Recombinant Macaca cyclopis Agouti-signaling protein (ASIP)

What is Macaca cyclopis ASIP and how does it differ from other primate ASIP proteins?

Macaca cyclopis (Taiwanese or Formosan macaque) is the only non-human primate endemic to Taiwan Island. The Agouti-signaling protein (ASIP) from M. cyclopis shares the characteristic structure of mammalian ASIP, consisting of a glycosylation site in the N-terminus, a central basic domain rich in positively charged amino acids, and a cysteine-rich Agouti domain containing five disulfide bonds at the C-terminus .

The cysteine-rich Agouti domain serves as the interaction site for melanocortin-1 receptor (MC1R) and is conserved between ASIP and its paralog AgRP . While the complete M. cyclopis ASIP sequence analysis isn't fully detailed in current literature, the protein likely maintains the highly conserved Lys58-Ser59 Corin cleavage site identified in human ASIP, given that this region is evolutionarily conserved across mammals . The evolutionary divergence of M. cyclopis from Chinese rhesus macaques (M. mulatta lasiota) approximately 1.8 million years ago may have resulted in species-specific variations that could be of research interest.

What are the functional domains of M. cyclopis ASIP and their significance in experimental design?

Recombinant M. cyclopis ASIP likely contains three main functional domains, each critical for experimental considerations:

• N-terminal domain: Contains glycosylation sites that affect protein stability and half-life

• Basic domain: Contains the Lys58-Ser59 Corin cleavage site, which is highly conserved across mammals

• C-terminal cysteine-rich domain: Contains five disulfide bonds critical for MC1R binding and signaling activity

When designing experiments, researchers should consider:

• Whether to use full-length or processed ASIP

• The impact of post-translational modifications (especially glycosylation)

• The importance of disulfide bonds for proper folding and function

• The effect of the conserved Corin cleavage site at Lys58-Ser59

Expression systems for recombinant ASIP production should be selected based on their ability to properly process post-translational modifications, particularly glycosylation and disulfide bond formation.

What are the optimal expression systems for producing recombinant M. cyclopis ASIP?

The selection of an appropriate expression system for recombinant M. cyclopis ASIP should be guided by the protein's structural requirements. Based on the known features of ASIP, the following expression systems can be considered:

Table 1: Comparison of Expression Systems for Recombinant M. cyclopis ASIP Production

For recombinant M. cyclopis ASIP, mammalian or insect cell systems would likely yield the most biologically active protein due to their ability to properly form the five disulfide bonds in the cysteine-rich domain and provide appropriate glycosylation at the N-terminus . This is particularly important when studying ASIP's interaction with MC1R or investigating the Corin protease cleavage at Lys58-Ser59.

What purification strategies are most effective for isolating recombinant M. cyclopis ASIP?

Effective purification of recombinant M. cyclopis ASIP requires a multi-step approach that accounts for its structural features:

• Initial capture: Affinity chromatography using a fusion tag (His6, GST, or FLAG) positioned to avoid interference with functional domains

• Intermediate purification: Ion exchange chromatography exploiting the basic domain's positive charge (cation exchange) or heparin affinity chromatography

• Polishing step: Size exclusion chromatography to separate monomeric ASIP from aggregates and to perform buffer exchange

For biochemical and structural studies requiring high purity, the following protocol is recommended:

• Express ASIP with a cleavable N-terminal tag to facilitate initial purification

• Include reducing agents during initial steps to prevent non-native disulfide bond formation

• Allow controlled oxidative folding during the final purification stages

• Validate the proper folding using circular dichroism or limited proteolysis

To monitor purification progress, samples from each step should be analyzed by SDS-PAGE under both reducing and non-reducing conditions, as the migration pattern of properly folded ASIP differs significantly depending on the disulfide bond status .

How can the Corin protease cleavage of M. cyclopis ASIP be analyzed in experimental settings?

The analysis of Corin protease cleavage of M. cyclopis ASIP can be approached through several complementary methods:

• SDS-PAGE and Western blot analysis:

  • Incubate recombinant ASIP with and without Corin protease

  • Analyze samples under both reducing and non-reducing conditions

  • Full-length glycosylated ASIP migrates at approximately 14-16 kDa, while Corin-cleaved fragments appear at approximately 6.2 kDa (N-terminal) and 8 kDa (C-terminal)

  • Use antibodies against both N-terminal and C-terminal regions to detect specific cleavage products

• N-terminal sequencing:

  • Following in vitro digestion with Corin, perform Edman degradation to confirm the cleavage site

  • Expected sequencing results would reveal the mature N-terminus and a new N-terminus beginning at Ser59 (assuming conservation of the Lys58-Ser59 cleavage site)

• Fluorogenic peptide substrates:

  • Design fluorogenic peptides spanning the putative cleavage site (e.g., ALNKK↓SKQIGR)

  • Measure fluorescence intensity as an indicator of proteolytic activity

  • Compare activity against mutated sequences to confirm site specificity

• Mass spectrometry:

  • Analyze Corin-treated and untreated ASIP using LC-MS/MS

  • Identify cleaved peptides and map the exact cleavage site

  • Quantify the efficiency of cleavage under various conditions

Control experiments should include catalytically inactive Corin mutant (Corin mut) to confirm specificity of the observed cleavage .

What techniques are available for studying the binding kinetics between M. cyclopis ASIP and melanocortin receptors?

Several biophysical techniques can be employed to characterize the binding kinetics and affinity between recombinant M. cyclopis ASIP and melanocortin receptors:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified melanocortin receptors (particularly MC1R and MC4R) on a sensor chip

  • Flow various concentrations of recombinant ASIP over the surface

  • Measure association (ka) and dissociation (kd) rate constants

  • Calculate equilibrium dissociation constant (KD = kd/ka)

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Determine binding stoichiometry, enthalpy (ΔH), and entropy (ΔS)

  • Calculate Gibbs free energy (ΔG) and dissociation constant (KD)

• Microscale Thermophoresis (MST):

  • Label either ASIP or receptor with a fluorescent dye

  • Measure changes in thermophoretic mobility upon binding

  • Determine KD values in solution with minimal protein consumption

• Functional cellular assays:

  • Measure cAMP levels in cells expressing MC1R after ASIP treatment

  • Quantify calcium flux using fluorescent indicators

  • Assess ERK1/2 phosphorylation through Western blotting

Table 2: Expected Binding Parameters for ASIP-Melanocortin Receptor Interactions

These techniques provide complementary data on both the kinetics and thermodynamics of ASIP-receptor interactions, which are essential for understanding species-specific differences in melanocortin signaling.

How does the genomic structure of M. cyclopis ASIP compare to that of other macaque species?

The genomic analysis of M. cyclopis can provide valuable insights into the evolutionary history and functional conservation of the ASIP gene across macaque species. Using the sequenced M. cyclopis genome, which consists of 2,846,042,475 base pairs distributed across 21 chromosomes , researchers can perform comparative genomic analyses.

Although specific ASIP gene structure in M. cyclopis is not directly reported in the search results, we can make informed comparisons based on evolutionary relationships. M. cyclopis diverged from Chinese rhesus macaques (M. mulatta lasiota) approximately 1.8 million years ago , suggesting that while the ASIP gene likely maintains core functional regions, some species-specific variations may exist.

Table 3: Predicted Genomic Comparison of ASIP Across Macaque Species

Genomic analysis should include:

• Sequence alignment of coding and regulatory regions

• Identification of conserved transcription factor binding sites

• Analysis of selection pressure on different domains (dN/dS ratio)

• Evaluation of intronic and regulatory elements that may influence expression patterns

The M. cyclopis genome, with its N50 of 2.66 megabases , provides a solid foundation for these comparative analyses.

What evolutionary insights can be gained from studying M. cyclopis ASIP in the context of primate pigmentation patterns?

Studying M. cyclopis ASIP in an evolutionary context can reveal important adaptations related to primate pigmentation patterns. The fascicularis group of macaques, to which M. cyclopis belongs, exhibits significant diversity in coat coloration across its geographic range . This presents an excellent model for investigating how ASIP variants contribute to adaptive pigmentation.

Several evolutionary insights can be gained:

• Natural selection on ASIP:

  • Analyze nucleotide diversity and selection signatures in ASIP coding regions

  • Determine if specific domains experience different selection pressures

  • Correlate ASIP variants with ecological factors (latitude, altitude, habitat type)

• Functional divergence:

  • Compare M. cyclopis ASIP activity with that of mainland macaques

  • Test for differences in antagonism potency against melanocortin receptors

  • Evaluate potential coevolution between ASIP and MC1R sequences

• Island effect on pigmentation genes:

  • Assess whether the geographic isolation of M. cyclopis on Taiwan Island has led to unique ASIP adaptations

  • Compare with other island-dwelling macaques versus mainland populations

  • Examine founder effects and genetic drift in ASIP regulatory regions

• Temporal expression patterns:

  • Investigate developmental timing of ASIP expression in macaque embryogenesis

  • Compare seasonal variation in ASIP expression across macaque species

  • Correlate with pelage color changes and reproductive cycles

The geographical isolation of M. cyclopis, combined with its divergence approximately 1.8 million years ago , makes it particularly valuable for understanding how pigmentation genes evolve in response to insular environments and local adaptation pressures.

How can recombinant M. cyclopis ASIP be utilized to study melanocortin receptor signaling pathways?

Recombinant M. cyclopis ASIP provides a valuable tool for dissecting melanocortin receptor signaling pathways through several sophisticated approaches:

• BRET/FRET-based signaling analysis:

  • Develop bioluminescence/fluorescence resonance energy transfer assays using tagged melanocortin receptors and signaling proteins

  • Monitor real-time conformational changes in receptors upon ASIP binding

  • Quantify recruitment of G proteins and β-arrestins

  • Compare M. cyclopis ASIP effects with those of α-MSH and synthetic analogues

• Biased signaling investigation:

  • Determine if M. cyclopis ASIP induces biased signaling at MC1R and MC4R

  • Measure multiple downstream pathways (cAMP, Ca²⁺, ERK1/2, β-arrestin)

  • Calculate bias factors to quantify pathway selectivity

  • Assess the role of the Corin-cleaved form versus the full-length protein

• Receptor trafficking studies:

  • Track melanocortin receptor internalization and recycling using fluorescently-labeled ASIP

  • Determine the kinetics of receptor desensitization and resensitization

  • Compare trafficking patterns induced by different ASIP variants

• Transcriptomic profiling:

  • Perform RNA-seq on melanocytes treated with M. cyclopis ASIP

  • Identify gene expression networks regulated by ASIP signaling

  • Compare with other primate ASIP proteins to detect species-specific responses

Table 4: Expected Signaling Outcomes with Recombinant M. cyclopis ASIP

These approaches would contribute significantly to understanding how evolutionary differences in ASIP structure affect melanocortin signaling across primate species.

What are the technical challenges in studying the interaction between M. cyclopis ASIP and Corin protease in vivo?

Investigating the in vivo interaction between M. cyclopis ASIP and Corin protease presents several technical challenges that researchers should address through carefully designed experimental approaches:

• Tissue-specific expression patterns:

  • Both ASIP and Corin show complex spatial and temporal expression patterns

  • Challenge: Determining where and when these proteins naturally interact

  • Solution: Develop tissue-specific reporter systems or conditional expression models

• Low abundance detection:

  • Native ASIP levels may be below detection limits of standard methods

  • Challenge: Quantifying low-abundance cleaved ASIP forms in tissue samples

  • Solution: Develop ultrasensitive immunoassays or proximity ligation assays specific for cleaved forms

• Specificity of proteolytic processing:

  • Multiple proteases may cleave ASIP in vivo

  • Challenge: Attributing observed cleavage specifically to Corin

  • Solution: Generate Corin knock-out/knock-in models with site-specific mutations at the Lys58-Ser59 site

• Temporal dynamics:

  • Cleavage events may be transient or regulated by physiological stimuli

  • Challenge: Capturing the dynamic nature of the interaction

  • Solution: Develop real-time biosensors that report on ASIP cleavage state

• Physiological significance:

  • The biological consequence of Corin-mediated ASIP cleavage remains unclear

  • Challenge: Linking processing to downstream phenotypic effects

  • Solution: Generate non-cleavable ASIP mutants and assess phenotypic consequences

Advanced techniques to overcome these challenges could include:

• CRISPR/Cas9-mediated knock-in of epitope-tagged ASIP at the endogenous locus

• Mass spectrometry imaging to visualize spatial distribution of cleaved versus uncleaved ASIP

• Intravital microscopy with fluorescent biosensors to monitor cleavage in real-time

• Single-cell proteomics to detect cell-type specific processing events

How can recombinant M. cyclopis ASIP be utilized to study pigmentation disorders and melanoma?

Recombinant M. cyclopis ASIP offers unique opportunities for investigating pigmentation disorders and melanoma through several research applications:

• Comparative oncology models:

  • Test effects of M. cyclopis ASIP on melanoma cell lines from different species

  • Compare with human ASIP to identify conserved anti-proliferative mechanisms

  • Assess differential regulation of MITF-dependent transcriptional programs

  • Investigate whether species-specific ASIP variants show different efficacy in melanoma suppression

• Pigmentation disorder mechanisms:

  • Utilize recombinant ASIP to rescue melanocyte functioning in vitro models of albinism or hyperpigmentation

  • Study the impact on melanosome biogenesis, maturation, and transfer

  • Compare effects on eumelanin versus pheomelanin production pathways

  • Assess whether the Corin-cleaved form of ASIP has distinct effects on pigmentation disorders

• Drug development platforms:

  • Screen for small molecules that modulate ASIP-MC1R interactions

  • Develop peptide mimetics based on the active domain of M. cyclopis ASIP

  • Test combination approaches targeting multiple points in the melanogenesis pathway

  • Utilize comparative approaches with other primate ASIP variants to identify optimal drug candidates

• Genetic therapy models:

  • Develop ASIP gene therapy approaches for hyperpigmentation disorders

  • Test regulated expression systems for controlled melanogenesis modulation

  • Evaluate off-target effects on other melanocortin receptors

  • Compare efficacy of M. cyclopis ASIP versus human ASIP in gene therapy vectors

The M. cyclopis ASIP, with its evolutionary adaptations for primate-specific pigmentation patterns, may provide insights into novel therapeutic approaches that more closely mimic natural regulatory mechanisms.

What potential exists for using M. cyclopis ASIP in comparative studies of metabolic regulation across primates?

Beyond its role in pigmentation, ASIP interacts with melanocortin-4 receptor (MC4R), which plays a central role in energy homeostasis and feeding behavior. This creates opportunities for using M. cyclopis ASIP in comparative metabolic research:

• Primate-specific metabolic adaptations:

  • Compare the affinity of M. cyclopis ASIP for MC4R with that of other primate species

  • Correlate differences with known metabolic adaptations to various ecological niches

  • Investigate whether the geographically isolated M. cyclopis shows unique metabolic adaptations

  • Assess evolutionary convergence/divergence in ASIP function related to dietary patterns

• Receptor selectivity profiles:

  • Determine the relative selectivity of M. cyclopis ASIP for MC1R versus MC4R

  • Compare with other primate ASIP proteins to identify species-specific differences

  • Investigate structure-function relationships underlying receptor selectivity

  • Map the evolutionary trajectory of melanocortin receptor interactions

Table 5: Predicted Comparative Effects of ASIP on Metabolic Parameters

• Translational applications:

  • Develop MC4R-selective modulators based on comparative ASIP structure analysis

  • Identify regions of ASIP that determine central versus peripheral effects

  • Design ASIP-based therapeutics with optimized pharmacokinetic properties

  • Test species-specific variations in ASIP as templates for obesity treatments

The study of M. cyclopis ASIP in metabolic contexts benefits from the species' well-characterized genome and evolutionary history, providing a unique perspective on how melanocortin signaling has adapted across primate lineages.

What strategies can be employed to study the three-dimensional structure of recombinant M. cyclopis ASIP?

Understanding the three-dimensional structure of M. cyclopis ASIP is crucial for elucidating its function and evolution. Several complementary approaches can be employed:

• X-ray crystallography:

  • Challenges: Obtaining diffraction-quality crystals may be difficult due to glycosylation and flexible regions

  • Strategies:

    • Express truncated domains (particularly the cysteine-rich C-terminus)

    • Use fusion partners to aid crystallization (e.g., T4 lysozyme)

    • Co-crystallize with antibody fragments or receptor domains

    • Deglycosylate the protein while maintaining disulfide integrity

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Advantages: Can work with proteins in solution; provides dynamic information

  • Approaches:

    • ¹⁵N/¹³C-labeling in optimized expression systems

    • Domain-specific analysis, focusing on the functional C-terminal region

    • Hydrogen-deuterium exchange to map solvent-accessible regions

    • Study the structural changes upon Corin cleavage at Lys58-Ser59

• Cryo-electron microscopy:

  • Best suited for complexes of ASIP with its receptors

  • Consider:

    • Expressing ASIP-receptor fusion proteins to ensure stoichiometric complexes

    • Using nanobodies to stabilize flexible regions

    • Comparing structures before and after Corin processing

• Computational methods:

  • Homology modeling based on known structures of related proteins

  • Molecular dynamics simulations to study flexibility and binding mechanisms

  • Integration with experimental data from limited proteolysis or crosslinking

Table 6: Expected Structural Elements in M. cyclopis ASIP

Integrating these approaches would provide comprehensive structural insights into how M. cyclopis ASIP functions and how its structure compares with ASIP from other species.

How do post-translational modifications influence the functional properties of recombinant M. cyclopis ASIP?

Post-translational modifications (PTMs) play crucial roles in determining the functional properties of ASIP. For recombinant M. cyclopis ASIP, several PTMs warrant detailed investigation:

• N-linked glycosylation:

  • Impact on function:

    • Affects protein stability and half-life in circulation

    • May influence receptor binding kinetics and specificity

    • Could protect against proteolytic degradation

  • Experimental approaches:

    • Compare native and deglycosylated ASIP in receptor binding assays

    • Analyze site-directed mutants at predicted glycosylation sites

    • Perform glycoprofiling using mass spectrometry to identify species-specific patterns

• Disulfide bond formation:

  • The five disulfide bonds in the cysteine-rich domain are critical for:

    • Maintaining the tertiary structure required for receptor recognition

    • Stabilizing the inhibitory cystine-knot motif

    • Determining specificity between MC1R and MC4R binding

  • Research strategies:

    • Create single cysteine mutants to map disulfide connectivity

    • Compare oxidative folding pathways with other mammalian ASIP proteins

    • Analyze disulfide bond contribution to thermal and pH stability

• Proteolytic processing:

  • The Corin cleavage at Lys58-Ser59 potentially affects:

    • Bioavailability and tissue distribution

    • Receptor selectivity (MC1R vs. MC4R)

    • Signaling properties and downstream effects

  • Approaches for investigation:

    • Compare functional properties of full-length and Corin-cleaved ASIP

    • Develop cleavage-resistant mutants to assess physiological importance

    • Investigate tissue-specific processing patterns

• Other potential modifications:

  • Phosphorylation, acetylation, or other modifications may regulate:

    • Protein-protein interactions

    • Subcellular localization

    • Turnover and degradation

  • Methodologies:

    • Phosphoproteomic analysis of native and recombinant ASIP

    • Site-directed mutagenesis of predicted modification sites

    • Pulse-chase experiments to determine modification-dependent half-life

Understanding these PTMs is essential for producing recombinant M. cyclopis ASIP with native-like properties and for interpreting experimental results accurately, particularly in comparative studies with ASIP from other primate species.

What novel research applications might emerge from studying recombinant M. cyclopis ASIP in primate evolutionary genomics?

The study of recombinant M. cyclopis ASIP opens up several exciting avenues for primate evolutionary genomics research:

• Reconstruction of ancestral pigmentation patterns:

  • Using the M. cyclopis genome sequence alongside other primate ASIP genes to infer ancestral states

  • Expressing reconstructed ancestral ASIP proteins to test functional properties

  • Correlating genomic changes with known primate migration patterns and habitat shifts

  • Developing models of how pigmentation genes co-evolved with other traits

• Adaptive evolution analysis:

  • Leveraging M. cyclopis' geographical isolation to study island-specific adaptations

  • Comparing selection signatures on ASIP between island and mainland macaque populations

  • Investigating whether the reduced endogenous viral element burden in M. cyclopis correlates with unique ASIP regulatory mechanisms

  • Assessing whether the divergence from Chinese M. mulatta lasiota 1.8 million years ago led to functional specialization

• Gene-environment interaction studies:

  • Examining how ASIP variants correlate with specific environmental factors

  • Comparing expression patterns across different macaque species inhabiting varied ecological niches

  • Investigating whether M. cyclopis ASIP shows specific adaptations to Taiwanese climate and habitat

  • Developing models that predict pigmentation patterns based on environmental variables

• Genomic regulatory network analysis:

  • Mapping species-specific enhancers and suppressors that regulate ASIP expression

  • Comparing the architecture of ASIP regulatory elements across primates

  • Testing hypotheses about the evolution of pleiotropy in pigmentation genes

  • Investigating how ASIP regulation interfaces with other developmental networks

The M. cyclopis genome, with its draft of 2,846,042,475 base pairs distributed across 21 chromosomes , provides an excellent foundation for these evolutionary studies, particularly given the species' unique island evolutionary history.

How might computational approaches enhance our understanding of M. cyclopis ASIP structure and function?

Advanced computational methods offer powerful tools for investigating M. cyclopis ASIP beyond what experimental approaches alone can achieve:

• AI-based structure prediction:

  • Apply AlphaFold2 or RoseTTAFold to predict the full three-dimensional structure

  • Generate models of ASIP-receptor complexes to guide experimental design

  • Compare predicted structures across primate species to identify functionally important differences

  • Simulate the structural consequences of Corin cleavage at the Lys58-Ser59 site

• Molecular dynamics simulations:

  • Investigate the conformational flexibility of different ASIP domains

  • Simulate receptor binding and identify key interaction residues

  • Model the effects of glycosylation on protein dynamics

  • Compare the dynamic properties of full-length versus Corin-cleaved ASIP

• Systems biology modeling:

  • Develop mathematical models of melanocortin signaling networks

  • Incorporate species-specific ASIP parameters to predict phenotypic outcomes

  • Simulate the effects of ASIP variants on melanogenesis pathways

  • Model the evolutionary trajectories of ASIP function across primates

• Machine learning applications:

  • Identify subtle patterns in ASIP sequence-function relationships across species

  • Predict optimal conditions for recombinant expression and purification

  • Develop algorithms to identify novel ASIP-interacting proteins

  • Create classification systems for ASIP variants related to pigmentation phenotypes

Table 7: Computational Resources for M. cyclopis ASIP Research