ZADH2 Human

What is ZADH2 and what are its basic molecular characteristics?

ZADH2, also known as PRG-3, is a zinc-binding alcohol dehydrogenase domain-containing protein 2 that belongs to the zinc-containing alcohol dehydrogenase family . The protein consists of 377 amino acids with a molecular mass of approximately 39kDa . The human ZADH2 gene maps to chromosome 18q22.3, a region that houses over 300 protein-coding genes within nearly 76 million bases .

The recombinant form of ZADH2 is typically produced as a single polypeptide chain containing 368 amino acids (positions 33-377) . Its full amino acid sequence includes characteristic motifs of zinc-binding alcohol dehydrogenases, suggesting a role in redox reactions that likely utilize zinc as a cofactor.

What expression systems are available for studying ZADH2?

Researchers have multiple options for expressing ZADH2 in laboratory settings:

Recombinant protein expression: ZADH2 human recombinant protein can be produced in E. coli expression systems. Commercially available recombinant ZADH2 is typically fused to a 23 amino acid His-tag at the N-terminus and purified by proprietary chromatographic techniques . The protein is generally formulated in phosphate buffer saline (pH 7.4) with 20% glycerol for stability .

Expression plasmids: Human ZADH2 cDNA clones are available in vectors such as pCMV6-XL5 with ampicillin selection markers (100 μg/mL) for bacterial propagation . These untagged clones have been fully sequenced to confirm the open reading frame, making them suitable for mammalian cell expression studies .

Storage considerations: For recombinant ZADH2, storage at 4°C is recommended if the entire vial will be used within 2-4 weeks. For longer periods, storage at -20°C is advised, with the addition of a carrier protein (0.1% HSA or BSA) to maintain stability . Multiple freeze-thaw cycles should be avoided to preserve protein integrity.

What antibodies and detection methods are available for ZADH2 research?

Anti-ZADH2 antibodies for research applications include:

Prestige Antibodies: Affinity-isolated polyclonal antibodies produced in rabbit are available for ZADH2 detection. These antibodies have been validated for immunohistochemistry and immunoblotting applications .

Recommended concentrations:

• Immunoblotting: 0.04-0.4 μg/mL

• Immunohistochemistry: 1:50-1:200 dilution

The antibodies are typically provided in buffered aqueous glycerol solutions and have been enhanced validated through recombinant expression techniques . The immunogen sequence used for antibody production corresponds to a specific region of the ZADH2 protein: "RLSPNFREAVTLSRDCPVPLPGDGDLLVRNRFVGVNASDINYSAGRYDPSVKPPFDIGFEGIGEVVALGLSASARYTVGQAVAYMAPGSFAEYTVVPASIATPVPS" .

How can researchers modulate ZADH2 expression for functional studies?

Several approaches are available for modulating ZADH2 expression in experimental systems:

siRNA knockdown: Small interfering RNA (siRNA) targeting ZADH2 can be used to reduce its expression in cellular models. Commercial siRNAs like sc-155428 are available for mouse models .

Expression plasmids: For overexpression studies, researchers can use expression vectors containing the ZADH2 cDNA sequence. Vectors like pCMV6-XL5 with the full ZADH2 sequence are commercially available .

Sequence considerations: When designing expression constructs or knockdown strategies, researchers should note that the RefSeq transcript and protein sequences for ZADH2 were derived from transcript and genomic sequences to maintain consistency with the reference genome . Clone variation with respect to the reference sequence (NM_175907.4) should be considered, such as the c=>t variation at position 225 noted in one commercial clone .

What is known about ZADH2's role in metabolic pathways?

While the complete functional characterization of ZADH2 remains ongoing, thermal proteome profiling studies have provided insights into its potential roles:

Prostaglandin metabolism: ZADH2 has been identified as a marker protein for prostaglandin metabolism pathways . Thermal proteome profiling studies have shown that ZADH2 is affected when these pathways are perturbed.

Phosphatidylinositol phosphate pathways: Mass spectrometry analysis of cells treated with the kinase inhibitor volasertib revealed that ZADH2 is involved in phosphatidylinositol phosphate metabolism pathways .

Immune response and fatty acid metabolism: Inhibition of ZADH2 is known to impact both immune responses and fatty acid metabolism . This suggests that ZADH2 may play a regulatory role at the intersection of these two crucial biological processes.

What is the significance of ZADH2 as an off-target of kinase inhibitors?

Recent research has highlighted ZADH2 as an important off-target of certain therapeutic compounds:

Volasertib off-target effects: Thermal proteome profiling (TPP) identified ZADH2 as one of approximately 200 potential off-targets of volasertib, an ATP-competitive small molecule PLK1 inhibitor that reached phase III clinical trials for acute myeloid leukemia .

Drug specificity: Importantly, ZADH2 was not affected by another PLK1 inhibitor called onvansertib, suggesting that ZADH2 is a true off-target specific to volasertib rather than a general consequence of PLK1 inhibition . This finding has significant implications for drug development and understanding the mechanism of side effects observed in clinical trials.

Clinical implications: The inhibition of off-targets including ZADH2 may explain some of the severe side effects seen in volasertib-treated patients that limited its clinical use . This highlights the importance of comprehensive off-target profiling during drug development.

How can thermal proteome profiling be applied to study ZADH2 interactions?

Thermal proteome profiling (TPP) has proven valuable for identifying ZADH2 interactions and can be further applied in research:

Methodology overview: TPP is based on the principle that binding of a ligand typically stabilizes a protein against thermal denaturation. In the context of ZADH2 research, this technique can:

• Identify small molecules that directly interact with ZADH2 by monitoring shifts in its thermal stability profile

• Discover proteins that associate with ZADH2 within specific pathways

• Reveal how ZADH2 stability changes under different cellular conditions

Application to drug discovery: The identification of ZADH2 as an off-target of volasertib demonstrates how TPP can be used to comprehensively profile drug interactions beyond the intended target . This approach is particularly valuable for understanding mechanisms of side effects and developing more specific inhibitors.

Experimental design considerations: When applying TPP to study ZADH2:

• Include appropriate controls, such as known ZADH2 ligands or structurally related compounds

• Consider dose-response experiments to establish binding affinities

• Combine with cellular assays to correlate thermal stability shifts with functional outcomes

What approaches can be used to investigate ZADH2's enzymatic function?

As a member of the zinc-containing alcohol dehydrogenase family, investigating ZADH2's enzymatic function requires specific experimental approaches:

Substrate screening: Researchers should consider testing potential substrates relevant to the pathways ZADH2 has been implicated in, particularly prostaglandin and fatty acid metabolism intermediates.

Recombinant protein assays: Purified recombinant ZADH2 protein can be used in enzymatic assays to:

• Determine cofactor requirements (likely NAD+/NADH or NADP+/NADPH)

• Establish optimal reaction conditions (pH, temperature, ionic strength)

• Measure kinetic parameters with candidate substrates

Structure-function analysis: The zinc-binding domain is critical for catalytic activity. Site-directed mutagenesis of predicted catalytic residues can help elucidate the enzymatic mechanism.

Metabolomics integration: Combining enzymatic assays with targeted metabolomics can help identify physiological substrates by monitoring changes in metabolite profiles when ZADH2 activity is modulated.

What are the current limitations in ZADH2 research?

Despite progress in understanding ZADH2, several significant research challenges remain:

Limited functional characterization: The precise enzymatic function and physiological substrates of ZADH2 remain incompletely characterized. While it belongs to the zinc-containing alcohol dehydrogenase family, its specific catalytic activities and preferred substrates in vivo need further investigation.

Regulatory mechanisms: How ZADH2 expression and activity are regulated under different physiological and pathological conditions remains largely unknown. Transcriptomic approaches, such as those used in studying induced pluripotent stem cells , may provide insights but have not specifically focused on ZADH2 regulation.

Structural information: Detailed structural data for ZADH2, particularly in complex with substrates or inhibitors, would greatly advance understanding of its function but is currently lacking in the literature.

Tissue-specific roles: While antibodies for tissue expression studies are available , comprehensive analysis of ZADH2's tissue-specific expression patterns and functions has not been fully documented.

What future research directions might advance understanding of ZADH2?

Several promising research directions could significantly advance ZADH2 knowledge:

Systems biology approaches: Integration of proteomics, metabolomics, and transcriptomics data could help position ZADH2 within broader cellular networks and clarify its role in prostaglandin and phosphatidylinositol metabolism pathways.

Structural biology: Determining the three-dimensional structure of ZADH2 through X-ray crystallography or cryo-electron microscopy would provide crucial insights into its mechanism and facilitate structure-based drug design for specific modulators.

Genetic association studies: Investigating potential associations between ZADH2 genetic variants and human diseases, particularly those involving chromosome 18q22.3, might reveal previously unrecognized clinical relevance.

Development of specific inhibitors: Creating selective ZADH2 inhibitors would provide valuable research tools and potentially lead to therapeutic applications, especially given its involvement in immune response and fatty acid metabolism pathways .

In vivo models: Generating and characterizing ZADH2 knockout or conditional knockout animal models would help elucidate its physiological functions in a whole-organism context.