Recombinant Mouse Archaemetzincin-1 (Amz1)

What is Mouse Archaemetzincin-1 and how does it relate to the archaemetzincin family?

Mouse Archaemetzincin-1 (Amz1) belongs to the archaemetzincin family of metalloproteases, which represents a unique evolutionary bridge between archaeal and vertebrate proteolytic systems. This family is characterized by a distinctive catalytic domain containing a zinc-binding motif and conserved cysteine residues not found at equivalent positions in other metalloproteases. Phylogenetic analysis indicates that archaemetzincins have undergone a complex evolutionary process involving lateral gene transfer, gene loss, and genetic duplication events .

The archaemetzincin family is widely distributed in Archaea and vertebrates but notably absent in many model organisms from bacteria to nematodes. Mouse Amz1 shares significant homology with human AMZ1, with approximately 62% sequence identity in key regions . This conservation suggests important functional roles that have been maintained throughout vertebrate evolution.

What are the key structural features that distinguish Mouse Amz1 from other metalloproteases?

Mouse Amz1, like its human counterpart, contains several distinctive structural features:

• A catalytic domain with the core motif HEXXHXXGX3CX4CXMX17CXXC

• An archetypal zinc-binding site critical for enzymatic activity

• A characteristic methionine residue found in all metzincins

• Four conserved cysteine residues absent in other metalloproteases

The zinc-binding histidine residues and catalytic glutamate are particularly crucial for proteolytic function, as demonstrated through mutational studies in related archaemetzincins. For instance, mutations in the corresponding residues in archaeal AMZ-tk (H182D, H186D, H192D, and E183Q) significantly impact enzymatic activity .

What expression patterns does Mouse Amz1 exhibit in different tissues?

While the search results don't provide specific expression data for mouse Amz1, human AMZ1 shows a distinctive tissue distribution pattern with predominant expression in liver and heart, with lower but detectable levels in other tissues . The mouse ortholog likely follows a similar expression pattern given the conservation between species, though researchers should verify this through Northern blot analysis or quantitative PCR in mouse tissues.

Expression patterns can provide valuable insights into potential physiological functions. For example, the high expression in liver may suggest roles in protein processing or degradation relevant to metabolic functions.

What are the optimal conditions for assessing Mouse Amz1 proteolytic activity in vitro?

Based on studies with related archaemetzincins, researchers should consider the following parameters when working with recombinant Mouse Amz1:

The presence of zinc ions is critical for Amz1 activity, as the enzyme is a zinc-dependent metalloprotease. Chelating agents like EDTA abolish enzymatic activity by removing essential zinc ions from the catalytic site .

How can I produce and purify active Recombinant Mouse Amz1 for experimental use?

Production of active recombinant Mouse Amz1 can be achieved using prokaryotic expression systems such as Escherichia coli. Based on successful protocols for human AMZ1 and archaeal AMZ-tk:

• Clone the full-length mouse Amz1 coding sequence into an appropriate expression vector (e.g., pET system)

• Transform into E. coli BL21(DE3) or similar expression strain

• Induce protein expression (typically with IPTG) under optimized conditions

• Lyse cells and purify using affinity chromatography (His-tag or other suitable tag)

• Confirm protein purity by SDS-PAGE and Western blotting

• Verify enzymatic activity using standard protease assays (e.g., casein degradation)

To ensure proper folding and activity of the recombinant protein, supplementation with zinc during purification and storage buffers may be beneficial. Additionally, researchers should consider removing any fusion tags if they interfere with enzymatic activity, although this requires careful validation.

What substrates should be used to assess Mouse Amz1 proteolytic activity?

Several substrates can be used to assess the proteolytic activity of Mouse Amz1:

• Casein: A general protease substrate useful for initial activity screening

• Neurogranin: Human AMZ1 exhibits aminopeptidase activity against neurogranin in vitro

• Synthetic peptide substrates: Custom peptides containing known cleavage motifs

When setting up activity assays, it's important to note that Mouse Amz1 does not hydrolyze all peptide substrates. For example, human AMZ1 does not hydrolyze angiotensin-2 , suggesting substrate specificity that should be considered when designing experiments.

Proteolytic activity can be quantified by measuring:

• Release of chromogenic or fluorogenic groups from synthetic substrates

• Appearance of proteolytic fragments by SDS-PAGE

• Release of amino acids or peptides detectable by spectrophotometry at 280 nm

How can mutations in catalytic residues be designed to study Mouse Amz1 function?

Site-directed mutagenesis of key catalytic residues provides valuable insights into the structure-function relationship of Mouse Amz1. Based on studies with archaeal AMZ-tk, the following mutations would be informative:

• Zinc-binding histidines: Mutating conserved histidine residues in the HEXXH motif to aspartate (H→D) would disrupt zinc binding

• Catalytic glutamate: Substituting the catalytic glutamate with glutamine (E→Q) would maintain the residue size but eliminate the catalytic capability

• Conserved cysteines: Mutations in the conserved cysteine residues would help understand their role in structural stability

A systematic mutagenesis approach should include:

The mutant proteins should be expressed, purified, and characterized using the same methodologies as wild-type protein to directly compare their properties .

What approaches can be used to identify novel physiological substrates of Mouse Amz1?

Identifying physiological substrates of Mouse Amz1 requires a multi-faceted approach:

• Degradomics approaches:

  • TAILS (Terminal Amine Isotopic Labeling of Substrates)

  • PICS (Proteomic Identification of Cleavage Sites)

  • COFRADIC (Combined Fractional Diagonal Chromatography)

• In vitro screening:

  • Peptide libraries

  • Bioactive peptide panels

  • Protein arrays

• Cell-based approaches:

  • Comparison of secretomes or membrane proteomes between wild-type and Amz1-knockout cells

  • Proximity labeling combined with proteomics

  • Substrate-trapping mutants

• In silico predictions:

  • Sequence motif analysis based on known substrates

  • Structural docking simulations

  • Evolutionary conservation analysis of potential cleavage sites

When validating candidate substrates, researchers should demonstrate direct cleavage by purified Mouse Amz1 in vitro, identify precise cleavage sites by mass spectrometry, and confirm the physiological relevance through cell-based or in vivo studies .

How does Mouse Amz1 compare with AMZ2 and other metalloproteases functionally?

Mouse Amz1 belongs to the archaemetzincin family alongside AMZ2, but they exhibit distinct expression patterns and potentially different physiological roles:

Unlike many well-characterized metalloproteases (MMPs, ADAMs, etc.), archaemetzincins like Mouse Amz1 have unique evolutionary origins and structural features. Their distinct tissue distribution patterns suggest specialized physiological roles that may not overlap with other metalloprotease families .

What are the considerations for using Recombinant Mouse Amz1 in structural biology studies?

For structural biology studies of Mouse Amz1, researchers should consider:

• Protein production optimization:

  • Expression in eukaryotic systems may provide better folding

  • Addition of zinc during expression and purification

  • Careful buffer optimization to maintain stability

• Crystallization considerations:

  • The presence of multiple cysteine residues may cause oxidation issues

  • Addition of reducing agents (DTT, β-mercaptoethanol) may be necessary

  • Co-crystallization with inhibitors or substrate analogs can stabilize conformation

• NMR studies:

  • Isotopic labeling (¹⁵N, ¹³C) for structural determination

  • Consider domain-specific constructs if the full-length protein is challenging

• Cryo-EM approach:

  • May be suitable if Mouse Amz1 forms oligomers or complexes

  • Consider antibody fragments to increase particle size if needed

Structural studies would significantly advance understanding of Mouse Amz1's substrate specificity, catalytic mechanism, and evolutionary relationships to both archaeal and mammalian proteases.

How can I design knockout/knockdown studies to investigate Mouse Amz1 function in vivo?

When designing knockout or knockdown studies for Mouse Amz1:

• CRISPR/Cas9 gene editing approach:

  • Target essential domains (zinc-binding region)

  • Design multiple gRNAs to increase efficiency

  • Consider conditional knockout systems if complete knockout is lethal

• siRNA/shRNA knockdown strategies:

  • Design multiple targeting sequences

  • Validate knockdown efficiency by qPCR and Western blot

  • Use scrambled sequences as controls

• Phenotypic analysis considerations:

  • Liver and heart should be primary tissues for investigation (based on expression patterns)

  • Examine metabolic parameters given liver expression

  • Assess cardiac function and structure

  • Screen for defects in protein homeostasis

• Molecular analysis:

  • Proteomics to identify accumulated substrates

  • Transcriptomics to detect compensatory mechanisms

  • Metabolomics to identify downstream effects

Given the evolutionary conservation of Amz1, researchers should also consider potential developmental roles and compensatory mechanisms through related proteases like AMZ2 .

What is known about the thermostability and pH stability of Mouse Amz1?

While specific data for Mouse Amz1 is not available in the search results, insights from the archaeal homolog AMZ-tk suggest:

• Thermostability:

  • Archaeal AMZ-tk retains activity after 4 hours at 70°C in the presence of EDTA

  • Mouse Amz1 is likely less thermostable than archaeal homologs but may retain significant stability

  • Zinc binding likely contributes to structural stability

• pH stability:

  • Archaeal AMZ-tk is active across pH 7.0-10.0, with optimal activity at pH 8.0

  • Mouse Amz1 likely functions optimally under physiological to slightly alkaline conditions

  • Activity testing across different buffer systems is recommended

Researchers working with Mouse Amz1 should empirically determine its stability profile, as mammalian orthologs may differ significantly from archaeal homologs despite sequence conservation in catalytic domains.

Does Mouse Amz1 exhibit antibacterial properties similar to archaeal homologs?

Archaeal AMZ-tk demonstrates antibacterial activity against several bacterial species . While not directly confirmed for Mouse Amz1, this property raises interesting questions:

• Could Mouse Amz1 contribute to innate immunity, particularly in the liver where it is highly expressed?

• Does Mouse Amz1 target bacterial peptides or proteins with structural similarities to its endogenous substrates?

• Could the antibacterial activity observed in archaeal homologs represent an evolutionarily conserved function?

To investigate potential antibacterial properties of Mouse Amz1, researchers could:

• Test purified recombinant Mouse Amz1 against various bacterial strains

• Determine minimal inhibitory concentrations

• Investigate mechanisms of bacterial growth inhibition

• Compare activity with human AMZ1 and archaeal homologs

What are common issues in recombinant Mouse Amz1 expression and how can they be resolved?

Common challenges in recombinant Mouse Amz1 expression and potential solutions include:

Self-cleavage activity is a particular concern with metalloproteases like AMZ1. The archaeal homolog AMZ-tk exhibits self-cleavage that requires zinc, suggesting Mouse Amz1 may behave similarly . Researchers should verify the integrity of their purified protein before functional studies.

How can I distinguish between Mouse Amz1 activity and contaminating proteases?

To ensure that observed proteolytic activity is specifically from Mouse Amz1:

• Control experiments:

  • Include metal chelators (EDTA, 1,10-phenanthroline) that should abolish Amz1 activity

  • Test catalytically inactive mutants (e.g., E→Q in the HEXXH motif)

  • Use specific metalloprotease inhibitors with known profiles

• Substrate specificity:

  • Test known AMZ1 substrates versus general protease substrates

  • Verify that angiotensin-2 is not hydrolyzed (as reported for human AMZ1)

  • Compare cleavage patterns with predicted sites based on sequence specificity

• Purification quality control:

  • Perform silver staining to detect minor contaminants

  • Consider additional purification steps (ion exchange, size exclusion)

  • Analyze purified protein by mass spectrometry to identify any co-purifying proteases

These approaches collectively provide strong evidence that the observed activity is specifically from Mouse Amz1 rather than contaminating proteases .