Recombinant Hylobates lar Microcephalin (MCPH1), partial

What is the domain structure of MCPH1 and how does it compare between human and non-human primates?

MCPH1 contains three BRCA1 C-terminal (BRCT) domains - one at the N-terminus and two tandem domains at the C-terminus. The human MCPH1 gene encodes an 835-amino acid protein . While the complete Hylobates lar MCPH1 sequence shows evolutionary conservation, the most preserved regions across species are the BRCT domains, which typically show around 80% identity between species (as observed in human-mouse comparison) .

The N-terminal BRCT domain is particularly critical as it:

• Regulates chromosome condensation to ensure coordinate mitotic entry

• Mediates centrosomal localization in vertebrates

• Contains binding sites for interaction partners like SET protein

The C-terminal tandem BRCT domains mediate interaction with phosphorylated H2AX at DNA damage sites . This domain structure is functionally significant as mutations in the N-terminal BRCT domain are specifically associated with premature chromosome condensation and microcephaly phenotypes.

What is the expression pattern of MCPH1 in developing tissues and how might this inform recombinant protein applications?

MCPH1 shows a tissue-specific expression pattern that provides important context for recombinant protein studies:

• Fetal brain: High expression, particularly in the developing cerebral cortex and walls of lateral ventricles where progenitor cells divide to produce neurons that migrate to form the cerebral cortex

• Fetal liver and kidney: Similar expression levels to fetal brain

• Other fetal tissues: Detectable but lower expression levels

• Adult tissues: Variable expression across multiple tissues

This expression pattern suggests recombinant MCPH1 applications should consider:

• Developmental timing when designing experiments with recombinant protein

• Tissue-specific effects when using the protein in cellular models

• Potential non-neuronal functions when interpreting experimental results

Mouse in situ hybridization experiments confirm high expression in developing forebrain, particularly in the progenitor-rich ventricular zones, aligning with MCPH1's role in neurogenesis .

Why focus on Hylobates lar MCPH1 specifically rather than human MCPH1 for certain research applications?

Comparative studies between human and non-human primate MCPH1 provide evolutionary insights into cortical development:

These comparative studies may reveal whether mutations affecting brain size in humans have different effects in closely related primates with different brain development trajectories.

What expression systems are most effective for producing functionally active recombinant MCPH1?

Based on published methodologies for MCPH1 research, the following expression systems have proven effective:

For functional studies of MCPH1, mammalian expression systems are often preferred. Research protocols show successful expression of SFB-tagged (S-protein, FLAG, and streptavidin-binding peptide) MCPH1 constructs in 293T cells . This approach allows for tandem affinity purification of MCPH1 and associated proteins, which has been instrumental in identifying interaction partners like SET protein.

For domain-specific studies, researchers should consider expressing individual domains (N-BRCT, middle region, or C-terminal tandem BRCT domains) separately, as different domains mediate different functions and interactions .

What purification strategies maximize yield and maintain functionality of recombinant MCPH1?

Effective purification of recombinant MCPH1 requires careful consideration of the protein's properties and intended applications:

• Tandem Affinity Purification (TAP) approach:

  • Effectively used with SFB-tagged MCPH1 constructs

  • Protocol: Cells expressing SFB-tagged MCPH1 are lysed with NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl at pH 8.0, 0.5% Nonidet P-40)

  • Cleared lysates are incubated with streptavidin-conjugated beads

  • Bound proteins are eluted with NETN buffer containing 2 mg/ml biotin

• GST-fusion protein approach:

  • Useful for domain-specific studies and in vitro binding assays

  • Successfully applied for MCPH1 N-terminal BRCT domain studies

  • Allows direct testing of protein-protein interactions through GST pulldown assays

• Considerations for maintaining functionality:

  • Include protease inhibitors in all buffers

  • Maintain samples at 4°C during purification

  • Consider phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Verify protein folding and activity after purification

The choice of affinity tag and purification strategy should align with downstream applications, with multi-step purification recommended for functional studies and simpler approaches sufficient for immunization or antibody production.

How can researchers effectively design truncation constructs to study specific functional domains of MCPH1?

When designing truncation constructs for MCPH1 functional studies, researchers should consider the following structure-function relationships:

• N-terminal BRCT domain (residues 1-130):

  • Essential for preventing premature chromosome condensation

  • Mediates interaction with SET protein

  • Associated with microcephaly-causing mutations (T27R, H49Q, V50G, I51V, S72L)

  Design strategy: Create constructs containing residues 1-130 for specific N-BRCT studies

• Middle region:

  • Contains condensin II-binding domain

  • May mediate other protein interactions

  Design strategy: Create constructs with the middle region but lacking N-terminal and C-terminal BRCT domains

• C-terminal tandem BRCT domains:

  • Mediate interaction with phosphorylated H2AX

  • Function in DNA damage response

  Design strategy: Create constructs containing only the C-terminal tandem BRCT domains

Functional testing of these domains has revealed that the N-terminus, not the middle condensin II-binding domain, is responsible for preventing premature chromosome condensation (PCC) in MCPH1-deficient cells . This type of domain analysis is crucial for understanding the distinct functions of each protein region.

When designing truncation constructs, researchers should also consider:

• Including flexible linkers between domains and tags

• Preserving natural boundaries between structured domains

• Testing multiple constructs with different boundaries around predicted domains

• Including positive controls (full-length protein) in all functional assays

How can researchers effectively assess MCPH1-SET protein interactions using recombinant proteins?

The interaction between MCPH1 and SET protein is crucial for regulating chromosome condensation. To assess this interaction:

• Co-immunoprecipitation (Co-IP) approach:

  • Transfect cells with tagged versions of MCPH1 and SET

  • Perform immunoprecipitation with antibodies against one protein

  • Detect co-precipitated partner by Western blotting

  • This approach confirmed that FLAG-tagged SET co-immunoprecipitates with SFB-tagged full-length MCPH1 and N-BRCT fragment, but not with ΔN-MCPH1

• GST pulldown assays:

  • Express GST-fused MCPH1 domains (full-length, N-BRCT, ΔN) and purify

  • Incubate with cell lysates containing SET or with purified SET protein

  • Detect binding through Western blotting

  • This technique confirmed direct interaction between SET and the N-terminal domain of MCPH1

• Reciprocal binding assays:

  • Express GST-fused SET protein

  • Test binding with different MCPH1 constructs

  • This approach verified that SET specifically associates with the N-terminus of MCPH1

• Functional validation:

  • Assess the impact of disrupting this interaction on chromosome condensation

  • DOWN-regulation of SET in wild-type MEFs leads to a prophase-like PCC phenotype identical to that seen in Mcph1-/- MEFs, with about 10-15% of cells showing premature chromosome condensation

These methods collectively provide robust validation of protein-protein interactions and their functional significance.

What methods are effective for analyzing the impact of MCPH1 mutations on protein function?

Comprehensive analysis of MCPH1 mutations requires multiple complementary approaches:

• Functional complementation assays:

  • Introduce wild-type or mutant MCPH1 into MCPH1-deficient cells

  • Assess rescue of phenotypes (e.g., premature chromosome condensation)

  • This approach revealed that MCPH1 V50G/I51V missense mutations fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1-/- mouse embryonic fibroblasts

• Protein interaction studies:

  • Test if mutations affect binding to known partners (e.g., SET protein)

  • Use co-IP or pulldown assays with mutant proteins

  • MCPH1 V50G/I51V missense mutations were shown to impair binding to SET protein

• Cellular localization analysis:

  • Examine if mutations affect MCPH1 subcellular distribution

  • Use immunofluorescence with tagged constructs

  • Assess co-localization with interaction partners or cellular structures

• Patient-derived cell studies:

  • Analyze cells from patients with specific MCPH1 mutations

  • Example: MCPH1 S25X mutation was found to use an alternative translational start site producing a truncated protein with partial deletion of the N-terminal BRCT domain

• Biochemical characterization:

  • Assess protein stability, folding, and post-translational modifications

  • Compare wild-type and mutant protein properties

How can comparative studies between human and Hylobates lar MCPH1 reveal evolutionary insights into brain development?

Comparative studies between human and gibbon MCPH1 can provide crucial evolutionary insights through these approaches:

These comparative approaches could reveal how evolutionary changes in MCPH1 correlate with primate brain size differences, potentially illuminating the molecular basis of human brain expansion.

How can researchers effectively use recombinant MCPH1 to study its role in DNA damage response pathways?

MCPH1 plays important roles in DNA damage-induced S and G2/M checkpoints. Advanced approaches to study these functions include:

• In vitro kinase assays:

  • Use recombinant MCPH1 as substrate for checkpoint kinases (ATM, ATR, CHK1, CHK2)

  • Identify phosphorylation sites through mass spectrometry

  • Create phospho-specific antibodies to track MCPH1 modification after DNA damage

• DNA binding assays:

  • Test direct binding of recombinant MCPH1 to damaged DNA structures

  • Assess the role of specific domains in DNA recognition

  • The C-terminal tandem BRCT domains interact with phosphorylated H2AX , suggesting a mechanism for MCPH1 recruitment to DNA damage sites

• Reconstitution of DNA damage response complexes:

  • Use recombinant proteins to reconstitute MCPH1-containing complexes

  • Assess binding to other DNA damage response proteins

  • MCPH1 ionizing radiation-induced foci co-localize with MDC1 and phosphorylated H2AX

• Single-molecule approaches:

  • Track the dynamics of fluorescently labeled recombinant MCPH1 at sites of DNA damage

  • Measure binding kinetics and residence times

  • Compare wild-type and mutant proteins

• Structural studies of damage-recognition complexes:

  • Solve structures of MCPH1 domains bound to damaged DNA or partner proteins

  • Focus on the C-terminal tandem BRCT domains and their interaction with phosphopeptides

These approaches can elucidate how MCPH1 coordinates DNA damage response with cell cycle progression and chromosome condensation, potentially revealing new therapeutic targets for conditions with dysregulated DNA damage response.

What chromatin immunoprecipitation (ChIP) approaches can be used to study genomic binding of recombinant MCPH1?

Advanced chromatin immunoprecipitation approaches can map MCPH1 binding across the genome:

• ChIP-seq with recombinant tagged MCPH1:

  • Express tagged recombinant MCPH1 in appropriate cell types

  • Perform ChIP using antibodies against the tag

  • Sequence precipitated DNA to identify genome-wide binding sites

  • Compare binding profiles before and after DNA damage induction

• CUT&RUN or CUT&Tag approaches:

  • Use antibodies against MCPH1 or its tag for in situ protein-DNA complex cleavage

  • These techniques offer higher signal-to-noise ratio than traditional ChIP

  • Particularly useful for factors with transient chromatin interactions

• ChIP-MS (Mass Spectrometry):

  • Identify proteins co-occupying MCPH1-bound chromatin regions

  • Characterize the composition of MCPH1-containing complexes at chromatin

  • Reveal context-specific interaction partners

• Sequential ChIP (Re-ChIP):

  • Perform successive immunoprecipitations with antibodies against MCPH1 and other factors

  • Identify genomic loci where MCPH1 co-localizes with specific partners

  • Useful for studying MCPH1 association with phosphorylated H2AX or condensin complexes

• ChIP-qPCR validation:

  • Verify binding at specific genomic loci of interest

  • Compare wild-type and mutant MCPH1 binding

  • Assess how binding changes during cell cycle progression or after DNA damage

These genomic approaches can identify direct target genes of MCPH1, potentially revealing its role in transcriptional regulation beyond its functions in chromosome condensation and DNA damage response.

How can researchers design experiments to distinguish between the roles of MCPH1 in chromosome condensation versus DNA damage response?

Distinguishing between MCPH1's dual functions requires carefully designed experiments:

• Domain-specific replacement studies:

  • Replace endogenous MCPH1 with constructs lacking specific domains

  • N-terminal BRCT domain is required for preventing premature chromosome condensation

  • C-terminal tandem BRCT domains mediate DNA damage response

  • This approach allows separation of functions through domain inactivation

• Temporal dissection experiments:

  • Study MCPH1 function in synchronized cell populations at different cell cycle stages

  • Separate DNA damage response (throughout interphase) from chromosome condensation regulation (primarily G2/M transition)

  • Use cell synchronization methods combined with MCPH1 depletion/replacement

• Interaction partner manipulation:

  • Selectively disrupt specific protein interactions

  • SET knockdown specifically affects chromosome condensation, resembling the phenotype of Mcph1-/- MEFs

  • Disrupting phospho-H2AX interaction would specifically affect DNA damage response

• Point mutation analysis:

  • Introduce mutations that specifically affect one function but not the other

  • MCPH1 V50G/I51V mutations impair SET binding and chromosome condensation regulation

  • Identify mutations that disrupt DNA damage response without affecting condensation

• Condensin manipulation experiments:

  • Test if condensin II knockdown rescues condensation defects without affecting DNA damage response

  • Condensin II knockdown was shown to rescue abnormal chromosome condensation in SET-depleted cells

• Quantitative phenotype assessment:

  • Develop assays that separately quantify each function:

    • Premature chromosome condensation (PCC): Measure percentage of cells with prophase-like chromosomes in interphase (10-15% in SET knockdown cells)

    • DNA damage response: Measure formation of ionizing radiation-induced foci or checkpoint activation

These approaches can delineate the mechanistic independence or interdependence of MCPH1's diverse cellular functions.

What are common challenges in expressing recombinant MCPH1 and how can they be addressed?

Researchers may encounter several challenges when working with recombinant MCPH1:

• Protein solubility issues:

  • Challenge: Full-length MCPH1 may show low solubility

  • Solution: Express individual domains separately, particularly the N-BRCT (residues 1-130) or C-terminal BRCT domains

  • Alternative: Use solubility-enhancing tags (MBP, SUMO) or optimize buffer conditions

• Low expression levels:

  • Challenge: Poor yield of recombinant protein

  • Solution: Optimize codon usage for expression system

  • Alternative: Test different promoters or expression conditions

  • Example: For mammalian expression, stable cell lines expressing SFB-tagged MCPH1 constructs can be selected using 2 μg/ml puromycin

• Protein degradation:

  • Challenge: Proteolytic degradation during expression/purification

  • Solution: Include protease inhibitors in all buffers

  • Alternative: Identify and remove unstructured regions prone to degradation

• Improper folding:

  • Challenge: Misfolded protein with impaired function

  • Solution: Express in mammalian cells for proper post-translational modifications

  • Alternative: Use chaperone co-expression in bacterial systems

• Functional verification:

  • Challenge: Confirming activity of purified protein

  • Solution: Develop robust functional assays (e.g., SET binding for N-BRCT domain)

  • Alternative: Include known functional mutants as negative controls (e.g., V50G/I51V mutations that impair SET binding)

When troubleshooting expression issues, systematic optimization of expression conditions and construct design is essential for obtaining functional recombinant MCPH1 suitable for downstream applications.

How should researchers interpret conflicting data between in vitro and cellular studies of MCPH1 function?

When faced with discrepancies between in vitro and cellular studies:

• Consider contextual factors:

  • Cellular environment provides cofactors, post-translational modifications, and appropriate concentrations

  • In vitro systems may lack essential components

  • Example: MCPH1's function in chromosome condensation depends on interaction with SET protein and condensin II

• Evaluate protein state:

  • Recombinant proteins may not fully recapitulate native structure/modifications

  • Patient-derived cells may express unexpected protein variants

  • Example: MCPH1 S25X patient cells unexpectedly produce a truncated MCPH1 protein through alternative translation initiation

• Apply multiple complementary approaches:

  • Confirm key findings using diverse methodologies

  • Establish functional assays that bridge in vitro and cellular systems

  • Example: Both direct in vitro binding assays and cellular co-immunoprecipitation confirmed MCPH1-SET interaction

• Assess physiological relevance:

  • Consider if experimental conditions reflect physiological context

  • Evaluate if protein concentrations match endogenous levels

  • Example: Expression patterns in developing brain provide context for MCPH1 function in neurogenesis

• Systematically identify variables:

  • Test if specific buffer components, cell types, or experimental conditions explain discrepancies

  • Isolate variables through controlled experiments

  • Develop quantitative assays to measure effects precisely

Resolution of conflicting data often leads to deeper mechanistic insights, as exemplified by the discovery that MCPH1's N-terminus, rather than its condensin II-binding domain, prevents premature chromosome condensation through SET protein interaction .

What controls are essential when studying species-specific differences in MCPH1 function?

When investigating differences between human and Hylobates lar MCPH1:

• Expression level controls:

  • Ensure comparable expression of different species' proteins

  • Quantify protein levels by Western blotting

  • Use inducible expression systems to test concentration-dependent effects

• Domain-specific controls:

  • Include chimeric proteins (human-gibbon domain swaps)

  • Test individual domains separately

  • Focus on the highly conserved BRCT domains versus more divergent regions

• Cellular context controls:

  • Test function in both human and non-human primate cells when possible

  • Consider species-specific interaction partners

  • Example: Confirm SET protein interaction is conserved across species

• Mutation analysis controls:

  • Test equivalent mutations in both species' proteins

  • Include known functional mutations (e.g., V50G/I51V in N-terminal BRCT domain)

  • Create "humanized" versions of gibbon protein and vice versa

• Evolutionary context:

  • Include additional primate species for evolutionary trajectory analysis

  • Consider convergent/divergent evolution patterns

  • Compare to outgroups (e.g., mouse MCPH1 shows 57% identity to human but 80% in BRCT domains)

• Phenotypic readout controls:

  • Use quantitative assays that work equally well for both species' proteins

  • Measure multiple functional outcomes (e.g., chromosome condensation, protein interactions)

  • Example: Quantify percentage of cells with premature chromosome condensation as done for SET knockdown (10-15%)

These controls help distinguish genuine species-specific functional differences from experimental artifacts, enabling accurate evolutionary interpretations of MCPH1 function across primates.