Recombinant Mouse Transmembrane protein C9orf71 homolog

What is the Recombinant Mouse Transmembrane protein C9orf71 homolog and how does it relate to C9orf72?

The Recombinant Mouse Transmembrane protein C9orf71 homolog (UniProt: Q8C353) is a mouse protein that shares sequence homology with the human C9orf71 gene product. While specific research on C9orf71 is more limited, it's important to understand that C9orf72, another member of the chromosome 9 open reading frame family, has been extensively studied due to its relevance to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) . Both proteins belong to the same gene family, though they have distinct functions. C9orf72 contains a DENN domain that places it in the FLCN branch of the DENN domain family, suggesting a role in membrane trafficking processes .

What are the recommended storage and handling protocols for Recombinant Mouse Transmembrane protein C9orf71 homolog?

The recombinant protein should be stored in a Tris-based buffer with 50% glycerol at -20°C, or at -80°C for extended storage periods . Repeated freeze-thaw cycles should be avoided to maintain protein integrity. For working applications, prepare smaller aliquots and store at 4°C for up to one week . When handling the protein, maintain sterile conditions and use appropriate personal protective equipment. The protein's stability may vary depending on experimental conditions, so preliminary validation of activity in your specific experimental system is recommended before conducting extensive studies.

How can researchers effectively validate interactions between C9orf71 homolog and potential binding partners?

To rigorously validate protein-protein interactions involving the C9orf71 homolog, researchers should employ multiple complementary techniques. Co-immunoprecipitation (Co-IP) assays can identify interactions in cell lysates, as demonstrated with C9orf72 and its binding partners . For in situ validation, proximity ligation assays (PLA) offer visualization of interactions within intact cells with high sensitivity and specificity . GST pulldown assays using recombinant proteins can confirm direct binding and map interaction domains . For higher resolution analysis, structural biology approaches such as X-ray crystallography or cryo-electron microscopy would provide detailed information about binding interfaces. Validation across multiple cell types and under different physiological conditions is crucial for establishing biological relevance.

What experimental approaches can determine if C9orf71 homolog functions in autophagy pathways similar to C9orf72?

To investigate a potential role of C9orf71 homolog in autophagy, researchers should implement a systematic approach combining genetic manipulation and functional assays. Begin with knockdown or knockout studies using siRNA or CRISPR-Cas9 in relevant cell lines, then assess autophagy markers like LC3-II conversion and p62 accumulation via Western blotting . Fluorescence microscopy with EGFP-LC3 can visualize autophagosome formation under basal conditions and following treatment with autophagy inducers such as rapamycin or Torin1 . Flux assays using bafilomycin A1 would distinguish between enhanced autophagosome formation and impaired clearance. Additionally, investigate potential interactions with known autophagy regulators like the ULK1 complex through co-immunoprecipitation assays . Compare findings directly with parallel experiments on C9orf72 to establish similarities or differences in function.

How should researchers design experiments to investigate the subcellular localization of C9orf71 homolog?

A comprehensive investigation of C9orf71 homolog subcellular localization requires multiple complementary approaches. Begin with immunofluorescence microscopy using validated antibodies against the recombinant protein in fixed cells, co-staining with markers for different cellular compartments (lysosomes, endosomes, Golgi, ER). Confirm findings with live-cell imaging using fluorescently tagged C9orf71 construct, while ensuring the tag doesn't disrupt protein localization or function. For higher resolution, employ super-resolution microscopy techniques such as STED or STORM. Biochemical fractionation of cellular components followed by Western blotting provides quantitative data on protein distribution across compartments. Based on insights from C9orf72 research, particular attention should be paid to lysosomal localization, as C9orf72 functions at lysosomes as part of a complex with SMCR8 and WDR41 proteins .

What is known about the structural domains of mouse C9orf71 homolog and how do they compare to C9orf72?

The mouse Transmembrane protein C9orf71 homolog has a known amino acid sequence of MQNRTGLILCALSLLTGFLMICLGGFFIS, indicating its transmembrane nature . Unlike C9orf72, which possesses a well-characterized DENN domain implicated in membrane trafficking processes , the specific structural domains of C9orf71 have not been as extensively characterized in the available literature. C9orf72 shows structural homology to the FLCN branch of the DENN domain family, particularly close to FLCN Interacting Proteins 1 and 2 (FNIP1/2) . To further elucidate the structural domains of C9orf71 homolog, researchers should perform bioinformatic analyses including sequence alignments with related proteins, secondary structure predictions, and homology modeling based on proteins with solved structures. Experimental approaches such as limited proteolysis combined with mass spectrometry would help identify stable domains.

How can researchers differentiate between the functions of C9orf71 homolog and C9orf72 in experimental settings?

To experimentally distinguish between C9orf71 homolog and C9orf72 functions, researchers should implement a comparative functional analysis approach. First, generate specific knockdowns or knockouts for each protein individually and in combination, followed by rescue experiments with each protein to identify unique and overlapping functions. Conduct comprehensive interactome analyses using techniques such as immunoprecipitation followed by mass spectrometry to identify distinct binding partners. C9orf72 interacts with Rab1a and the ULK1 complex in autophagy regulation , so researchers should test if C9orf71 shares these interactions or has unique binding partners. Perform domain swap experiments between the two proteins to determine which regions confer specific functions. Monitor phenotypic outcomes across multiple cellular processes including autophagy, lysosomal function, and membrane trafficking, as C9orf72 has been implicated in these pathways .

What experimental techniques are most appropriate for investigating the potential role of C9orf71 homolog in lysosomal function?

To investigate lysosomal function of C9orf71 homolog, researchers should employ a multi-faceted experimental approach. Begin with lysosomal staining using LysoTracker or immunofluorescence for LAMP1/LAMP2 in cells with C9orf71 knockout or overexpression to assess changes in lysosomal number, size, and distribution. Measure lysosomal pH using ratiometric probes to determine if C9orf71 affects acidification. Assess lysosomal enzyme activity (e.g., cathepsins) using fluorescent substrates in live cells or enzymatic assays in cell lysates. To connect with potential autophagy roles, monitor autophagic flux by measuring LC3-II and p62 levels in the presence of lysosomal inhibitors . Investigate mTORC1 signaling, as C9orf72 has been implicated in this pathway at lysosomes . For a direct comparison with C9orf72, perform parallel experiments and complementation assays to determine functional overlap or divergence in lysosomal regulation.

How can researchers develop and validate antibodies specific to mouse C9orf71 homolog for immunological applications?

Developing specific antibodies against mouse C9orf71 homolog requires a strategic approach to ensure specificity and functionality across applications. Begin by identifying unique epitopes through comparative sequence analysis with related proteins, particularly C9orf72, to avoid cross-reactivity. Utilize the known amino acid sequence (MQNRTGLILCALSLLTGFLMICLGGFFIS) to design peptide antigens from unique regions, preferably from predicted exposed domains. Generate both monoclonal and polyclonal antibodies for complementary applications. Rigorous validation should include Western blotting comparing wild-type and knockout/knockdown samples, immunoprecipitation efficiency testing, immunofluorescence with appropriate subcellular markers, and cross-reactivity assessment against related proteins. For experiments requiring quantitative analysis, establish standard curves using the recombinant protein at known concentrations. Due to the challenges with antibody specificity noted in C9orf72 research , consider alternative approaches such as epitope tagging of endogenous protein using CRISPR-Cas9 when appropriate.

What are the critical considerations when designing CRISPR-Cas9 knockout models for studying C9orf71 homolog function?

When designing CRISPR-Cas9 knockout models for C9orf71 homolog, researchers must address several critical considerations to ensure valid experimental outcomes. First, perform comprehensive guide RNA design with multiple bioinformatic tools to identify target sequences with minimal off-target effects, preferably targeting early exons or essential domains. Include appropriate controls: wild-type cells, cells transfected with non-targeting guides, and independent knockout clones generated with different guide RNAs. Validation of knockout should combine genomic verification (sequencing of the targeted region), transcript analysis (RT-qPCR), and protein detection (Western blotting if reliable antibodies are available). If complete knockout causes cell lethality, consider inducible or conditional systems. Based on observations from C9orf72 knockout studies, which revealed phenotypes in immune cells and neurons , researchers should be prepared to characterize cell type-specific effects in various tissues and potentially develop tissue-specific knockout models to distinguish between primary and secondary effects.

How can researchers investigate whether C9orf71 homolog functions as part of a protein complex similar to C9orf72?

To determine if C9orf71 homolog functions within a protein complex similar to the C9orf72-SMCR8-WDR41 complex , implement a systematic biochemical and proteomic approach. Begin with co-immunoprecipitation followed by mass spectrometry to identify potential binding partners in an unbiased manner. Validate high-confidence interactions with reciprocal co-immunoprecipitation, proximity ligation assays, and in vitro binding assays using recombinant proteins. Assess complex stoichiometry through analytical size exclusion chromatography or native mass spectrometry. For structural characterization, employ cryo-electron microscopy or X-ray crystallography of the purified complex. Functional studies should include simultaneous knockdown of multiple complex components to identify collaborative functions. Based on C9orf72 research, specifically investigate potential interactions with SMCR8 and WDR41 , as well as the ULK1 autophagy initiation complex components (ULK1, FIP200, ATG13) . Examine whether complex formation is regulated by cellular conditions such as nutrient availability, similar to observations with C9orf72 complexes.

How should researchers correlate findings from mouse C9orf71 studies to human disease mechanisms?

When translating findings from mouse C9orf71 homolog research to human disease contexts, researchers must implement a rigorous cross-species validation approach. Begin with comparative sequence and structural analysis between mouse and human proteins to identify conserved domains and potential functional differences. Conduct parallel experiments in both mouse and human cellular models (including primary cells and iPSC-derived cells) to confirm conservation of molecular mechanisms. For disease relevance, examine expression patterns of C9orf71 in human patient samples across different tissues, particularly in diseases where related proteins like C9orf72 play a role, such as ALS and FTD . Investigate genetic associations through database mining of genome-wide association studies and exome sequencing data. Consider generating humanized mouse models expressing the human C9orf71 variant for in vivo validation. Given C9orf72's role in autophagy and lysosomal function , special attention should be paid to whether C9orf71 influences similar pathways relevant to neurodegenerative diseases characterized by protein aggregation.

What methodological approaches can determine if C9orf71 homolog has GTPase regulatory functions similar to other DENN domain-containing proteins?

To investigate potential GTPase regulatory functions of C9orf71 homolog, researchers should implement biochemical and cell-based assays specific for GEF (guanine nucleotide exchange factor) or GAP (GTPase-activating protein) activity. Begin with in vitro nucleotide exchange assays using purified recombinant C9orf71 protein and candidate GTPases (particularly Rab family members, given C9orf72's interaction with Rab1a ). Measure GTP binding and hydrolysis rates using fluorescent or radioactive GTP analogs. In cellular contexts, employ FRET-based biosensors to monitor GTPase activation states in response to C9orf71 manipulation. Perform co-localization studies with specific GTPases in their active and inactive forms. Based on C9orf72's structural similarity to DENN domain proteins and its role as a Rab1a effector , systematically test interactions with Rab GTPases using proximity ligation assays and co-immunoprecipitation. For suspected GTPase targets, compare the effects of constitutively active and dominant-negative GTPase mutants in cells with and without C9orf71 to establish functional relationships.