Recombinant ATPase ASNA1 homolog (PC000665.03.0)

What is ASNA1 and how is it evolutionarily conserved across species?

ASNA1 (Arsenite-stimulated ATPase 1) is a highly conserved ATPase found across all three domains of life - eukarya, archaea, and prokarya. Originally identified as a homolog of the bacterial ArsA ATPase, it functions as the catalytic component of an oxyanion pump responsible for resistance to arsenicals and antimonials . The mouse homolog exhibits approximately 27% sequence identity with the bacterial ArsA ATPase .

ASNA1 is also known as TRC40 in some contexts, particularly when discussing its role in tail-anchored protein insertion into the endoplasmic reticulum . Homologs have been characterized in organisms ranging from bacteria to humans, with functional conservation demonstrated through cross-species rescue experiments. For example, human ASNA1 can rescue cisplatin hypersensitivity in C. elegans asna-1 mutants .

Methodological approach: Evolutionary conservation can be studied through:

• Sequence alignment and phylogenetic analysis using bioinformatics tools

• BLAST searches against genomic databases (e.g., GenBank, ToxoDB)

• Cloning and sequencing of ASNA1 from different organisms followed by comparative analysis

• Functional complementation assays across species

What experimental methods are most effective for ASNA1 gene amplification and cloning?

Based on established protocols, the following methodological workflow is recommended for ASNA1 amplification and cloning:

• Template preparation:

  • Extract total RNA from tissues/cells of interest

  • Synthesize first-strand cDNA using reverse transcriptase

• PCR amplification:

  • Design primers containing appropriate restriction sites (e.g., EcoRI and SalI as used for EtASNA1)

  • Recommended PCR conditions: Initial denaturation (95°C, 3 min); 32 cycles of denaturation (95°C, 45s), annealing (62°C, 45s), and extension (72°C, 2 min); final extension (72°C, 10 min)

• Product verification and purification:

  • Analyze PCR products using 1% agarose gel electrophoresis

  • Purify target bands using commercial gel purification kits (e.g., Qiagen)

• Cloning:

  • Subclone purified PCR products into an intermediate vector (e.g., pGEM-T-easy)

  • Transform into competent E. coli cells

  • Verify positive recombinants through restriction analysis and sequencing

• Expression vector construction:

  • Digest verified constructs with appropriate restriction enzymes

  • Ligate into expression vectors (e.g., pGEX-6P-1 for GST-fusion proteins)

This approach has been successfully applied to ASNA1 homologs from multiple species and provides a reliable framework for molecular characterization studies.

How can researchers effectively analyze ASNA1 sequence and structural features?

Comprehensive ASNA1 sequence analysis requires a multi-faceted bioinformatic approach:

• Primary sequence analysis:

  • BLAST searches against GenBank and organism-specific databases (e.g., ToxoDB for Eimeria species)

  • Determination of molecular mass and theoretical isoelectric point using ProtParam tools

• Structural feature prediction:

  • Signal peptide prediction using SignalP

  • Transmembrane domain identification with TMHMM

  • Protein motif detection using Motifscan

  • Identification of conserved ATPase domains and CXXC di-cysteine motifs critical for function

• Functional domain characterization:

  • Multiple sequence alignment to identify conserved regions across species

  • Homology modeling based on crystal structures of related proteins

  • Analysis of metal-binding sites that interact with substrates like arsenite and antimonite

Methodological significance: Thorough sequence analysis provides the foundation for experimental design, particularly for site-directed mutagenesis studies targeting functional domains and the interpretation of phenotypic outcomes in genetic studies.

What is the optimal protocol for recombinant ASNA1 protein expression and purification?

Based on published methodologies, the following optimized protocol is recommended for recombinant ASNA1 production:

• Expression vector construction:

  • Clone ASNA1 coding sequence into pGEX-6P-1 or similar expression vector

  • Transform into E. coli BL21(DE3) strain for protein expression

• Induction conditions optimization:

  • Grow transformed bacteria to OD600 of 0.6

  • Induce expression with 1.0 mM IPTG

  • Incubate for 6 hours post-induction

• Cell lysis and protein extraction:

  • Harvest cells by centrifugation

  • Disrupt cells by sonication

  • Analyze lysates by 12% SDS-PAGE to confirm expression

• Purification strategy:

  • For GST-tagged proteins: Use glutathione affinity chromatography

  • For MBP-fusions (as used with C. elegans ASNA-1): Use amylose resin purification

  • For specific applications: Gel extraction purification

• Quality control:

  • Verify protein purity by SDS-PAGE

  • Determine concentration using BCA protein assay

  • Store purified protein in aliquots at -20°C

Troubleshooting considerations:

• Expression level optimization may require testing different temperatures and induction times

• Protein solubility issues may be addressed by adjusting lysis buffer composition

• For functional studies, ensuring proper folding and activity is critical, particularly for ATPase assays

How does ASNA1 contribute to drug resistance mechanisms in parasites and cancer cells?

ASNA1 appears to play a significant role in multiple drug resistance contexts:

• Parasite drug resistance:

  • In Eimeria tenella, EtASNA1 is significantly upregulated in drug-resistant strains

  • Transcriptome analysis revealed log2 expression ratios of:

    • DZR/DS (diclazuril-resistant/drug-sensitive): 2.45

    • MRR/DS (maduramicin-resistant/drug-sensitive): 2.27

  • Expression levels increase proportionally with drug concentration exposure

• Cancer chemoresistance:

  • ASNA1 overexpression is observed in cisplatin-resistant melanoma and ovarian carcinoma cells

  • Experimental ASNA1 blockage increases sensitivity to cisplatin, carboplatin, and oxaliplatin

• Mechanistic models:

  • Drug efflux: Similar to its role in metalloid export, ASNA1 may participate in active drug efflux

  • Cell survival promotion: ASNA1 may enhance survival pathways, indirectly contributing to drug resistance

  • Stress response modulation: ASNA1's role in integrated stress response may help cells withstand drug-induced stress

Methodological approach: To investigate ASNA1's role in drug resistance:

• Compare expression in sensitive versus resistant cell lines (qRT-PCR, Western blotting)

• Perform genetic manipulation (knockdown/overexpression) followed by drug sensitivity assays

• Use model organisms like C. elegans for in vivo validation

• Evaluate combination therapy approaches targeting ASNA1 alongside conventional drugs

What expression patterns of ASNA1 are observed during development and in disease states?

ASNA1 exhibits distinct expression patterns with important developmental and pathological implications:

• Developmental expression:

  • Essential during early embryonic development (knockout causes lethality between E3.5-E8.5 in mice)

  • Particularly important in pancreatic progenitor cells, where conditional knockout leads to pancreatic agenesis

  • High expression in second-generation merozoites of E. tenella, potentially associated with parasite propagation and development

• Disease-associated expression:

  • Upregulated in drug-resistant cancer cell lines (melanoma, ovarian carcinoma)

  • Differentially expressed in drug-resistant parasite strains

• Cell-type specific functions:

  • In pancreatic multipotent progenitor cells (MPCs): Required for survival and differentiation

  • In parasite sporozoites: May be involved in invasion processes

Methodological considerations:

• Temporal expression patterns require stage-specific sampling and analysis

• Cell-type specific expression is best evaluated through immunohistochemistry or single-cell RNA-seq

• For developmental studies, conditional knockout approaches are necessary due to embryonic lethality of complete knockout

How does ASNA1's ATPase activity correlate with its biological functions across species?

ASNA1's ATPase activity demonstrates species-specific and substrate-dependent characteristics that correlate with its diverse biological functions:

Key findings across experimental systems:

• The ATPase activity is directly stimulated by metalloids in C. elegans ASNA-1

• In pancreatic development, the ATPase activity is specifically required for:

  • Maintaining Golgi integrity

  • Proper localization of syntaxin 5 (TA SNARE protein)

  • Prevention of p53-mediated apoptosis

• Rescue experiments demonstrate that the CXXC di-cysteine motif works in concert with ATPase activity to ensure cellular functions

Methodological implications: When designing experiments to study ASNA1 function, researchers must consider:

• Species-specific variations in ATPase activity and regulation

• The need for appropriate substrates when assessing enzymatic activity

• Potential differences in cofactor requirements across species

• The value of cross-species complementation to assess functional conservation

What are the molecular mechanisms by which ASNA1 mediates resistance to metalloids and platinum-based drugs?

The molecular mechanisms underlying ASNA1-mediated resistance appear to involve several interrelated pathways:

• Direct detoxification mechanisms:

  • Functions as part of an oxyanion pump that actively exports arsenicals and antimonials

  • Reduces intracellular concentration of toxic compounds to subtoxic levels

  • ATP hydrolysis likely provides energy for metalloid transport

• Structural evidence:

  • Arsenite (As(III)) and antimonite (Sb(III)) directly stimulate ATPase activity

  • Metal-binding sites likely evolved to recognize both naturally occurring metalloids and platinum-based drugs

  • The CXXC di-cysteine motif may provide critical coordination sites for metal binding

• Cellular pathways affected:

  • Protection against apoptosis through modulation of p53-mediated pathways

  • Maintenance of Golgi integrity and vesicular transport systems

  • Potential role in stress response pathways activated by metalloid exposure

Experimental approach for dissecting mechanisms:

• Structure-function analysis using site-directed mutagenesis of metal-binding sites

• Real-time tracking of labeled metalloids or drugs in cells with normal or reduced ASNA1

• Identification of ASNA1-interacting proteins under metalloid stress conditions

• Separation of distinct functions through domain-specific mutations and rescue experiments

How can researchers overcome challenges in studying ASNA1 given its essential role in development?

The embryonic lethality of ASNA1 knockout presents significant challenges for functional studies, requiring sophisticated experimental approaches:

• Conditional gene inactivation strategies:

  • Tissue-specific Cre-loxP systems to delete ASNA1 in specific cell types

  • Temporal control using inducible systems (e.g., Tamoxifen-inducible CreERT2)

  • Combined approach used successfully for pancreatic progenitor-specific deletion

• Partial loss-of-function approaches:

  • RNAi for transient and potentially incomplete knockdown

  • Hypomorphic alleles that reduce but don't eliminate function

  • Chemical inhibitors of ASNA1 activity (if available)

• Domain-specific functional analysis:

  • Generation of point mutations affecting specific functions

  • Rescue experiments with mutated forms of ASNA1 to identify critical domains

  • Successfully demonstrated in pancreatic development studies where ATPase activity and CXXC motif were separately evaluated

• Model organism advantages:

  • C. elegans provides valuable insights due to:

    • Ease of genetic manipulation

    • Transparency for in vivo imaging

    • Rapid development

    • Demonstrated conservation of ASNA1 function across species

Decision matrix for experimental approach selection:

What novel therapeutic applications might emerge from targeting ASNA1?

Based on current understanding of ASNA1 functions, several therapeutic applications warrant investigation:

• Overcoming chemoresistance in cancer:

  • ASNA1 inhibition sensitizes resistant cells to platinum-based drugs

  • Potential combination therapy using ASNA1 inhibitors alongside conventional chemotherapeutics

  • Biomarker potential: ASNA1 expression levels could predict platinum drug response

• Anti-parasitic strategies:

  • EtASNA1's differential expression in drug-resistant E. tenella suggests it as a target for novel anti-coccidial agents

  • Inhibiting ASNA1 might restore sensitivity to existing drugs in resistant parasites

  • Cross-resistance mechanisms suggest ASNA1 inhibitors might address multiple drug resistance issues simultaneously

• Metalloid toxicity treatment:

  • Understanding ASNA1's role in arsenite and antimonite detoxification provides insights for treating acute poisoning

  • Potential applications in environmental health contexts where metalloid exposure is common

• Development of specific inhibitors:

  • Structure-based drug design targeting ASNA1's ATPase domain

  • Allosteric modulators affecting metal binding sites

  • Peptide inhibitors disrupting protein-protein interactions required for ASNA1 function

Methodological considerations for therapeutic development:

• Target validation using genetic approaches before chemical intervention

• Careful assessment of toxicity given ASNA1's essential developmental roles

• Development of tissue-specific delivery to avoid systemic effects

• Evaluation of resistance mechanisms that might emerge against ASNA1-targeting drugs

What are the most critical unanswered questions about ASNA1 for future research?

Several compelling research directions emerge from current ASNA1 literature:

• Structure-function relationships:

  • Complete structural characterization of mammalian ASNA1

  • Identification of critical residues for specific functions

  • Understanding how ASNA1 recognizes diverse substrates including metalloids and drugs

• Regulatory mechanisms:

  • How ASNA1 expression and activity are regulated in different tissues

  • Post-translational modifications affecting ASNA1 function

  • Transcriptional control mechanisms explaining differential expression in drug resistance

• Protein interaction networks:

  • Comprehensive identification of ASNA1 binding partners

  • How these interactions change under different stress conditions

  • Potential for targeting specific protein-protein interactions therapeutically

• Translational applications:

  • Development of specific ASNA1 inhibitors

  • Biomarker potential in predicting treatment response

  • Therapeutic targeting strategies that avoid developmental toxicity