Recombinant Human Uncharacterized protein C1orf115 (C1orf115)

What is C1orf115 and what is its structural composition?

C1orf115 (chromosome 1 open reading frame 115) is a protein-coding gene located on human chromosome 1. Structurally, C1orf115 consists of an N-terminal intrinsically disordered region (IDR) and a C-terminal α-helix region . The C-terminal region can be further divided into:

• A positively charged amphipathic helix (AH) region

• A hydrophobic helix (HH) region

The positively charged amphipathic α-helix specifically binds phosphoinositides, particularly PI(4,5)P₂, which contributes to its plasma membrane localization . This structural organization is crucial for its cellular functions, particularly its involvement in clathrin-mediated endocytosis.

How is C1orf115 expressed in different tissues?

C1orf115 demonstrates a wide expression pattern across human tissues with notable tissue-specific abundance:

• Most abundant in the small intestine

• Expressed at variable levels in other tissues

This differential expression pattern, particularly the enrichment in intestinal tissue, correlates with its functional role in intestinal processes such as cholesterol efflux via ABCA1 and drug resistance mechanisms . The tissue-specific expression profile suggests potential specialized functions in intestinal physiology that may not be present in other tissues.

What homologs of C1orf115 exist in other species?

C1orf115 has identified homologs in several species:

Additionally, C1orf115 shares structural and functional similarities with FACI/C11orf86 . These similarities include:

• Conserved C-terminal α-helix region

• Ability to bind clathrin adaptors

• Involvement in endocytosis mechanisms

The conservation of C1orf115 across species suggests essential biological functions that have been maintained through evolution.

What are the known functions of C1orf115?

C1orf115 has multiple identified cellular functions:

• Endocytosis regulation: Functions as a potential endocytic adaptor that interacts with clathrin adaptors to undergo clathrin-mediated endocytosis (CME) .

• Drug resistance modulation: Regulates multidrug resistance through interaction with ATP-dependent membrane transporter ABCB1/MDR1. C1orf115 promotes ABCB1 translocation from the plasma membrane to the cytosol .

• Cholesterol efflux promotion: Induces ABCA1 transcription, subsequently promoting ABCA1-mediated cholesterol efflux in enterocytes, which contributes to intestinal HDL biogenesis .

• Membrane protein trafficking: May serve as an endocytic adaptor for specific cargo proteins, facilitating their internalization through CME .

These functions highlight C1orf115's importance in cellular homeostasis and response to external agents.

How does C1orf115 interact with clathrin adaptors and what is the functional significance?

C1orf115 contains specific motifs that mediate its interaction with clathrin adaptor proteins:

• An acidic dileucine-like motif "ExxxIL" within C1orf115 binds with the AP2 complex

• This interaction mediates C1orf115 localization to clathrin-coated pits (CCPs)

• Similar to FACI, C1orf115 binds directly to both the AP2 complex and PI(4,5)P₂

Functional significance:
This interaction is critical for C1orf115's role in drug resistance. Experimental evidence shows that:

• Ablation of the AP2-binding site on C1orf115 or direct knockdown of AP2 leads to abnormal CME of C1orf115

• This impairs its drug sensitivity phenotype and increases cellular drug resistance

• C1orf115 knockout Caco2 cells exhibit better survival under pyrvinium treatment compared to parental cells

• AP2-knockdown C1orf115-rescue cells show increased cell viability under pyrvinium treatment compared to control C1orf115-rescue cells

These findings suggest that C1orf115 could function as an endocytic adaptor, bridging the endocytic machinery and specific cargo proteins, similar to the function of FACI .

What is the role of C1orf115 in multidrug resistance mechanisms?

C1orf115 has been identified in CRISPR knockout screens as a critical regulator of multidrug resistance with several lines of evidence supporting this role:

• Loss-of-function phenotype: Loss of C1orf115 results in cellular resistance to multiple drugs, including anti-cancer drugs paclitaxel, vincristine, vinorelbine, and anti-helminthic drug pyrvinium .

• ABCB1 interaction model:

  • C1orf115 physiologically interacts with ABCB1/MDR1

  • It promotes ABCB1 translocation from the plasma membrane to the cytosol

  • In C1orf115-deficient cells, ABCB1 accumulates in the plasma membrane

  • Increased presence of ABCB1 in the plasma membrane leads to greater drug efflux

  • This enhances cellular resistance to drugs in C1orf115-deficient cells

• Experimental validation:

  • C1orf115 knockout Caco2 cells exhibited better survival under pyrvinium treatment

  • Overexpression of C1orf115 sensitized Caco2 cells to pyrvinium cytotoxicity

  • CME-deficient cells (Caco2-ΔC1) showed increased survival compared to Caco2-C1orf115 cells under pyrvinium treatment

This model explains how C1orf115 modulates drug resistance through ABCB1 and aligns with observations that abolishing the CME of C1orf115 enhances cellular drug resistance .

How does C1orf115 influence cholesterol efflux through ABCA1?

C1orf115 plays a significant role in cholesterol metabolism through its effects on ABCA1:

• Transcriptional regulation: RNA-seq analysis revealed that C1orf115 induces intestinal transcription of ABCA1, an ATP-dependent transporter .

• Tissue-specific effects: C1orf115 is most abundant in the small intestine, suggesting a specialized role in intestinal cholesterol homeostasis .

• Functional outcome:

  • Upregulation of ABCA1 promotes ABCA1-mediated cholesterol efflux in enterocytes

  • This process contributes to intestinal high-density lipoprotein (HDL) biogenesis

  • The mechanism represents a novel pathway for regulating cholesterol metabolism

• Broader implications: This function connects C1orf115 to lipid metabolism disorders and potentially to cardiovascular health, as HDL is known to have protective effects against atherosclerosis .

These findings suggest that C1orf115 could be a potential therapeutic target for disorders involving dysregulated cholesterol metabolism or impaired HDL biogenesis.

What is the connection between C1orf115 and neurodegenerative diseases?

Evidence from transcriptomic analysis has revealed a potential link between C1orf115 and Parkinson's disease (PD):

• Circular RNA associations:

  • A study examining circular RNA (circRNA) expression profiles in peripheral exosomes from PD patients identified differential expression of 99 circRNAs

  • Correlation analysis revealed that hsa-SCMH1_0001 has strong clinical relevance to PD

• Target gene identification:

  • Through computational analysis using multiple databases (TargetScanHuman, miRDB, and miRTarBase), 17 potential binding miRNAs of hsa-SCMH1_0001 with 149 potential target genes were identified

  • ARID1A and C1orf115 were found at the intersection of predicted target genes and differentially expressed mRNAs obtained by sequencing

• Expression pattern in disease:

  • Both hsa-SCMH1_0001 and its target genes, including C1orf115, were downregulated in PD patients

  • The expression levels of hsa-SCMH1_0001 negatively correlated with MDS-UPDRS III scores, indicating a relationship with disease severity

• Potential mechanism:

  • The study suggested that downregulation of hsa-SCMH1_0001 and its target genes ARID1A and C1orf115 may be involved in the pathogenesis of PD

  • This may represent a novel molecular pathway contributing to PD development

These findings suggest that C1orf115 could be a novel biomarker or therapeutic target for neurodegenerative diseases, particularly Parkinson's disease.

How does subcellular localization affect C1orf115 function?

C1orf115 demonstrates a complex subcellular distribution pattern that directly impacts its functionality:

• Multiple localization compartments:

  • Plasma membrane, particularly in clathrin-coated pits (CCPs)

  • Nucleolus and nuclear speckles within the nucleus

  • This multi-compartment distribution suggests diverse functions

• Structural determinants of localization:

  • The C-terminal α-helix dictates its localization to plasma membrane, nucleolus, and nuclear speckles

  • The positively charged amphipathic helix (AH) region specifically binds phosphoinositides, particularly PI(4,5)P₂, mediating plasma membrane association

• Post-translational regulation:

  • Acetylation of the positively charged amphipathic α-helix redistributes C1orf115 from the plasma membrane and nucleolus to nuclear speckles

  • This post-translational modification serves as a regulatory switch for C1orf115 localization and consequently its function

• Functional implications:

  • Plasma membrane localization is essential for its role in clathrin-mediated endocytosis and drug resistance modulation

  • Nuclear localization may be related to its roles in gene regulation, potentially explaining its effect on ABCA1 transcription

  • Dynamic shuttling between compartments allows C1orf115 to perform distinct functions based on cellular needs

This dynamic localization pattern provides a mechanism by which cells can regulate C1orf115 function through controlling its subcellular distribution.

What experimental approaches are recommended for studying C1orf115 function?

Based on successful methodologies documented in the literature, several experimental approaches are recommended for investigating C1orf115:

• Genetic manipulation techniques:

  • CRISPR-Cas9 gene editing to generate knockout cell lines

  • Stable overexpression of tagged C1orf115 (Flag-C1orf115 or mEmerald-C1orf115) for localization and functional studies

• Transcriptomic analysis:

  • RNA-seq analysis to identify genes regulated by C1orf115

  • Quantitative polymerase chain reaction (qPCR) for validation of expression changes

• Protein interaction studies:

  • Affinity-purification and mass spectrometry (AP-MS) to identify protein-protein interactions

  • SAINT (Significance Analysis of INTeractome) analysis for statistical validation of protein interactions

  • Co-immunoprecipitation experiments to confirm direct interactions

• Functional assays:

  • Cell viability assays under drug treatment to assess impact on drug resistance

  • Cholesterol efflux assays to measure ABCA1-mediated cholesterol transport

  • siRNA knockdown of interaction partners (e.g., AP2) to study the functional significance of protein-protein interactions

• Microscopy techniques:

  • Fluorescence microscopy to visualize subcellular localization

  • Live-cell imaging to monitor dynamic trafficking events (as evidenced by Video S2 mentioned in the literature)

• Biochemical analysis:

  • Western blotting to assess protein expression levels

  • Protein modification analysis to study post-translational modifications like acetylation

These methodologies provide a comprehensive toolkit for investigating the structural, molecular, and functional aspects of C1orf115 in various cellular contexts.

What are the challenges in expressing and purifying recombinant C1orf115?

Researchers working with recombinant C1orf115 should be aware of several challenges:

• Structural complexity:

  • The N-terminal intrinsically disordered region (IDR) may lead to aggregation and instability during expression and purification

  • The C-terminal α-helix region requires proper folding for functionality

• Solubility concerns:

  • Based on studies of related proteins, mutations in the conserved residues can lead to insolubility indicating inefficient folding

  • Special solubilization strategies may be required to maintain protein in solution

• Post-translational modifications:

  • Acetylation of the positively charged amphipathic α-helix affects C1orf115 localization and function

  • Expression systems may not reproduce native post-translational modifications, potentially affecting protein behavior

• Expression system selection:

  • E. coli may not be optimal for expressing mammalian proteins with complex folding requirements

  • Eukaryotic expression systems like insect cells or mammalian cells may better preserve native structure and function

• Purification strategy optimization:

  • Affinity tags should be carefully selected to minimize interference with protein function

  • Multi-step purification protocols may be necessary to achieve high purity while maintaining native conformation

• Functional assay development:

  • Establishing reliable assays to verify the functionality of purified recombinant C1orf115

  • Assessment of binding to known partners like AP2 complex or phosphoinositides as functional readouts

Addressing these challenges requires optimization of expression conditions, careful selection of expression systems, and potentially the use of solubility tags or fusion partners to enhance expression and solubility.

How might C1orf115 be targeted therapeutically in disease states?

Based on its roles in drug resistance and cholesterol metabolism, C1orf115 represents a potential therapeutic target:

• Cancer therapy applications:

  • Enhancing C1orf115 function could potentially increase tumor cell sensitivity to chemotherapeutic drugs

  • Targeting C1orf115 might reverse multidrug resistance in cancer cells, particularly those with upregulated ABCB1/MDR1

• Metabolic disease applications:

  • Modulating C1orf115 activity could potentially enhance cholesterol efflux through ABCA1

  • This approach might benefit conditions characterized by impaired reverse cholesterol transport or reduced HDL levels

• Neurodegenerative disease applications:

  • Rescuing C1orf115 expression or function might be beneficial in Parkinson's disease, where it is downregulated

  • Understanding the relationship between C1orf115 and neurodegeneration could reveal novel therapeutic strategies

• Potential therapeutic approaches:

  • Small molecule modulators that enhance C1orf115 activity or stability

  • Peptide-based therapeutics that mimic C1orf115 binding to key partners

  • Gene therapy approaches to restore C1orf115 expression in deficient states

  • Targeting post-translational modifications like acetylation to modulate C1orf115 function

• Challenges in therapeutic development:

  • Achieving specificity given C1orf115's multiple cellular roles

  • Developing delivery strategies that target relevant tissues (intestine, brain, etc.)

  • Balancing effects on drug resistance and cholesterol metabolism pathways

These therapeutic strategies remain theoretical at present, and additional research is needed to validate C1orf115 as a viable drug target in specific disease contexts.