Recombinant Human UPF0420 protein C16orf58 (C16orf58)

What is the basic structure and characteristics of the C16orf58 protein?

C16orf58, also known as RUSF1 or RUS family member 1, is a human protein encoded by the C16orf58 gene spanning 18892 bp . The gene produces an mRNA transcript of 2760 bp that encodes a protein consisting of 468 amino acids . The protein contains a conserved domain of unknown function called DUF647, which is preserved across diverse species . Current predictive analyses suggest that C16orf58 is a cytoplasmic protein, likely residing in the endoplasmic reticulum, though its precise subcellular localization patterns may vary depending on cell type and physiological conditions . Despite being identified and sequenced, the tertiary structure and functional domains of C16orf58 remain largely uncharacterized, presenting significant opportunities for structural biology investigations.

What expression patterns does C16orf58 demonstrate across tissues and developmental stages?

While the search results don't provide comprehensive expression data for C16orf58 across different tissues and developmental stages, the evolutionary conservation of this protein suggests it may be expressed in multiple tissue types . The protein has been identified in the Cape elephant shrew (Elephantulus edwardii), classified as a "chromosome unknown open reading frame, human C16orf58" . Researchers investigating C16orf58 expression patterns would typically employ techniques such as quantitative PCR, RNA sequencing, or protein detection methods like Western blotting and immunohistochemistry across various tissues and developmental timepoints. Given recent findings about ER proteins and their dynamic regulation during cellular differentiation processes, including neuronal differentiation, C16orf58 expression may potentially be regulated during development and differentiation . A systematic analysis of expression patterns would provide valuable insights into potential tissue-specific functions of this conserved but poorly characterized protein.

What are the most effective protocols for expression and purification of recombinant C16orf58?

While no specific protocols for C16orf58 expression and purification are detailed in the search results, researchers can adapt established methods for similar proteins. For recombinant expression of human proteins that form inclusion bodies in E. coli, a protocol similar to that used for rhGM-CSF could be effective . This would involve: (1) Cloning the C16orf58 cDNA minus any signal sequence into an expression vector like pET40b(+), incorporating a C-terminal His-tag for purification ; (2) Transforming into E. coli and inducing expression, followed by inclusion body isolation ; (3) Solubilizing inclusion bodies in a buffer containing 6 M guanidine-HCl and 10 mM DTT ; (4) Refolding through dilution into a buffer containing arginine and a glutathione redox system ; (5) Purification using a combination of ion-exchange chromatography and size-exclusion chromatography . Protein purity can be assessed using SDS-PAGE and identity confirmed through mass spectrometry of peptide digests . This methodological approach would likely yield milligram quantities of purified recombinant C16orf58 from a single liter of E. coli culture, providing sufficient material for functional and structural studies.

How might C16orf58 be involved in endoplasmic reticulum function and autophagy pathways?

Recent research suggests a potential connection between C16orf58 and endoplasmic reticulum (ER) functions . While C16orf58 is not specifically mentioned in the proteomics studies of ER remodeling during neuronal differentiation described in the search results, its predicted localization to the ER and evolutionary conservation suggest it may play a role in fundamental ER processes . The ER undergoes significant remodeling during cellular differentiation, with different ER-shaping proteins showing distinct expression patterns . Autophagy pathways, particularly selective ER-phagy, contribute to this remodeling process . Given that ATG12-deficient neurons show accumulation of specific ER proteins, C16orf58 might potentially be regulated through similar pathways . Researchers investigating C16orf58 function should consider designing experiments to assess its potential roles in: (1) ER membrane shaping and remodeling; (2) Selective autophagy of ER components; (3) ER stress responses; and (4) Protein trafficking through the secretory pathway. Techniques such as co-immunoprecipitation, proximity labeling, and quantitative proteomics in autophagy-deficient systems would be valuable for elucidating C16orf58's functional interactions.

What structural features might explain the absence of C16orf58 orthologs in reptiles, birds, and amphibians?

The unusual evolutionary distribution of C16orf58, with apparent absence in reptiles, birds, and amphibians despite conservation from mammals to fish, plants, and fungi, presents an intriguing research question . Several hypotheses could explain this pattern: (1) Divergent Evolution – The protein may have evolved so dramatically in these lineages that sequence-based detection methods fail to identify the orthologs; (2) Functional Redundancy – Another protein may have assumed C16orf58's function in these lineages; (3) Gene Loss – The ortholog might have been lost in the common ancestor of reptiles, birds, and amphibians if its function became dispensable; (4) Technical Limitations – Incomplete genome assemblies or annotation errors could result in failure to detect existing orthologs . Researchers could address this question through: (1) More sensitive sequence- and structure-based homology searches using methods like PSI-BLAST, HHpred, or AlphaFold predictions; (2) Synteny analysis to identify genomic regions where C16orf58 orthologs would be expected; (3) Functional complementation studies to identify proteins in these species that can rescue C16orf58 deficiency in experimental models. Understanding this evolutionary puzzle could provide insights into both C16orf58 function and unique aspects of reptile, bird, and amphibian cell biology.

What are optimal strategies for generating functional C16orf58 knockout models?

Creating functional C16orf58 knockout models requires careful consideration of the gene's evolutionary conservation and potential fundamental cellular role . For mammalian systems, CRISPR-Cas9 genome editing offers the most precise approach. Researchers should design multiple guide RNAs targeting conserved regions within early exons of C16orf58, preferably within the DUF647 domain . To account for potential embryonic lethality, conditional knockout strategies using Cre-loxP or inducible degradation systems (e.g., auxin-inducible degron) may be preferable . Given C16orf58's potential role in the endoplasmic reticulum, researchers should monitor ER morphology and function in knockout models using fluorescent ER markers, electron microscopy, and ER stress reporters . For cross-species validation of phenotypes, researchers could leverage the evolutionary conservation of C16orf58, creating parallel knockout models in organisms like zebrafish (53% sequence identity) or Drosophila (40% sequence identity) to compare phenotypic effects . A complementation approach, where human C16orf58 is expressed in knockout models from different species, would provide valuable insights into functional conservation. As C16orf58 may interact with autophagy pathways, knockout phenotypes should be assessed under both basal conditions and stress conditions that activate autophagy .

How can protein-protein interaction partners of C16orf58 be effectively identified?

Identifying protein-protein interaction partners is crucial for understanding C16orf58 function. A multi-pronged approach combining different methodologies would provide the most comprehensive and reliable results. Researchers should consider implementing: (1) Proximity-dependent labeling methods such as BioID or TurboID, where C16orf58 is fused to a biotin ligase to biotinylate nearby proteins, which are then isolated and identified by mass spectrometry ; (2) Co-immunoprecipitation followed by mass spectrometry, using epitope-tagged C16orf58 expressed at near-endogenous levels ; (3) Yeast two-hybrid screening, which can detect both strong and weak interactions; (4) Crosslinking mass spectrometry to identify transient or context-dependent interactions . Given C16orf58's predicted ER localization, researchers should pay particular attention to interactions with known ER proteins, especially those involved in ER shaping (RTNs, REEPs, ATLs) and ER-phagy receptors (FAM134B, SEC62, TEX264) . Validation of identified interactions through reciprocal co-immunoprecipitation, FRET/BRET approaches, and functional studies in cells with C16orf58 depletion would strengthen the reliability of interaction data. Researchers should also consider performing these experiments under various cellular conditions, such as ER stress or nutrient deprivation, which might reveal context-dependent interactions .

What approaches can be used to determine the three-dimensional structure of C16orf58?

Determining the three-dimensional structure of C16orf58 is essential for understanding its molecular function. Researchers should consider a comprehensive structural biology approach: (1) X-ray crystallography remains the gold standard for high-resolution protein structures, requiring purified recombinant C16orf58 at concentrations of 5-20 mg/mL and screening of various crystallization conditions ; (2) Cryo-electron microscopy (cryo-EM) has emerged as a powerful alternative, particularly useful if C16orf58 forms larger complexes or if crystallization proves challenging ; (3) Nuclear magnetic resonance (NMR) spectroscopy could provide valuable information about protein dynamics and might be suitable for structured domains within C16orf58 . For optimal expression and purification, researchers should adapt protocols used for other difficult-to-express human proteins like GM-CSF, using E. coli expression systems with appropriate refolding from inclusion bodies or eukaryotic expression systems . To enhance crystallization success, researchers might consider using truncated constructs focusing on the conserved DUF647 domain, surface entropy reduction mutations, or fusion partners like T4 lysozyme . Computational approaches, including AlphaFold2 predictions, can provide valuable starting models to guide experimental design and interpret partial structural data. The structure should be validated using orthogonal approaches such as hydrogen-deuterium exchange mass spectrometry or small-angle X-ray scattering .

What can be inferred about C16orf58 function from its conserved domains across species?

The presence of the conserved DUF647 (Domain of Unknown Function 647) in C16orf58 across diverse species provides a starting point for functional inference despite its current status as a functionally uncharacterized domain . Researchers investigating this domain should pursue several complementary approaches: (1) Structure prediction using AlphaFold or RoseTTAFold, which might reveal structural similarity to functionally characterized domains despite low sequence similarity; (2) Conservation mapping to identify absolutely conserved residues that likely play critical roles in the domain's function; (3) Charge distribution and hydrophobicity analysis to predict potential interaction surfaces or membrane association properties . The evolutionary conservation of this domain from humans to plants suggests it likely performs a fundamental cellular function rather than a specialized multicellular role . Given C16orf58's predicted localization to the endoplasmic reticulum, the DUF647 domain might function in membrane association, protein-protein interactions within the ER, or even enzymatic activities related to ER function . Targeted mutagenesis of conserved residues within the DUF647 domain, followed by functional assays, would help validate hypothesized functions. Additionally, researchers should explore whether the domain undergoes post-translational modifications that might regulate its function, particularly in response to ER stress or altered cellular states that affect ER morphology and function .

What is the significance of C16orf58 conservation in the Cape elephant shrew (Elephantulus edwardii)?

The identification of a C16orf58 ortholog in the Cape elephant shrew (Elephantulus edwardii) adds an important data point to our understanding of this protein's evolutionary history . Elephant shrews (also called sengis) belong to the Afrotheria superorder of mammals, which diverged from other placental mammals approximately 90 million years ago . This phylogenetic positioning makes the elephant shrew C16orf58 ortholog particularly valuable for comparative analysis. Researchers should pursue: (1) Detailed sequence comparison between human and elephant shrew C16orf58 to identify conserved and divergent regions; (2) Analysis of selective pressures acting on different regions of the protein in this evolutionary lineage; (3) Investigation of whether the genomic context (neighboring genes) is also conserved, which would suggest conserved regulatory mechanisms . Given that elephant shrews have unusual physiological characteristics including high metabolic rates and precocial development, studying C16orf58 in this context might provide insights into whether the protein functions in metabolic regulation or developmental processes . Comparative expression studies examining C16orf58 expression patterns in elephant shrew tissues compared to human or mouse tissues could reveal conserved or divergent expression patterns. This evolutionary comparison, bridging across mammalian orders, could help distinguish essential functions of C16orf58 from lineage-specific adaptations.

How might C16orf58 be involved in ER-phagy and cellular stress response pathways?

Recent research on endoplasmic reticulum remodeling through selective autophagy (ER-phagy) provides a compelling framework for investigating potential C16orf58 functions . While C16orf58 is not specifically mentioned in the proteomics studies described in the search results, its predicted ER localization makes it a candidate for involvement in ER homeostasis and remodeling pathways . Researchers should design experiments to determine whether: (1) C16orf58 expression or localization changes during ER stress or autophagy induction; (2) C16orf58 interacts with known ER-phagy receptors (FAM134B, SEC62, TEX264) or core autophagy machinery ; (3) C16orf58 depletion affects ER morphology or the selective degradation of ER components. Methodologically, researchers could employ: (1) Live-cell imaging with fluorescently tagged C16orf58 and ER markers under various stress conditions; (2) Quantitative proteomics comparing wild-type and autophagy-deficient cells (e.g., ATG12-/- cells) to determine if C16orf58 accumulates when autophagy is impaired ; (3) Proximity labeling approaches to identify C16orf58 interaction partners under basal and stress conditions. Understanding C16orf58's potential role in ER-phagy would not only illuminate its function but could also provide insights into fundamental mechanisms of ER homeostasis, which are critically important in various diseases including neurodegenerative disorders and cancer.

What functional insights can be gained through CRISPR screening approaches targeting C16orf58?

CRISPR-based functional genomics approaches offer powerful strategies for uncovering C16orf58 functions through systematic phenotypic analysis. Researchers should implement: (1) Genome-wide CRISPR synthetic lethality screens in C16orf58-depleted backgrounds to identify genes whose loss exacerbates C16orf58 deficiency phenotypes, potentially revealing functional pathways and redundant mechanisms ; (2) Domain-focused CRISPR scanning, using guide RNAs targeting throughout the C16orf58 gene to identify critical functional regions through differential phenotypic impacts; (3) CRISPR activation (CRISPRa) and interference (CRISPRi) screens to identify genes whose up- or down-regulation modifies phenotypes of C16orf58 perturbation . Given the protein's evolutionary conservation and potential ER localization, researchers should focus on cellular phenotypes related to ER function, protein secretion, and autophagy when designing screens . Additionally, since C16orf58 is conserved in simple eukaryotes like yeast, parallel genetic interaction screens in model organisms could provide complementary insights . Analysis should pay particular attention to genetic interactions with ER-shaping proteins (RTNs, REEPs, ATLs) and autophagy factors, which might reveal functional relationships . Integration of CRISPR screening data with proteomic interaction data would provide a multi-dimensional view of C16orf58 function, highlighting both physical and functional interaction networks.