Recombinant Pongo abelii Abhydrolase domain-containing protein 16A (ABHD16A)

What is ABHD16A and what are its basic structural features in Pongo abelii?

ABHD16A (Abhydrolase domain-containing protein 16A) is a 63 kDa transmembrane protein containing 558 amino acid residues in Pongo abelii (Sumatran orangutan). It is also known as HLA-B-associated transcript 5 (BAT5). The protein contains a characteristic α/β hydrolase domain and four transmembrane regions located at residues 59-85, 91-113, 204-229, and 350-365 . The full amino acid sequence of Pongo abelii ABHD16A includes highly conserved functional domains, including the alpha/beta hydrolytic enzyme domain, acyltransferase motif HXXXXD, and lipase-like motifs GXSXXG . Similar to other ABHD family members, it possesses a typical α/β-hydrolase fold with 8 β-strands and 6 α-helices, where the hydrolytic enzyme active center is formed by histidine residues surrounded by helices and loops .

How is ABHD16A conserved across different species compared to Pongo abelii?

ABHD16A is highly conserved across multiple species, though with notable variations in protein length due to alternative splicing. While Pongo abelii ABHD16A is 558 amino acids long (UniProt ID: Q5R6S0), comparable to the human protein, significant differences exist in other species . Despite these length variations, the functional domains remain remarkably conserved, particularly the catalytic residues in the hydrolase domain . Phylogenetic analysis reveals that ABHD16A is genetically distant from other ABHD family members across species, suggesting unique evolutionary pressure to maintain its specific functions . Comparison of Pongo abelii ABHD16A with homologs from human, mouse, pig, and zebrafish shows conservation of key structural features while maintaining species-specific interaction capabilities, particularly with IFITM proteins .

What are the recommended storage and handling conditions for Recombinant Pongo abelii ABHD16A?

For optimal stability and activity, Recombinant Pongo abelii ABHD16A should be stored at -20°C, and for extended storage, conserved at -20°C or -80°C . The protein is typically supplied in a Tris-based buffer with 50% glycerol, optimized for stability . Researchers should avoid repeated freeze-thaw cycles, as this significantly reduces protein activity . Working aliquots can be stored at 4°C for up to one week . The shelf life depends on several factors including storage conditions and buffer composition, but generally, liquid preparations maintain integrity for approximately 6 months at -20°C/-80°C, while lyophilized forms remain stable for up to 12 months . When handling the protein, maintain sterile conditions and use appropriate buffer systems compatible with downstream applications.

What assays can be used to measure ABHD16A enzymatic activity?

Several methodological approaches are available for assessing ABHD16A enzymatic activity:

The APEGS assay is particularly valuable for studying ABHD16A's role in protein palmitoylation cycles, where palmitoylated proteins are labeled with methoxy polyethylene glycol maleimide (mPEG-Mal) and detected by Western blotting . When designing enzymatic assays, researchers should consider that ABHD16A shows higher specific activity with phosphatidylserine than with lysophospholipids or neutral lipids .

How can protein-protein interactions with ABHD16A be effectively studied?

Multiple complementary approaches can be employed to investigate ABHD16A interactions:

• Yeast Two-Hybrid (Y2H) Assay: This system has been successfully used to identify interactions between ABHD16A and IFITM proteins across species. For Pongo abelii ABHD16A, cotransformed yeast with potential interacting partners should be cultured on selective media (SD/−Leu/−Trp/−Ade/−His) to screen for positive interactions .

• Co-immunoprecipitation (Co-IP): This technique can confirm interactions identified through Y2H. When studying Pongo abelii ABHD16A, epitope tags (such as His-tag) can be leveraged for pull-down experiments, followed by Western blot detection of interacting partners .

• Bimolecular Fluorescence Complementation (BiFC): This method enables visualization of protein interactions in living cells, providing spatial information about where interactions occur within cellular compartments .

• Proximity Ligation Assay (PLA): Though not explicitly mentioned in the search results, this technique offers high sensitivity for detecting endogenous protein interactions with minimal background.

When investigating interactions, researchers should be mindful that ABHD16A shows species-specific interaction patterns, particularly with IFITM family proteins .

What enzymatic activities does ABHD16A possess and how do they compare across species?

ABHD16A demonstrates multiple enzymatic activities that appear conserved across species, including Pongo abelii:

• Acylglycerol Lipase Activity: ABHD16A functions as a lipase with preference for medium-chain and long-chain fatty acids, particularly long-chain unsaturated monoglycerides and 15-deoxy-Δ12,14-prostaglandin J2–2-glycerol ester (15d-PGJ2-G) .

• Phosphatidylserine Lipase Activity: The protein shows higher specific activity with phosphatidylserine than with hydrolyzing lysophospholipids, suggesting a specialized role in phospholipid metabolism .

• S-Depalmitoylase Activity: ABHD16A functions as a critical S-depalmitoylase in the palmitoylation/depalmitoylation cycle of IFITM proteins, directly affecting their localization and function .

Comparative studies suggest these enzymatic activities are well-conserved across mammals, with the ABHD16A from Pongo abelii likely showing similar substrate preferences and catalytic efficiencies to human ABHD16A . The conservation of catalytic sites, including the nucleophile centers (Ser, Cys or Asp) and the alpha/beta hydrolytic enzyme domain, supports functional conservation across species .

How does ABHD16A regulate the palmitoylation and function of IFITM proteins?

ABHD16A serves as a critical regulator in the palmitoylation/depalmitoylation cycle of IFITM proteins with significant implications for cellular antiviral responses. Research using the APEGS assay has demonstrated that ABHD16A functions as an S-depalmitoylase of IFITM proteins across human, pig, and mouse species . The interaction between ABHD16A and IFITMs affects the subcellular localization and antiviral functions of IFITM proteins .

Mechanistically, ABHD16A controls the palmitoylation state of IFITMs through direct interaction, with species-specific patterns: swine ABHD16A interacts with sIFITM1, sIFITM2, and sIFITM3; human ABHD16A primarily interacts with hIFITM1; while mouse ABHD16A interacts with mIFITM3 . By modulating the palmitoylation status of these proteins, ABHD16A regulates their membrane localization and consequently their ability to restrict viral entry and infection . This regulatory mechanism represents a potential target for broad-spectrum antiviral strategies through pharmacological control of ABHD16A activity.

What is the relationship between ABHD16A and immune regulation?

ABHD16A has significant connections to immune regulation through multiple mechanisms:

• Genomic Context: The gene location in the main histocompatibility complex (MHC) III gene cluster, alongside TNF and HSP70, strongly suggests immunomodulatory functions .

• Lysophosphatidylserine Regulation: ABHD16A regulates immunomodulatory lysophosphatidylserines (lyso-PSs), affecting the release of lipopolysaccharide-induced proinflammatory cytokines from macrophages .

• IFITM Protein Regulation: By controlling the palmitoylation status and function of IFITM proteins, ABHD16A influences antiviral immunity. This regulatory axis represents an important component of the innate immune response to viral infections .

• Prostaglandin Metabolism: ABHD16A functions as an immune-balancing regulator that catalyzes the hydrolysis of prostaglandin-glycerol (PG-G) in neutrophils, further supporting its role in inflammatory regulation .

• Disease Associations: Polymorphisms and haplotypes of ABHD16A show associations with the genetic predisposition to Kawasaki disease and coronary artery aneurysm, both conditions with significant immune and inflammatory components .

These findings collectively establish ABHD16A as an important mediator in immune and inflammatory processes, with potential implications for understanding and treating immune-related disorders.

How can ABHD16A be therapeutically targeted and what are the implications for disease treatment?

ABHD16A represents a promising therapeutic target with several potential intervention strategies:

• Selective Inhibitors: Several selective inhibitors of ABHD16A have been identified through comparative activity-based protein profiling analyses . These compounds could be developed to modulate ABHD16A's enzymatic activities in conditions where its overactivity contributes to pathology.

• Gene Expression Modulation: Controlling ABHD16A expression levels could provide another therapeutic approach, particularly in contexts where abnormal expression correlates with disease states.

• Targeting Protein-Protein Interactions: Disrupting specific interactions, such as those between ABHD16A and IFITM proteins, represents a more precise intervention strategy that could yield fewer off-target effects.

The therapeutic implications span multiple disease areas:

• Viral Infections: Pharmacological intervention targeting ABHD16A could enhance IFITM-mediated antiviral effects, potentially providing broad-spectrum protection against multiple viral pathogens .

• Inflammatory Disorders: Modulating ABHD16A activity could regulate lysophosphatidylserine levels and inflammatory cytokine production, offering potential benefits in inflammatory conditions .

• Neurodegenerative Diseases: Given the association between ABHD family proteins and neuroinflammation (particularly ABHD12 and PHARC disease), ABHD16A inhibition might have neuroprotective effects .

• Metabolic Disorders: The correlation between ABHD16A SNPs and metabolic traits (like back fat thickness in pigs) suggests potential applications in metabolic disease management .

What are the current challenges in ABHD16A research and future directions?

Despite significant progress, several challenges and knowledge gaps remain in ABHD16A research:

• Structural Characterization: Complete crystal structures of ABHD16A, particularly of Pongo abelii origin, would facilitate structure-based drug design and deeper understanding of its mechanism.

• Physiological Substrates: While in vitro enzymatic activities have been characterized, the full spectrum of physiologically relevant substrates in different cellular contexts requires further investigation.

• Regulatory Mechanisms: How ABHD16A itself is regulated at transcriptional, post-transcriptional, and post-translational levels remains insufficiently understood.

• Species-Specific Functions: Although ABHD16A is conserved across species, the functional implications of species-specific variations need further elucidation, particularly for using animal models in translational research.

Future research directions should focus on:

• Developing more potent and selective ABHD16A modulators with favorable pharmacokinetic properties

• Establishing conditional knockout models to study tissue-specific functions

• Investigating the role of ABHD16A in newly identified disease associations

• Exploring potential applications in immunotherapy and antiviral drug development

• Understanding the interplay between ABHD16A and other ABHD family members in complex physiological processes

How does ABHD16A interact with the broader ABHD protein family network in lipid metabolism?

ABHD16A functions within a complex network of ABHD family proteins that collectively regulate lipid metabolism and signaling:

• ABHD16A-ABHD12 Axis: A particularly important relationship exists between ABHD16A and ABHD12 in regulating lysophosphatidylserine levels. While ABHD16A generates lyso-PS by hydrolyzing PS, ABHD12 degrades lyso-PS, creating a dynamic regulatory system for these bioactive lipids .

• Complementary Substrate Specificities: Different ABHD family members show preferences for distinct lipid substrates, creating a comprehensive network for lipid metabolism regulation. For example, while ABHD16A preferentially hydrolyzes PS, ABHD6 acts as a monoacylglycerol hydrolase with different substrate preferences .

• Shared Structural Features: Most ABHD proteins contain the characteristic α/β hydrolase fold, though with variations in substrate binding regions that determine their specific functions .

• Disease Relevance: Mutations in ABHD12 cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract), highlighting how disruption of this network can lead to disease . The interrelationship between different ABHD proteins suggests that alterations in ABHD16A might influence pathways affected in ABHD12-related disorders.

• Evolutionary Conservation: Phylogenetic analysis indicates that while ABHD family members share common ancestry, ABHD16A is genetically distant from other members, suggesting specialized functions within the broader network .

Understanding these network interactions is crucial for developing targeted interventions that modify specific aspects of lipid metabolism without disrupting the entire system.

What evidence links ABHD16A to neurodegenerative and inflammatory diseases?

Multiple lines of evidence connect ABHD16A to neurodegenerative and inflammatory conditions:

• Regulation of Neuroinflammatory Lipids: ABHD16A's role in controlling lysophosphatidylserine levels directly impacts neuroinflammatory processes. Lysophosphatidylserines are bioactive lipids that influence microglial activation and neuroinflammation .

• ABHD12-ABHD16A Pathway: The interplay between ABHD16A and ABHD12 is particularly relevant, as ABHD12 mutations cause PHARC, a neurodegenerative condition. ABHD16A potentially influences pathways affected in this disorder through its opposing action in the same metabolic pathway .

• Kawasaki Disease Association: Genetic studies have identified associations between ABHD16A polymorphisms and haplotypes with Kawasaki disease and coronary artery aneurysm, demonstrating its relevance to inflammatory vascular conditions .

• MHC III Localization: ABHD16A's gene location in the MHC III region, alongside genes involved in inflammation like TNF, supports its potential role in inflammatory processes relevant to neurodegenerative diseases .

• IFITM Protein Regulation: Through its impact on IFITM proteins, ABHD16A may influence neuroinflammatory responses to viral infections, which have been implicated in some neurodegenerative conditions .

While direct causative relationships remain to be established, these associations suggest ABHD16A as a potential therapeutic target in neuroinflammatory and neurodegenerative disorders.

How might genetic variations in ABHD16A contribute to disease susceptibility?

Genetic variations in ABHD16A can influence disease susceptibility through several mechanisms:

• Single Nucleotide Polymorphisms (SNPs): Specific SNPs in ABHD16A have been associated with disease predisposition. For example, analysis of ABHD16A polymorphisms revealed associations with genetic predisposition to coronary artery aneurysm and Kawasaki disease .

• Alternative Splicing: Significant differences in the length of ABHD16A polypeptide chains across species and within species (such as human isoforms a and b having 558 and 525 amino acids, respectively) suggest variable splicing during post-transcriptional processing . These splicing variations might influence protein function and disease susceptibility.

• Expression Regulation: While not explicitly mentioned in the search results, variations in regulatory regions could affect ABHD16A expression levels, potentially contributing to disease states where abnormal enzyme activity affects lipid metabolism or immune regulation.

• Metabolic Traits: In Sus scrofa (pig), a correlation was observed between SNPs of the ABHD16A gene and back fat thickness, suggesting genetic variations might influence metabolic traits with potential relevance to human metabolic disorders .

These genetic variations provide important insights for personalized medicine approaches, potentially identifying individuals at higher risk for certain conditions or who might respond differently to therapeutics targeting ABHD16A pathways.