Recombinant Mouse Protein angel homolog 1 (Angel1)

Advanced Research Questions

• What are the optimal expression systems for producing functional recombinant Mouse Angel1?

Several expression systems have been successfully used to produce recombinant Mouse Angel1, each with advantages depending on research requirements:

For functional studies requiring proper protein folding and post-translational modifications, mammalian expression systems like HEK-293 are recommended. When optimizing recombinant Angel1 expression, researchers should consider:

• Codon optimization for the expression host

• Selection of appropriate purification tags that don't interfere with protein function

• Addition of protease inhibitors during purification

• Validation of protein activity post-purification

• Storage conditions to maintain protein stability

Validation of recombinant protein quality should include SDS-PAGE, Western blotting, and activity assays appropriate to Angel1's putative functions .

• How can Angel1's phosphatase activity be characterized and what are its substrates?

Characterizing Angel1's phosphatase activity requires systematic biochemical approaches:

• Enzyme kinetics analysis:

  • Use synthetic substrates like p-nitrophenyl phosphate to determine basic kinetic parameters

  • Compare activity with the related enzyme Angel2, which hydrolyzes terminal 2′,3′-cyclic phosphates on RNA molecules

  • Determine optimal pH, temperature, and ion requirements

• Substrate identification:

  • Test various RNA substrates with different end modifications

  • Perform RNA immunoprecipitation followed by sequencing (RIP-seq) to identify bound RNA targets

  • Use phosphatase-dead mutants as negative controls

• Structural analysis:

  • Identify catalytic residues through sequence alignment with characterized phosphatases

  • Generate point mutations of conserved residues to confirm their role in catalysis

  • Consider the E121A mutation which abolishes activity in Drosophila Angel

• In cellulo validation:

  • Express wild-type and phosphatase-dead mutants in cells

  • Analyze global changes in RNA phosphorylation status

  • Perform rescue experiments in Angel1-depleted cells

This methodological approach will help determine whether Angel1's phosphatase activity is similar to Angel2's ability to hydrolyze terminal 2′,3′-cyclic phosphates on RNA molecules.

• What is the relationship between Angel1 and eIF4E, and how does this interaction affect translation regulation?

Angel1 has been identified as a novel eIF4E-binding protein through a refined screening approach . Key findings about this interaction include:

• Angel1 interacts with eIF4E both in vitro and in vivo

• The interaction is mediated through a consensus eIF4E-binding motif

• Point mutations in this motif disrupt the interaction

• Angel1 partially co-localizes with eIF4E and eIF4G, but not with 4E-BP

• Unlike other eIF4E-binding proteins, manipulating Angel1 levels has no effect on global translation rates, suggesting a more specific function

To investigate the functional significance of this interaction, researchers should:

• Map the precise binding regions through deletion constructs and point mutations

• Perform polysome profiling with Angel1 overexpression or knockdown

• Analyze translational efficiency of specific mRNAs using ribosome profiling

• Investigate whether Angel1 competes with other eIF4E-binding proteins

• Determine if the interaction is regulated by cellular conditions (stress, nutrient availability, etc.)

This Angel1-eIF4E interaction appears distinct from canonical translation regulators, potentially representing a specialized regulatory mechanism for specific subsets of mRNAs.

• How does Angel1 expression change in response to environmental chemicals and what are the epigenetic mechanisms involved?

Search result provides extensive gene-chemical interaction annotations for Angel1 in rat models, which can inform similar studies in mouse. These data reveal complex patterns of Angel1 regulation in response to environmental exposures:

To investigate these effects in mouse Angel1, researchers should:

• Design exposure studies using mouse models or cell lines

• Employ qPCR and Western blotting to measure expression changes

• Analyze DNA methylation using bisulfite sequencing

• Perform ChIP-seq to examine histone modifications

• Use CRISPR-based epigenetic editing to confirm causality between epigenetic changes and expression

These approaches would help determine if the epigenetic regulation of Angel1 is conserved between rat and mouse models, and identify potential species-specific differences.

• What are the methodological considerations for designing CRISPR-based knockout and knockin studies for Angel1?

When designing CRISPR-based genetic manipulation of Angel1, researchers should consider:

• Guide RNA design:

  • Target exons that are critical for protein function

  • Avoid regions with high homology to Angel2 to prevent off-target effects

  • Use multiple bioinformatic tools to predict guide efficiency and specificity

  • Consider targeting the consensus eIF4E-binding motif to specifically disrupt this interaction

• Knockout validation approaches:

  • Genomic PCR and sequencing to confirm mutations

  • RT-PCR and Western blotting to verify loss of expression

  • Immunofluorescence to confirm absence of localization patterns

  • Functional assays relevant to Angel1's putative roles

• Knockin considerations:

  • For tagging, avoid N-terminal tags that might disrupt mitochondrial targeting

  • Consider the E121A mutation (or mouse equivalent) that abolishes activity in Drosophila Angel

  • Design knockin constructs with selectable markers that can be subsequently removed

  • Include silent mutations in the repair template to prevent re-cutting

• Phenotypic analysis:

  • Assess subcellular localization changes

  • Examine interactions with known partners like eIF4E

  • Investigate effects on translation of specific mRNAs

  • Evaluate mitochondrial parameters if targeting outer mitochondrial membrane functions

This methodological framework accounts for Angel1's unique characteristics while applying standard CRISPR genome editing principles.

• How can researchers investigate the potential role of Angel1 in aging and longevity?

Angel1 has been implicated in longevity studies according to search result , which identified Angel1 SNPs among 281 SNPs that discriminate between centenarians and controls in a genome-wide association study. To investigate Angel1's potential role in aging:

• Comparative expression analysis:

  • Measure Angel1 expression across different age groups in mouse tissues

  • Compare expression between short-lived and long-lived mouse strains

  • Analyze expression changes in response to lifespan-extending interventions (caloric restriction, rapamycin)

• Genetic manipulation approaches:

  • Generate tissue-specific or inducible Angel1 knockout or overexpression mouse models

  • Assess lifespan and healthspan parameters

  • Measure biomarkers of aging (telomere length, senescence markers, etc.)

• Molecular pathway analysis:

  • Investigate interaction with known longevity pathways (mTOR, insulin/IGF-1, sirtuins)

  • Perform RNA-seq and proteomics to identify affected pathways

  • Analyze mitochondrial function given Angel1's potential localization to the outer mitochondrial membrane

• Human genetic studies:

  • Validate the SNPs identified in centenarian studies

  • Perform functional analysis of these variants

  • Investigate population-specific differences in Angel1 polymorphisms

These methodological approaches would help establish whether Angel1 has a causal role in aging regulation or is merely associated with longevity-related processes.

• What approaches can be used to develop Angel1 as a potential therapeutic target?

Developing Angel1 as a therapeutic target would require a systematic research approach:

• Target validation:

  • Comprehensive characterization of Angel1's physiological functions

  • Identification of disease contexts where Angel1 modulation might be beneficial

  • Confirmation of Angel1's role through genetic models and pharmacological tools

• Druggability assessment:

  • Structural analysis to identify potential binding pockets

  • In silico screening to identify potential chemical starting points

  • Development of robust biochemical assays for Angel1 activity

• Compound screening strategies:

  • High-throughput screening of compound libraries against Angel1's phosphatase activity

  • Fragment-based approaches to identify chemical scaffolds with binding potential

  • Structure-based drug design if crystal structures become available

• Development of biological approaches:

  • Evaluation of antisense oligonucleotides or siRNAs for Angel1 knockdown

  • Assessment of PROTAC approaches for targeted degradation

  • Investigation of antibody-based approaches for specific contexts

• Specificity considerations:

  • Development of assays to distinguish between effects on Angel1 versus Angel2

  • Assessment of off-target effects on related phosphatases

  • Tissue-specific delivery strategies based on expression patterns

This framework provides a roadmap for potential therapeutic development, though substantial basic research on Angel1's functions would be required before pursuing therapeutic applications.