Recombinant Nomascus leucogenys UPF0767 protein C1orf212 homolog

What is Nomascus leucogenys and why is its genome significant for research?

Nomascus leucogenys (northern white-cheeked gibbon) is a critically endangered species of small apes endemic to the forests of southern China, Laos, and Vietnam. This species is classified as Critically Endangered on the IUCN Red List and is considered ecologically extinct in many regions . The genome of N. leucogenys is significant because gibbons represent a sister lineage to the "great apes" (Pongo, Gorilla, Pan, and Homo) in primate phylogeny, having diverged after the split from Old World Monkeys . Recent high-quality genome assembly efforts have produced chromosome-scale haplotype-phased assemblies with scaffold/contig N50 values of 124.2/102.2 Mb for Haplotype 1 and 121.2/85.67 Mb for Haplotype 2, with BUSCO assessment indicating completeness scores exceeding 95% . The genome contains approximately 18,925 protein-coding genes (23,783 mRNAs) with about 50% comprising repetitive elements . This genomic data offers valuable resources for studying primate evolution, genomic plasticity, and conservation genetics.

What is the UPF0767 protein C1orf212 homolog and what do we know about its structure?

The UPF0767 protein C1orf212 homolog in Nomascus leucogenys is a protein of currently unknown function ("UPF" designation) encoded by a gene homologous to the human C1orf212 (Chromosome 1 open reading frame 212). Based on available data, the full-length protein consists of 92 amino acids with the sequence: "MWPVFWTVVRTYAPYVTFPVAFVVGAVGYHLEWFIRGKDPQPVEEEKSISERREDRKLDE LLGKDHTQVVSLKDKLEFAPKAVLNRNRPEKN" . The protein is cataloged in UniProt with the accession number G1S9B8 . While the specific structure of this particular protein hasn't been fully characterized, insights might be drawn from related C1orf family proteins. For example, human C1ORF123 (a different member of C1orf proteins) has a structure with 2-fold internal symmetry and contains a zinc-binding domain in its N-terminal half that interacts with a zinc ion near a potential ligand binding cavity .

How does the Nomascus leucogenys UPF0767 protein compare to homologs in other species?

Comparative analysis across species reveals conservation patterns that may provide functional insights. While specific comparative data for the UPF0767 protein C1orf212 homolog is limited in the provided sources, we can observe that homologs exist across mammals, including rats . C1orf family proteins generally show evolutionary conservation, suggesting important biological functions. For context, when examining other proteins from Nomascus leucogenys, high degrees of conservation are often observed with human counterparts. For instance, GPIHBP1 from Nomascus leucogenys is 94% identical to human GPIHBP1, with specific amino acid differences that may affect protein processing (e.g., Arg38 in human GPIHBP1 corresponds to Gly38 in the Nomascus leucogenys version) . This high degree of conservation suggests that functional studies of the UPF0767 protein in accessible model systems may provide relevant insights about the Nomascus leucogenys homolog.

What expression systems are suitable for producing recombinant Nomascus leucogenys UPF0767 protein?

Several expression systems can be considered for producing the recombinant Nomascus leucogenys UPF0767 protein C1orf212 homolog, each with specific advantages depending on research objectives:

• Bacterial expression systems: While E. coli systems offer high yield and simplicity, they may not provide proper post-translational modifications that might be essential for the protein's function. This limitation should be considered when interpreting functional assays.

• Insect cell expression: Based on experiences with other primate proteins, Drosophila S2 cells have proven effective for expressing recombinant proteins from gibbons. For example, truncated versions of GPIHBP1 from primates have been successfully expressed using Drosophila S2 cells as fusion proteins with tags that facilitate purification . This system provides eukaryotic post-translational modifications while maintaining reasonable yields.

• Mammalian expression systems: These may offer the most physiologically relevant post-translational modifications but typically with lower yields. For studying protein-protein interactions or functional assays, this system may be preferable despite yield considerations.

When designing expression constructs, researchers should consider including purification tags that can be cleaved without altering the native N-terminus, as demonstrated in the successful expression strategy for gibbon GPIHBP1 . Additionally, identifying and potentially modifying protease-sensitive sites (as illustrated by the R38G modification made in GPIHBP1 to prevent unwanted cleavage) may be necessary to improve protein yield and integrity .

What purification strategies are effective for recombinant UPF0767 protein from gibbon sources?

Based on successful approaches with other recombinant proteins from gibbons, a multi-step purification process is recommended:

• Affinity chromatography: Using an N-terminal or C-terminal tag (His-tag, GST, or fusion partners like uPAR domain III) for initial capture . The tag selection should consider the physiochemical properties of the UPF0767 protein.

• Tag removal: Incorporating a specific protease cleavage site (e.g., enterokinase recognition sequence) between the tag and the target protein allows for tag removal while preserving the native termini .

• Ion-exchange chromatography: For the UPF0767 protein, cation-exchange chromatography may be effective for separating the target protein from contaminants and truncated versions, as demonstrated with gibbon GPIHBP1 .

• Size-exclusion chromatography: Critical for verifying the monomeric state of the purified protein and removing any aggregates or oligomers. This step is particularly important as some recombinant proteins from gibbons have shown susceptibility to multimerization, which can affect functional studies .

The purification protocol should include analytical methods to verify protein integrity, including mass spectrometry to confirm the exact molecular weight and N-terminal sequencing to ensure the preservation of the native N-terminus if that's critical for functional studies.

What analytical methods are essential for characterizing the recombinant UPF0767 protein?

Complete characterization of recombinant Nomascus leucogenys UPF0767 protein requires multiple analytical approaches:

• Structural integrity assessment:

  • SDS-PAGE for purity and apparent molecular weight

  • Size-exclusion chromatography to analyze oligomeric state and detect potential aggregation

  • Circular dichroism spectroscopy to evaluate secondary structure content

  • Thermal shift assays to assess protein stability under various buffer conditions

• Chemical characterization:

  • Mass spectrometry for accurate molecular weight determination and potential post-translational modifications

  • N-terminal sequencing to confirm intact N-terminus

  • If cysteine residues are present, analysis of disulfide bond formation

• Functional characterization:

  • Based on studies of other UPF proteins, assessing potential enzymatic activities

  • Investigating potential zinc-binding capacity, as observed in other C1orf family proteins

  • Analysis of potential binding partners through pull-down assays or surface plasmon resonance

• Biophysical characterization:

  • Dynamic light scattering for hydrodynamic radius measurement

  • Assessment of thermal stability using differential scanning calorimetry

  • If structural studies are planned, preliminary crystallization trials or NMR suitability tests

These analytical approaches will provide a comprehensive profile of the recombinant protein and serve as quality control metrics for subsequent functional studies.

What bioinformatic approaches can predict potential functions of the UPF0767 protein?

Given the limited direct experimental data on UPF0767 protein C1orf212 homolog function, computational approaches become critical for generating testable hypotheses:

• Sequence-based function prediction:

  • Search for conserved domains using tools like PFAM, SMART, or InterPro

  • Identify sequence motifs that suggest enzymatic activity, post-translational modification sites, or targeting signals

  • Predict secondary structure elements using tools like PSIPRED or JPred

• Structural bioinformatics:

  • Generate homology models based on proteins with similar sequences whose structures have been solved

  • Look for structural similarities to proteins of known function using tools like DALI or VAST

  • Identify potential binding pockets or catalytic sites using CASTp or similar tools

• Evolutionary analysis:

  • Perform phylogenetic analysis across primates and other mammals to identify patterns of selection

  • Use conservation analysis to identify functionally important residues

  • Examine synteny relationships to identify genomic context conservation

• Network-based predictions:

  • Use co-expression data from closely related species to predict functional associations

  • Analyze protein-protein interaction networks to place the protein in a functional context

  • Examine genomic context (neighboring genes) for functional clues

Based on analysis of other C1orf family proteins, potential functions might involve mitochondrial processes, as observed with C1ORF123 which shows involvement in mitochondrial oxidative phosphorylation . The presence of zinc-binding domains in some C1orf proteins also suggests potential roles in DNA/RNA binding or catalytic activities requiring metal cofactors .

How might evolutionary analysis of the UPF0767 protein inform functional studies?

Evolutionary analysis of the UPF0767 protein across primates can provide valuable insights into its function and importance:

• Conservation patterns:

  • Highly conserved regions likely represent functionally critical domains

  • Variable regions may indicate species-specific adaptations or relaxed selection

  • Comparing gibbon UPF0767 to homologs in humans and other primates can highlight gibbon-specific features

• Selection pressure analysis:

  • Calculate dN/dS ratios across the protein sequence to identify regions under positive or purifying selection

  • Sites under positive selection may indicate adaptation to species-specific functions

  • Sites under purifying selection likely represent core functional domains

• Lineage-specific changes:

  • Identify amino acid substitutions unique to the gibbon lineage

  • Correlate these changes with known gibbon-specific physiological traits

  • Use the high-quality gibbon genome assembly (with 95.9% BUSCO completeness) to ensure accurate genomic context

• Structural implications:

  • Map conservation patterns onto predicted structural models

  • Identify conserved surface patches that may represent interaction sites

  • Analyze the impact of lineage-specific substitutions on protein folding and stability

This evolutionary context is particularly valuable given that Nomascus leucogenys represents a critically endangered species. Understanding the evolution of its proteins provides both basic scientific knowledge and potentially valuable insights for conservation genetics efforts aimed at protecting the remaining gibbon populations .

What experimental approaches can elucidate the biological role of the UPF0767 protein in cellular processes?

A comprehensive experimental strategy to determine the biological function of the UPF0767 protein should include:

• Localization studies:

  • Express fluorescently tagged versions in mammalian cells to determine subcellular localization

  • Perform fractionation studies followed by western blotting to confirm localization biochemically

  • Use proximity labeling approaches (BioID or APEX) to identify neighboring proteins in the cellular environment

• Interaction partner identification:

  • Perform co-immunoprecipitation studies followed by mass spectrometry

  • Use yeast two-hybrid or mammalian two-hybrid screens to identify direct binding partners

  • Validate key interactions using recombinant proteins and biophysical methods (isothermal titration calorimetry, surface plasmon resonance)

• Loss-of-function studies:

  • Use CRISPR-Cas9 to generate knockout cell lines for the homologous gene

  • Perform RNA interference to achieve transient knockdown

  • Analyze resulting phenotypes through transcriptomics, proteomics, and cellular assays

• Structure-function analysis:

  • Generate point mutations at conserved residues identified through evolutionary analysis

  • Create domain deletion constructs to identify functional regions

  • Test mutants in rescue experiments in knockout backgrounds

• Physiological context:

  • Based on findings from C1ORF123 studies, investigate potential roles in mitochondrial function

  • Examine potential zinc-binding properties and their functional significance, as observed in related proteins

  • Test involvement in specific cellular pathways identified through interactome studies

When designing these experiments, researchers should consider the ethical implications of working with proteins from endangered species. Using homologs from closely related, non-endangered species for initial functional characterization may be appropriate, followed by validation with the Nomascus leucogenys protein for gibbon-specific features.

What are the challenges in structural biology approaches for the UPF0767 protein and how can they be addressed?

Structural characterization of the UPF0767 protein presents several challenges that require specific methodological approaches:

These methodological considerations should be tailored based on preliminary characterization of the recombinant protein. The experience with other gibbon proteins suggests that careful construct design and expression system selection are critical first steps in successful structural biology projects involving proteins from Nomascus leucogenys .

How does studying the UPF0767 protein contribute to our understanding of gibbon biology and evolution?

Research on the UPF0767 protein C1orf212 homolog from Nomascus leucogenys contributes significantly to multiple scientific domains:

The comprehensive characterization of proteins from the northern white-cheeked gibbon contributes to our understanding of this critically endangered species at a molecular level . With gibbons representing a unique evolutionary lineage among primates, detailed studies of their proteins provide insights into primate evolution and adaptation . The availability of high-quality genome assemblies with over 95% BUSCO completeness scores for Nomascus leucogenys provides an excellent foundation for accurate protein sequence identification and evolutionary analysis .

Future research should focus on comparative functional genomics, examining the role of the UPF0767 protein across different primate species to understand evolutionary conservation and divergence. Additionally, structural studies combined with functional characterization will help assign a definitive biological role to this protein of currently unknown function. These efforts will contribute not only to basic scientific knowledge but also potentially to conservation strategies for this critically endangered species.