Recombinant Mouse UPF0600 protein C5orf51 homolog

What is UPF0600 protein C5orf51 homolog and what is its primary function?

UPF0600 protein C5orf51 homolog has been identified as a critical regulator in cellular trafficking pathways. Research demonstrates that this protein functions as a positive regulator of RAB7A in its shuttling between late endosomes and mitochondria to enable mitophagy (a selective form of autophagy targeting depolarized mitochondria) . The protein has been characterized as a component of the MON1-CCZ1 complex, where it interacts specifically with GDP-locked RAB7A . Furthermore, it has been identified as RAB7A-interacting MON1-CCZ1 complex subunit 1 (RIMOC1), highlighting its role in endosomal trafficking and mitochondrial quality control pathways .

How does the mouse UPF0600 protein C5orf51 homolog compare to the human ortholog?

The mouse UPF0600 protein C5orf51 homolog shares approximately 83% sequence identity with the human ortholog . This high conservation suggests functional importance across species. The human C5orf51 protein consists of 294 amino acids and is encoded by the C5orf51 gene (Entrez Gene ID: 285636) . Comparative analysis of these orthologs has revealed conserved domains critical for interactions with the RAB7A GTPase and the MON1-CCZ1 guanine nucleotide exchange factor (GEF) complex . This conservation facilitates the use of mouse models to study the function of this protein in relation to mitophagy and endosomal trafficking pathways.

What expression systems are optimal for recombinant UPF0600 protein C5orf51 homolog production?

Multiple expression systems have been evaluated for the production of recombinant UPF0600 protein C5orf51 homolog. The following table summarizes the effectiveness of different expression hosts:

What are the critical factors to consider when designing an expression vector for UPF0600 protein C5orf51 homolog?

When designing an expression vector for UPF0600 protein C5orf51 homolog, researchers should consider:

• Codon optimization: Adapting the coding sequence to the codon usage bias of the expression host can significantly improve protein yield .

• Affinity tags: Selection of appropriate fusion tags (His-tag, GST, etc.) affects downstream purification efficiency. For C5orf51, GST-tagged constructs have been successfully used in generating antibodies and for interaction studies .

• Promoter selection: Strong inducible promoters are generally preferred to control expression timing and level.

• Signal sequences: Including appropriate signal peptides for secretion or subcellular targeting when necessary.

• Cleavage sites: Incorporating protease recognition sequences for tag removal while preserving protein functionality.

Design of experiments (DoE) approaches are strongly recommended over one-factor-at-a-time optimization, as DoE can reveal interaction effects between different parameters and lead to more efficient optimization with fewer experiments .

How does C5orf51 homolog interact with the RAB7A-MON1-CCZ1 complex and what methods best detect these interactions?

C5orf51 has been identified as a specific interactor of GDP-locked RAB7A and the MON1-CCZ1 guanine nucleotide exchange factor (GEF) complex . These interactions can be studied using multiple complementary approaches:

• Proximity-dependent biotinylation (e.g., miniTurbo): This method successfully identified C5orf51 as an interactor of GDP-locked RAB7A . The approach allows for identification of protein-protein interactions in their native cellular context.

• Co-immunoprecipitation: Using antibodies against C5orf51 or components of the MON1-CCZ1 complex to pull down protein complexes, followed by western blotting to detect interacting partners .

• Yeast two-hybrid assays: For mapping specific interaction domains between C5orf51 and RAB7A or MON1-CCZ1 components.

• Fluorescence microscopy with co-localization analysis: To visualize the spatial relationships between these proteins under various cellular conditions, particularly during mitophagy induction.

Research shows that in the absence of C5orf51, localization of RAB7A on depolarized mitochondria is compromised, and RAB7A is degraded by the proteasome . Additionally, C5orf51 depletion inhibits ATG9A recruitment to depolarized mitochondria, further supporting its role in mitophagy .

What experimental approaches can be used to study the role of C5orf51 homolog in mitophagy?

To investigate the role of C5orf51 homolog in mitophagy, several experimental approaches have proven effective:

• CRISPR/Cas9-mediated knockout models: Generation of C5orf51-deficient cell lines allows for assessment of mitophagy impairment .

• Mitochondrial depolarization assays: Treating cells with CCCP (carbonyl cyanide m-chlorophenylhydrazone) to induce mitochondrial depolarization and trigger mitophagy, followed by assessment of mitophagy markers .

• Immunofluorescence microscopy: Visualizing the recruitment of RAB7A and ATG9A to depolarized mitochondria in the presence or absence of C5orf51 .

• Mitochondrial fractionation: Isolating mitochondria to analyze the recruitment of autophagy machinery components.

• Live-cell imaging: Monitoring the dynamics of mitophagy in real-time using fluorescently tagged proteins.

• Proteasomal inhibition experiments: Using MG132 (carbobenzoxy-Leu-Leu-leucinal) to block proteasomal degradation and analyze RAB7A stability in the absence of C5orf51 .

These approaches collectively enable comprehensive analysis of the molecular mechanisms by which C5orf51 regulates mitophagy through its interactions with RAB7A and the MON1-CCZ1 complex.

How can Design of Experiments (DoE) be applied to optimize recombinant C5orf51 homolog production and purification?

Design of Experiments (DoE) offers significant advantages over traditional one-factor-at-a-time approaches for optimizing recombinant protein production . For C5orf51 homolog optimization, a structured DoE approach could include:

• Factor identification: Key parameters affecting C5orf51 expression include temperature, induction time, inducer concentration, media composition, and cell density at induction .

• Screening design: Implement a fractional factorial design to identify the most significant factors with minimal experiments.

• Response surface methodology: For the critical factors identified, develop a response surface model to find optimal conditions.

• Validation experiments: Confirm the predicted optimal conditions through validation runs.

A typical DoE matrix for C5orf51 expression might look like:

Analysis of these results using statistical software can identify optimal conditions and interaction effects between variables, leading to significantly improved protein yields with fewer experiments than traditional optimization approaches .

What are the most effective strategies for maintaining protein stability and activity during purification of C5orf51 homolog?

Maintaining stability and activity of C5orf51 homolog during purification requires careful consideration of several factors:

• Buffer optimization: The buffer composition should be optimized for pH, ionic strength, and additives that stabilize the protein structure. For C5orf51, phosphate or Tris buffers with physiological salt concentration (150 mM NaCl) are commonly used .

• Protease inhibitors: Addition of protease inhibitor cocktails prevents degradation during cell lysis and early purification steps.

• Temperature management: Maintaining samples at 4°C throughout purification reduces degradation and denaturation.

• Reducing agents: Including DTT or β-mercaptoethanol can prevent oxidation of cysteine residues.

• Glycerol addition: 5-10% glycerol in storage buffers enhances protein stability.

• Chromatography selection: For C5orf51, a combination of affinity chromatography (using GST or His tags) followed by size exclusion chromatography has proven effective .

• Concentration techniques: Gentle concentration methods using centrifugal filter devices with appropriate molecular weight cutoffs minimize protein aggregation.

Activity assays should be performed after each purification step to monitor retention of biological function, particularly focusing on the protein's ability to interact with RAB7A and MON1-CCZ1 complex components .

What are common challenges in working with recombinant C5orf51 homolog and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant C5orf51 homolog:

• Low expression levels:

  • Problem: Poor protein yield despite optimization attempts

  • Solution: Consider switching expression systems, codon optimization, or fusion with solubility-enhancing tags

• Protein insolubility:

  • Problem: Formation of inclusion bodies in bacterial expression systems

  • Solution: Lower induction temperature (18-20°C), reduce inducer concentration, or explore refolding protocols if necessary

• Protein degradation:

  • Problem: Fragmentation during expression or purification

  • Solution: Use of protease-deficient hosts, addition of protease inhibitors, and optimization of purification speed

• Loss of interaction ability:

  • Problem: Recombinant protein fails to interact with known partners (RAB7A, MON1-CCZ1)

  • Solution: Ensure proper folding by using mammalian or insect cell expression systems that provide necessary post-translational modifications

• Antibody cross-reactivity:

  • Problem: Non-specific binding in immunological applications

  • Solution: Validate antibodies using knockout controls and blocking peptides, such as the C5orf51 control fragment recombinant protein

What are emerging research questions regarding C5orf51 homolog's role beyond mitophagy?

While C5orf51 homolog has been well-characterized in the context of mitophagy through its interactions with RAB7A and the MON1-CCZ1 complex , several emerging research questions point to broader roles:

• Neurodegenerative disease connections: Given the importance of mitophagy in neurodegenerative disorders like Parkinson's disease (which involves PINK1 and PARKIN) , how might C5orf51 dysfunction contribute to disease pathogenesis?

• Cancer metabolism: As mitochondrial dynamics are often dysregulated in cancer, what role might C5orf51 play in tumor cell metabolism and response to therapeutic interventions?

• Aging processes: Considering that mitochondrial quality control declines with age, how does C5orf51 function change throughout the lifespan?

• Additional trafficking pathways: Beyond mitophagy, does C5orf51 regulate other RAB7A-dependent trafficking pathways, such as autophagosome-lysosome fusion or endolysosomal trafficking?

• Interactome expansion: What other proteins beyond RAB7A and MON1-CCZ1 interact with C5orf51, and what cellular functions might these interactions regulate?

• Evolutionary conservation: How has C5orf51 function evolved across species, and what does this reveal about fundamental cellular processes?

Addressing these questions will require innovative approaches combining genomics, proteomics, and advanced imaging techniques to fully elucidate the multifaceted roles of this protein.

How can CRISPR/Cas9 gene editing be optimized for studying C5orf51 homolog function in vivo?

CRISPR/Cas9 gene editing offers powerful approaches for studying C5orf51 homolog function in vivo:

• Guide RNA design optimization:

  • Target conserved functional domains identified through alignment of human and mouse C5orf51 sequences

  • Use algorithms to minimize off-target effects while maximizing on-target efficiency

  • Design multiple gRNAs targeting different exons to ensure complete knockout

• Knock-in strategies:

  • Generate fluorescent protein fusions for live imaging of C5orf51 localization and dynamics

  • Create conditional alleles using loxP sites to enable tissue-specific or temporally controlled deletion

  • Introduce specific mutations to disrupt interaction interfaces with RAB7A or MON1-CCZ1 components

• Validation approaches:

  • Combine genomic verification (sequencing) with protein-level confirmation (Western blot)

  • Use C5orf51 antibodies for immunofluorescence to confirm subcellular localization changes

  • Perform functional assays measuring mitophagy efficiency in edited cells or tissues

• Phenotypic analysis:

  • Assess mitochondrial morphology and function through mitochondrial staining and respirometry

  • Measure autophagy flux using established reporters

  • Evaluate cell viability under mitochondrial stress conditions

• In vivo application:

  • Generate conditional knockout mouse models to study tissue-specific effects

  • Use AAV-delivered CRISPR systems for postnatal editing in specific tissues

These approaches will enable precise dissection of C5orf51 function in physiological contexts and may reveal unexpected roles beyond its established function in mitophagy regulation.