Recombinant Pongo abelii UPF0708 protein C6orf162 homolog

How does this protein relate to evolutionary studies in primates?

This protein provides valuable insights into primate evolution, particularly when comparing the Sumatran orangutan (Pongo abelii) with other great apes. The orangutan genome has undergone slower structural evolution compared to other great apes, with fewer rearrangements and less segmental duplication . The UPF0708 protein C6orf162 homolog serves as a useful marker for studying these evolutionary patterns.

Although Bornean and Sumatran orangutans diverged approximately 400,000 years ago, their proteins still share significant homology . Studying variations in this protein between the two species can provide insights into the speciation process and adaptive evolution. The Sumatran orangutan (Pongo abelii) genome shows greater diversity than its Bornean counterpart, which is reflected in species-specific variations in proteins like UPF0708 .

What is known about the gene expression patterns of SMIM8 in Pongo abelii?

The SMIM8 gene (which encodes the UPF0708 protein C6orf162 homolog) demonstrates tissue-specific expression patterns in primates. While direct data for Pongo abelii is limited, comparative analysis with other primates suggests differential expression across tissues. Alternative splicing and isoform regulation likely play important roles in the expression of this gene, as observed in the broader context of primate transcriptomes .

Recent tissue transcriptome analyses in primates have revealed that many genes undergo tissue-regulated alternative processing, producing multiple mRNA and protein isoforms with potentially diverse functions . For membrane proteins like SMIM8, these regulatory mechanisms can significantly impact protein localization and function.

What are the optimal conditions for recombinant expression of this protein?

For successful recombinant expression of Pongo abelii UPF0708 protein C6orf162 homolog, the following methodology has been established:

• Expression System: E. coli is the preferred expression system for this protein, providing high yields and relatively straightforward purification .

• Construct Design: For optimal expression, the full-length protein (amino acids 1-97) should be fused to an N-terminal histidine tag .

• Expression Conditions: Standard bacterial expression protocols with IPTG induction are typically used, with best results obtained at lower induction temperatures (16-18°C) to enhance proper folding.

• Purification Strategy: A two-step purification process is recommended:

  • Initial purification using Ni-NTA affinity chromatography

  • Further purification via size exclusion chromatography to achieve >90% purity

For membrane proteins like SMIM8, detergent selection is critical during purification to maintain native structure. Non-ionic detergents such as DDM or LDAO are often effective for maintaining stability during purification.

What reconstitution and storage protocols ensure optimal protein stability?

For reconstitution and storage of lyophilized Pongo abelii UPF0708 protein C6orf162 homolog, the following methodological approach is recommended:

• Reconstitution Protocol:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is optimal) for long-term storage

  • Aliquot to minimize freeze-thaw cycles

• Storage Conditions:

  • For long-term storage: -20°C to -80°C in storage buffer (Tris/PBS-based buffer, 6% Trehalose, pH 8.0)

  • For working stocks: 4°C for up to one week

  • Avoid repeated freeze-thaw cycles which significantly reduce protein stability

• Quality Control Metrics:

  • Purity assessment via SDS-PAGE (should be >90%)

  • Activity assays specific to membrane protein function

  • Circular dichroism to confirm proper secondary structure

These methodological guidelines ensure maximum retention of native structure and function for downstream experiments.

How can structural biology techniques be applied to understand the membrane topology of this protein?

Understanding the membrane topology of Pongo abelii UPF0708 protein C6orf162 homolog requires a multifaceted structural biology approach:

• Predictive Bioinformatics:

  • Hydropathy analysis suggests a single transmembrane domain spanning approximately 20-22 amino acids in the central portion of the protein

  • Secondary structure prediction indicates primarily alpha-helical structure in the transmembrane region

• Experimental Topology Mapping:

  • Cysteine scanning mutagenesis combined with accessibility assays

  • Protease protection assays to determine which regions are protected by the membrane

  • Epitope tagging at N and C termini to determine orientation

• Advanced Structural Analysis:

  • For membrane proteins like SMIM8, cryo-electron microscopy offers advantages over crystallography

  • Detergent micelles or nanodiscs can be used to stabilize the protein in a membrane-like environment

  • Nuclear magnetic resonance (NMR) spectroscopy of isotopically labeled protein can provide detailed structural information

These methods provide complementary data that can be integrated to develop a comprehensive structural model of the protein's membrane topology and orientation.

What are the challenges in functional characterization of this protein, and how can they be addressed?

The functional characterization of Pongo abelii UPF0708 protein C6orf162 homolog presents several methodological challenges:

• Challenge: Limited prior functional information on this protein family
  Solution: Employ comprehensive bioinformatic analyses including:

  • Profile-profile alignments to detect distant homologs with known functions

  • Protein-protein interaction predictions

  • Structural modeling to identify potential functional sites

• Challenge: Membrane protein expression and purification complexities
  Solution: Optimize expression conditions through:

  • Testing multiple detergents for extraction and purification

  • Using membrane mimetics (nanodiscs, liposomes) for functional studies

  • Employing cell-free expression systems designed for membrane proteins

• Challenge: Assessing protein-protein interactions in a membrane context
  Solution: Apply specialized techniques including:

  • Biolayer interferometry with reconstituted protein

  • Microscale thermophoresis for measuring binding affinities

  • Split-GFP complementation assays in heterologous expression systems

• Challenge: Connecting in vitro observations to physiological function
  Solution: Develop cellular models through:

  • CRISPR-Cas9 knockout/knockin studies in relevant cell lines

  • Rescue experiments with the orangutan variant versus human homolog

  • Tissue-specific expression analysis in orangutan samples when available

These methodological approaches can systematically address the challenges inherent in studying membrane proteins with unknown functions.

How does the UPF0708 protein differ between Sumatran and Bornean orangutans, and what might this reveal about their speciation?

Comparative analysis of the UPF0708 protein between Sumatran (Pongo abelii) and Bornean (Pongo pygmaeus) orangutans reveals insights into their evolutionary divergence:

The speciation between these two orangutan species was complex and likely influenced by:

• Geographic isolation: The rising sea levels separated Sumatra and Borneo islands

• Different ecological adaptations: The distinct environments of each island created different selective pressures

• Population dynamics: The Sumatran effective population size expanded exponentially after the split, while the Bornean population declined

Studying the specific amino acid differences in the UPF0708 protein between these species can reveal potential functional adaptations. These differences may reflect adaptations to different diets, environmental pressures, or mating systems between the two orangutan species.

What can comparative analysis with human UPF0708 protein reveal about primate evolution?

Comparative analysis between orangutan and human UPF0708 protein provides valuable insights into primate evolution:

These comparative analyses reveal that orangutans occupy a unique position in primate evolution, with their genome evolving more slowly than other great apes. This makes the Pongo abelii UPF0708 protein C6orf162 homolog particularly valuable for reconstructing ancestral states of primate proteins.

How can multi-omics approaches enhance our understanding of UPF0708 protein function?

A comprehensive multi-omics strategy provides the most robust approach to unraveling the function of the Pongo abelii UPF0708 protein:

• Genomics Integration:

  • Whole genome sequencing data from multiple orangutans reveals population-level variation

  • Comparative genomics across primates identifies conserved regulatory elements

  • Analysis of selection signatures can indicate functional constraints

• Transcriptomics Applications:

  • RNA-seq analysis across tissues reveals expression patterns

  • Alternative splicing analysis identifies tissue-specific isoforms

  • Single-cell RNA-seq can determine cell type-specific expression

• Proteomics Approaches:

  • Interaction proteomics (IP-MS) identifies binding partners

  • Post-translational modification analysis reveals regulatory mechanisms

  • Quantitative proteomics across tissues maps protein abundance

• Structural Biology Integration:

  • Cryo-EM or NMR structures provide atomic-level details

  • Molecular dynamics simulations predict conformational changes

  • In silico docking identifies potential ligand binding sites

• Functional Genomics Validation:

  • CRISPR-Cas9 editing assesses phenotypic consequences

  • Reporter assays validate regulatory elements

  • Cellular assays measure specific biochemical activities

This integrated approach would systematically build a functional model of the UPF0708 protein, connecting genomic variation to molecular mechanism and ultimately to biological function.

What are the most promising research directions for understanding the physiological role of this protein in orangutans?

The most promising research directions for elucidating the physiological role of the UPF0708 protein in orangutans include:

• Membrane Proteome Analysis:

  • Comprehensive characterization of the orangutan membrane proteome across tissues

  • Identification of tissue-specific interaction partners

  • Comparison with human membrane proteome to identify species-specific differences

• Evolutionary Energetics:

  • Investigation of potential roles in energy metabolism, as orangutans have extremely low energy usage for a eutherian mammal

  • Analysis of potential contributions to glycolipid metabolism, which shows signals of positive selection in orangutans

  • Examination of expression in tissues relevant to orangutan-specific metabolic adaptations

• Regulatory Network Mapping:

  • Identification of transcription factors controlling SMIM8 expression

  • Analysis of microRNA regulation of SMIM8 mRNA

  • Integration with epigenetic data to understand tissue-specific regulation

• Functional Conservation Testing:

  • Cross-species complementation studies to assess functional equivalence

  • Domain swapping experiments to identify species-specific functional regions

  • Comparative phenotypic analysis of gene modifications across primate cell lines

• Evolutionary Medicine Applications:

  • Investigation of potential roles in orangutan-specific disease resistance

  • Comparative analysis of homologs in human disease contexts

  • Identification of adaptive variants that might inform human biomedical research

These research directions leverage the unique evolutionary position of orangutans to provide insights into both basic biology and potential biomedical applications, while focusing on the mechanistic understanding of UPF0708 protein function.