for researchers.

What methods are available for studying ABHD14B's substrate specificity?

Researchers can employ several complementary methods to investigate ABHD14B's substrate specificity:

• Surrogate substrate screening: Test ABHD14B's activity against synthetic substrates such as p-nitrophenyl esters with varying acyl chain lengths (e.g., pNp-acetate, pNp-butyrate, pNp-octanoate) . This provides initial insights into the enzyme's preference for different acyl moieties.

• Peptide library screening: Utilize libraries of acetylated peptides with varying sequences around the acetylated lysine to determine sequence preferences for deacetylation by ABHD14B.

• Proteomic approaches:

  • Mass spectrometry analysis of proteins with altered acetylation status upon ABHD14B overexpression or knockdown

  • Acetyl-lysine antibody enrichment followed by mass spectrometry to identify potential ABHD14B substrates

• In vitro deacetylation assays: Test purified candidate substrate proteins with ABHD14B and measure deacetylation using specific antibodies or mass spectrometry.

• Structural modeling: Model potential substrates into the enzyme active site to identify potential binding interactions as mentioned in reports .

• Activity-based protein profiling (ABPP): Use activity-based probes like fluorophosphonate-rhodamine (FP-Rh) to assess ABHD14B's activity under different conditions or in the presence of potential substrates .

By combining these approaches, researchers can build a comprehensive understanding of ABHD14B's substrate preferences, potentially leading to the identification of its endogenous biological substrates.

How can researchers perform site-directed mutagenesis on ABHD14B, and what key residues should be targeted?

Researchers can perform site-directed mutagenesis on ABHD14B using standard Quik-Change protocols as reported in the literature . The process involves:

• Designing primers containing the desired mutation

• PCR amplification using a high-fidelity polymerase

• DpnI digestion to remove template DNA

• Transformation into competent cells

• Sequence verification of the mutant construct

• Expression in BL21(DE3) E. coli for protein production

Key residues that should be targeted for site-directed mutagenesis include:

The S111A mutation has already been studied and serves as an important negative control in activity assays . Systematic mutagenesis of additional residues would provide valuable insights into ABHD14B's catalytic mechanism and substrate specificity.

What are the challenges in identifying endogenous substrates of ABHD14B?

Identifying the endogenous substrates of ABHD14B presents several significant challenges:

• Widespread protein acetylation: Protein acetylation affects thousands of proteins in the human proteome, creating a vast pool of potential ABHD14B substrates .

• Redundant deacetylase activities: Multiple deacetylase families (HDACs, sirtuins, and ABHD14B) may exhibit overlapping substrate specificities, making it difficult to isolate ABHD14B-specific effects .

• Context-dependent activity: ABHD14B's activity might be regulated by cellular conditions, subcellular localization, or interaction partners, complicating substrate identification in different contexts.

• Transient enzyme-substrate interactions: The interaction between ABHD14B and its substrates is likely transient, making it challenging to capture using traditional protein-protein interaction methods.

• Validation limitations: While ABHD14B demonstrates lysine deacetylase activity in vitro, additional experiments are required to confirm this activity in vivo .

• Structural constraints: The unique active site architecture of ABHD14B, with its non-canonical SxS motif, might impose specific structural requirements on substrates that are difficult to predict .

• Technical limitations: Until recently, there was a lack of specific antibodies and activity probes for ABHD14B, limiting the tools available for substrate identification .

Overcoming these challenges requires integrative approaches combining biochemical assays, proteomic techniques, structural studies, and cellular experiments to identify and validate physiologically relevant ABHD14B substrates.

How does the S111A mutation affect ABHD14B's enzymatic activity?

The S111A mutation in ABHD14B has significant effects on the enzyme's activity as studied through Activity-Based Protein Profiling (ABPP) with fluorophosphonate-rhodamine (FP-Rh) activity probes . This mutation:

The systematic comparison between wild-type ABHD14B and the S111A mutant in various assays provides strong evidence for the catalytic role of S111 in the enzyme's lysine deacetylase activity .

What techniques can be used to study ABHD14B's interaction with the transcription factor TFIID?

To investigate the interaction between ABHD14B and the transcription factor TFIID (originally identified through a yeast two-hybrid screen ), researchers can employ several complementary techniques:

• Co-immunoprecipitation (Co-IP): Using selective ABHD14B antibodies to pull down ABHD14B from cell lysates and detect associated TFIID components, or vice versa.

• Chromatin Immunoprecipitation (ChIP): To determine if ABHD14B co-localizes with TFIID at specific genomic loci, potentially indicating a role in transcriptional regulation at specific genes.

• Proximity Ligation Assay (PLA): This technique can visualize protein-protein interactions in fixed cells with high sensitivity by producing a fluorescent signal only when the two proteins are in close proximity.

• Fluorescence techniques:

  • Bimolecular Fluorescence Complementation (BiFC)

  • Förster Resonance Energy Transfer (FRET)

  • Fluorescence Correlation Spectroscopy (FCS)

• Biophysical methods:

  • Surface Plasmon Resonance (SPR)

  • Isothermal Titration Calorimetry (ITC)

  • Microscale Thermophoresis (MST)

• Functional assays: Assess how ABHD14B affects TFIID's functions, such as its ability to acetylate histones or initiate transcription.

• Structural studies: X-ray crystallography or cryo-electron microscopy could determine the structure of the ABHD14B-TFIID complex.

Given that ABHD14B may activate transcription , understanding its interaction with TFIID could reveal novel regulatory mechanisms connecting protein deacetylation to transcriptional control.