Recombinant Pig Serine-protein kinase ATM (ATM), partial

What is porcine ATM protein and how does it compare structurally to human ATM?

Porcine ATM (Ataxia Telangiectasia Mutated) is a serine/threonine protein kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family, similar to its human counterpart. The protein contains characteristic structural modules including:

• An N-terminal substrate-binding domain (HEAT repeat domain) that binds to substrates such as NBS1, p53, and BRCA1

• A C-terminal kinase domain (KD) with significant homology to the catalytic domain of phosphatidylinositol 3-kinase (PI3K)

The porcine ATM gene has been characterized as a potential model for human ATM-related diseases. Research indicates that pig models can provide valuable insights into human ataxia-telangiectasia (A-T) since pigs share greater physiological similarities with humans than rodent models .

What are the primary functions of ATM protein in cellular processes?

ATM functions as a master regulator of cellular responses to DNA damage and genomic stability:

• Activates checkpoint signaling upon detection of double-strand breaks (DSBs)

• Phosphorylates >800 substrates involved in cell cycle checkpoints, DNA repair, and apoptosis

• Recognizes the substrate consensus sequence [ST]-Q

• Phosphorylates histone variant H2AX at Ser-139 at double-strand breaks, regulating DNA damage response mechanisms

• Plays roles in signal transduction, cell cycle control, and tumor suppression

• Contributes to pre-B cell allelic exclusion, enforcing clonality and monospecific recognition in B-lymphocytes

Why are researchers developing porcine models for ATM studies?

The domestic pig (Sus scrofa domestica) offers several advantages for ATM research:

• Established model for medical studies with greater physiological similarities to humans than rodents

• Provides insights into various human diseases including cancer, diabetes, and atherosclerosis

• Can reproduce neurological features and motor deficits seen in human A-T patients

• Supports studies where mouse models have limitations in replicating human conditions

• Allows for translational research more directly applicable to human medicine

Porcine ATM studies showed that ATM-deficient pigs demonstrated characteristic A-T phenotypes including:

• Reduced weights starting at ~3 months of age, reminiscent of growth retardation in human patients

• Motor coordination deficits on balance beam tests

• Reduced Purkinje cell numbers and increased inter-PC distance in cerebellum

What are the established methods for expressing recombinant pig ATM protein?

Several expression systems have been used for recombinant ATM production:

Insect Cell Expression:

• Baculovirus vector systems in insect cells have demonstrated success, albeit with low expression levels

• Allows for post-translational modifications closer to mammalian systems than bacterial expression

Mammalian Cell Expression:

• Human cell lines like 293T cells using episomal expression vectors

• FLAG-epitope tagging enables specific detection and purification

• Optimized transfection procedures for large (225-cm²) culture flasks yield approximately 1 μg of catalytically active F-ATM protein per flask

Alternative Expression Systems:

• Plant-based systems like wheat germ have been used for producing fragment ATM proteins (1-138 aa range) with ≥80% purity

• Pichia pastoris has been used for expressing other recombinant proteins in pig research, suggesting potential for ATM expression

What purification strategies provide optimal yield and activity for recombinant pig ATM?

Purification of recombinant ATM requires specialized approaches due to its large size (~370 kDa) and relatively low expression levels:

Affinity Chromatography:

• Anti-FLAG-agarose affinity chromatography for FLAG-tagged ATM proteins

• Allows purification to near homogeneity as judged by SDS-PAGE

• Provides catalytically active protein suitable for biochemical studies

Ion-Exchange Chromatography:

• Successfully used for purifying recombinant proteins in pig research

• Can be applied as a secondary purification step after affinity chromatography

General Considerations:

• Maintaining protein stability throughout purification is critical

• Use of protease inhibitors and appropriate buffer conditions to preserve kinase activity

• Low temperature operations to minimize degradation

• Careful handling during reconstitution as the protein may appear as a film at the bottom of storage vials

How can researchers validate the kinase activity of recombinant pig ATM in vitro?

Several established approaches can verify ATM kinase functionality:

In Vitro Kinase Assays:

• Use of recombinant substrates like CHK2 in direct phosphorylation assays

• Measurement of phosphorylation at [ST]-Q consensus sites using phospho-specific antibodies

• Detection of ATM autophosphorylation at Ser1981 (human equivalent) as an indicator of activation

Activation Methods:

• MRN (Mre11-Rad50-Nbs1) complex can directly activate ATM kinase in vitro without DNA

• DNA double-strand breaks can stimulate ATM activity

• Oxidative stress can activate ATM through disulfide bond formation

• APE1 protein has been shown to directly stimulate ATM kinase activity in vitro

Functional Complementation:

• Verification that recombinant ATM restores normal sensitivity to ionizing radiation and radiomimetic drugs in ATM-deficient cells

• Restoration of normal post-irradiation DNA synthesis (S-phase checkpoint)

What experimental approaches are used to study ATM-dependent phosphorylation events?

Substrate Identification and Validation:

• ATM recognizes and phosphorylates [ST]-Q motifs in >800 downstream substrates

• Mass spectrometry-based phosphoproteomics to identify ATM-dependent phosphorylation events

• Validation using phospho-specific antibodies against known ATM substrates including p53, BRCA1, and H2AX

Analytical Techniques:

• Immunoblotting using phospho-specific antibodies

• Immunofluorescence microscopy to detect phosphorylated ATM substrates at DNA damage sites

• Radioactive ATP-based kinase assays for direct measurement of phosphorylation

Experimental Design Considerations:

• Include ATM kinase inhibitors as controls

• Compare wild-type ATM with kinase-dead mutants

• Use ATM-deficient cells as negative controls

• Consider the timing of phosphorylation events (rapid/early vs. sustained/late)

What are the key considerations when working with kinase-dead (KD) pig ATM mutants?

Kinase-dead ATM mutants have revealed important biological functions distinct from ATM-null models:

Critical Design Considerations:

• Expression levels: KD-ATM should be expressed at levels comparable to wild-type protein

• Strategic mutations: N2875K (human equivalent) in the ATP-binding region abolishes kinase activity without significantly affecting ATM protein levels

• Control groups should include both wild-type and ATM-null conditions to distinguish between loss-of-function and dominant-negative effects

Biological Implications:

• KD mutants can have more severe phenotypes than complete ATM knockouts

• In mice, Atm^KD/- and Atm^KD/KD cause embryonic lethality with severe genomic instability, while Atm^-/- mice develop normally

• KD-ATM is not dominant-inhibitory for non-homologous end-joining (NHEJ) but affects homologous recombination (HR)

• ATM-KD protein suppresses CPT-induced DSB formation during replication and reduces sister chromatid exchanges (SCEs)

Experimental Applications:

• Studies using KD mutants revealed ATM's roles beyond its kinase activity

• KD mutants help distinguish between scaffolding and enzymatic functions of ATM

• Useful for studying the dominant negative effects of kinase domain missense mutations found in cancer

How does ATM respond differently to various types of DNA damage?

ATM activation mechanisms vary depending on the type of DNA damage:

Double-Strand Break Response:

• MRN complex recognizes DSBs and recruits ATM

• Intermolecular autophosphorylation occurs at multiple sites including Ser1981 (human)

• ATM activates cell cycle checkpoints and DNA repair pathways

Single-Strand Break Response:

• ATM can be activated by SSBs via distinct mechanisms

• APE1 (apurinic/apyrimidinic endonuclease 1) directly stimulates ATM kinase activity

• The N-terminal motif (NT34) and positively charged lysine residues of APE1 are critical for ATM activation

Replication Stress Response:

• ATM-KD protein physically blocks CPT-induced DSBs formation at replication forks after recruitment by MRN

• ATM kinase inhibitor prevents CPT-induced DSBs formation in an MRE11-dependent manner

• Differences in RAD51 foci formation between IR-induced and CPT-induced damage in ATM-KD cells

Oxidative Stress Response:

• ATM activation through disulfide bond formation (Cys2991-mediated) independent of DNA or MRN complex

• Represents a distinct activation mechanism from DNA damage-induced activation

What methodological approaches exist for studying pig ATM in a translational context?

Genome Editing Strategies:

• Targeting vector design with neomycin-resistant cassette and premature termination stop codon

• rAAV2/1 transduction into fetal fibroblasts from Yucatan miniature pigs

• Southern blotting to confirm targeting and absence of random integration

• SCNT (somatic cell nuclear transfer) to generate ATM-modified pigs

Phenotypic Analysis:

• Balance beam tests for motor coordination assessment

• Gait analysis using specialized equipment

• Histological analysis of cerebellar Purkinje cells with measurement of inter-PC distance

• Growth monitoring for developmental abnormalities

Translational Applications:

• Drug testing in a physiologically relevant model

• Testing of gene therapy approaches

• Assessment of radiation sensitivity and potential protective compounds

• Study of neurodegeneration mechanisms in a large animal model

What are common challenges in producing and maintaining stable recombinant pig ATM protein?

Expression Challenges:

• Inherent instability of the full-length ATM sequence

• Large size (~370 kDa) complicates expression and purification

• Relatively low expression levels in various systems

• Potential toxic effects of overexpression in certain cell types

Stability Considerations:

• ATM missense mutations can result in protein instability (e.g., Glu2904Gly)

• Proper storage conditions are critical for maintaining activity

• Freeze-thaw cycles may reduce activity

• Reconstitution requires careful handling as protein may appear as a film at vial bottom

Solutions and Strategies:

• Use of specialized vector/host combinations to overcome sequence instability

• Addition of epitope tags (e.g., FLAG) for improved detection and purification

• Expression of functional domains rather than full-length protein for certain applications

• Inclusion of protease inhibitors and appropriate buffer conditions during purification

How can researchers design experiments to study ATM signaling in pig models compared to human systems?

Experimental Design Considerations:

• Account for species-specific differences in ATM signaling networks

• Include appropriate controls (wild-type, ATM-null, ATM inhibitor-treated)

• Consider timing of signaling events and dose-response relationships

• Use validated antibodies that recognize porcine ATM and its substrates

Cross-Species Validation:

• Confirm key phosphorylation sites are conserved between pig and human

• Validate antibody cross-reactivity with porcine proteins

• Consider potential differences in protein-protein interactions

• Account for possible variations in response kinetics

Advanced Approaches:

• Use phosphoproteomics to comprehensively map ATM-dependent phosphorylation events

• Employ CRISPR/Cas9 to introduce specific ATM mutations seen in human diseases

• Develop isogenic cell lines with defined ATM modifications for controlled comparisons

• Consider organoid models to study tissue-specific ATM functions in a more physiological context

How might pig ATM models contribute to developing new therapeutic approaches for A-T and cancer?

Therapeutic Target Identification:

• Kinase-dead ATM is highly oncogenic, suggesting potential for targeting ATM in cancer therapy

• ATM kinase domain missense mutations identified as potent oncogenic events

• ATM status as a biomarker for Topoisomerase I inhibitor-based therapy

Drug Development Opportunities:

• Testing of ATM kinase inhibitors in physiologically relevant models

• Evaluation of synthetic lethality approaches (e.g., PARP inhibitors show increased effectiveness in ATM-deficient contexts)

• Development of agents that selectively target cells with kinase-dead ATM mutations

Gene Therapy Approaches:

• Pig models provide a platform for testing gene therapy approaches for A-T

• Assessment of delivery methods, expression levels, and functional restoration

• Evaluation of safety and efficacy prior to human trials

Neuroprotective Strategies:

• Understanding cerebellar Purkinje cell loss mechanisms in ATM-deficient pigs

• Testing neuroprotective compounds in a translational model

• Developing interventions to slow neurodegeneration in A-T patients

What new methodologies are emerging for studying ATM protein interactions and regulatory mechanisms?

Advanced Structural Biology Approaches:

• Cryo-EM studies of ATM complexes with activators and substrates

• Homology modeling using related PIKKs like mTOR (31% sequence identity in kinase and FATC domains)

• Computational prediction of interaction interfaces and regulatory regions

Proximity-Based Protein Interaction Mapping:

• BioID or APEX proximity labeling to identify ATM interactors at DNA damage sites

• iPOND (isolation of Proteins On Nascent DNA) to study ATM recruitment to stalled replication forks

• Cross-linking mass spectrometry (XL-MS) to map interaction interfaces

Real-Time Signaling Dynamics:

• Live-cell imaging of fluorescently tagged ATM and its substrates

• FRET-based sensors for monitoring ATM activation and substrate phosphorylation

• Optogenetic approaches to spatiotemporally control ATM activation

Integrative Multi-Omics:

• Combined proteomics, phosphoproteomics, and transcriptomics in ATM-modified systems

• Network analysis to identify critical nodes in ATM signaling pathways

• Machine learning approaches to predict ATM-dependent responses to DNA damage