Recombinant Mouse Uncharacterized protein C17orf78 homolog (Gm11437)

What is Gm11437 and what is its relationship to the newly discovered hormone famsin?

Gm11437 is a protein encoded by the Gm11437 gene in mice and serves as the precursor for the novel hormone famsin. According to recent research, famsin is a secreted protein released from Gm11437 after cleavage by furin. Famsin is secreted from the intestine and promotes metabolic adaptations during fasting periods .

The extracellular portion (amino acids 1-191) of Gm11437 constitutes the famsin hormone after cleavage. This was confirmed through mass spectrometry analysis which identified the precise amino acid sequence of the secreted hormone . Famsin derives its name from its protective effects during "famine" conditions, highlighting its physiological role in metabolic adaptation during nutritional scarcity .

Where is Gm11437 primarily expressed in mouse tissues?

Gm11437 shows highly tissue-specific expression patterns as demonstrated by qPCR analysis:

Within the intestinal tissue, Gm11437 is localized to the basolateral membrane of enterocytes and enteroendocrine cells (EECs), with enteroendocrine cells being the primary site of famsin production . Immunostaining and Gm11437-GFP knock-in mice confirmed this distribution pattern .

What is the homology between mouse Gm11437 and human C17orf78?

Mouse Gm11437 is the homolog of human C17orf78 (Chromosome 17 Open Reading Frame 78). The human C17orf78 gene is located on the long arm cytogenetic band 17q12, spanning base pair positions 37,375,985 to 37,392,708 on the forward strand . Similar to the expression pattern of mouse Gm11437, human C17orf78 is highly expressed in the small intestine, particularly in the duodenum .

Both proteins share similar structural organization and physiological functions. The tissue distribution of C17orf78, like Gm11437, shows high expression in the intestine with the human protein also being detectable at small expression levels in the testes and other tissues .

What is the mechanism of famsin cleavage from Gm11437 and which enzymes are involved?

Famsin is released from Gm11437 through a specific proteolytic cleavage process:

• Enzyme responsible: Furin, a proprotein convertase, is the primary enzyme that cleaves Gm11437 to release famsin .

• Cleavage site: The cleavage occurs at position K190/R191, as demonstrated by mutational analysis. A mutant form of Gm11437 (K190A/R191A, designated as AA) was resistant to furin-mediated cleavage .

• Cellular location of cleavage: Experimental evidence indicates that famsin can be cleaved from both plasma membrane-bound and intracellular Gm11437. This was demonstrated through biotin labeling of cell surface proteins, which detected biotin-labeled famsin in the medium, and through experiments showing that cellular Gm11437 could also produce famsin .

• Processing regulation: Inhibition of proprotein convertases abrogated famsin secretion, while overexpression of furin dramatically enhanced famsin levels. Other proprotein convertases did not show this effect, indicating the specificity of furin in this process .

The dual processing pathways (membrane and intracellular) suggest sophisticated regulation of famsin secretion in response to physiological stimuli.

How does famsin contribute to metabolic adaptation during fasting?

Famsin plays a crucial role in metabolic adaptation during fasting through several mechanisms:

• Gluconeogenesis promotion: Famsin enhances glucose production, which is essential during fasting to maintain blood glucose levels. This was demonstrated using purified famsin in experimental models .

• Gene expression regulation: Famsin administration restored the expression of genes involved in gluconeogenesis and ketogenesis in intestine-specific Gm11437 knockout (IKO) mice .

• Torpor induction: Famsin promotes torpor during fasting, a physiological state characterized by reduced metabolic rate and body temperature. Gm11437 intestine-specific knockout (IKO) mice showed decreased torpor-associated genes and were more susceptible to starvation during fasting than wild-type mice .

• Recovery of metabolic parameters: Administration of famsin to IKO mice restored:

  • Torpor response

  • Starvation resistance

  • Blood glucose levels

  • Expression of genes involved in gluconeogenesis and ketogenesis

These findings establish famsin as a fasting-induced hormone that plays a protective role during periods of nutritional scarcity by promoting metabolic adaptations that enhance survival.

What experimental design considerations are important when studying Gm11437/famsin function in vivo?

When designing experiments to study Gm11437/famsin function in vivo, researchers should consider the following key aspects:

• Experimental models:

  • Tissue-specific knockout models: Generate mice with tissue-specific knockout of Gm11437 in the intestine (IKO) or liver (LKO) to determine the contribution of different tissues to circulating famsin levels .

  • Protein expression models: Consider using Gm11437-GFP knock-in mice to track protein expression and localization .

• Experimental design structure:

  • Independent variable: For metabolic studies, consider fasting duration as the primary independent variable .

  • Dependent variables: Key metabolic parameters including blood glucose, plasma β-hydroxybutyrate, body temperature, and gene expression of gluconeogenic and ketogenic pathways .

  • Control groups: Include proper controls such as wild-type littermates with identical fasting protocols .

• Statistical considerations:

  • Determine appropriate sample sizes through power analysis before experimentation .

  • Use randomized block design to control for potential confounding variables like age or body weight .

• Validation approaches:

  • Rescue experiments: Test whether administration of recombinant famsin or adenoviral-mediated expression of wild-type Gm11437 can rescue phenotypes in knockout models .

  • Mutant controls: Include functionally impaired mutants (such as the K190A/R191A Gm11437 mutant) as negative controls in rescue experiments .

What are the recommended protocols for purifying recombinant Gm11437?

The purification of recombinant Gm11437 requires careful consideration of expression systems and purification strategies:

• Expression systems:

  • Insect cells: Sf9 or Hi-5 cells using baculovirus expression system have proven effective for producing glycosylated Gm11437/famsin .

  • Mammalian cells: HEK293T cells can be used for mammalian expression with proper post-translational modifications .

  • E. coli: Can be used for producing non-glycosylated protein with His tags for initial studies .

• Purification protocol:

  • For His-tagged protein:
    a. Express full-length (1-290 aa) mouse Gm11437 with an N-terminal His-tag in E. coli .
    b. Lyse cells in appropriate buffer (typically Tris-based).
    c. Purify using immobilized metal affinity chromatography (IMAC).
    d. Consider further purification using size exclusion chromatography if needed.

• Storage recommendations:

  • Store lyophilized protein at -20°C/-80°C upon receipt .

  • Reconstitute in Tris/PBS-based buffer, pH 8.0 with 6% Trehalose .

  • For working solutions, aliquot and store at 4°C for up to one week .

  • For long-term storage, add glycerol (final concentration 5-50%) and store at -20°C/-80°C .

  • Avoid repeated freeze-thaw cycles .

For studies requiring functionally active famsin, expression in insect or mammalian cells is recommended to ensure proper glycosylation, which appears essential for its biological activity .

How can researchers differentiate between membrane-bound Gm11437 and secreted famsin in experimental settings?

Differentiating between membrane-bound Gm11437 and secreted famsin requires specific methodological approaches:

• Antibody-based detection strategies:

  • Use antibodies specific to the extracellular domain (1-191 aa) to detect both Gm11437 and famsin.

  • Use antibodies against the C-terminal domain (post-cleavage portion) to specifically detect full-length or cleaved Gm11437 but not famsin .

  • In the Gm11437-GFP knock-in model, anti-GFP antibodies can detect the C-terminal fragment remaining after cleavage .

• Subcellular fractionation:

  • Separate membrane fractions from cytosolic and secreted proteins.

  • Western blotting of different fractions can reveal the distribution of full-length Gm11437 versus processed famsin .

• Cell surface biotinylation:

  • Label cell surface proteins with biotin.

  • Immunoprecipitate with streptavidin and detect with anti-famsin antibodies to identify membrane-derived famsin .

  • This approach successfully detected biotin-labeled famsin in medium, confirming cleavage from membrane-bound Gm11437 .

• Experimental protocol for distinguishing cellular vs. membrane processing:

  Step	Procedure	Expected Outcome
  1	Express Gm11437 in appropriate cells	Production of full-length protein
  2	Collect cell lysate and medium separately	Separation of cellular and secreted proteins
  3	Analyze both fractions by immunoblotting	Detect full-length Gm11437 in cells and famsin in medium
  4	Perform cell surface biotinylation	Label only surface-exposed proteins
  5	Analyze biotinylated proteins in medium	Detect famsin derived from membrane-bound Gm11437

This methodological approach was successfully used to demonstrate that famsin can be cleaved from both plasma membrane-bound and cellular Gm11437 .

What experimental controls should be included when studying Gm11437/famsin in metabolic research?

Robust experimental controls are essential when investigating Gm11437/famsin in metabolic research:

• Genetic controls:

  • Wild-type controls: Always include wild-type littermates as baseline controls .

  • Tissue-specific knockouts: Compare intestine-specific (IKO) with liver-specific (LKO) Gm11437 knockout mice to determine tissue-specific contributions .

  • Rescue controls: In gene knockout studies, include both:

    • Wild-type Gm11437 for rescue experiments

    • Mutant Gm11437 (K190A/R191A) that cannot produce famsin as a negative control

• Experimental design controls:

  • Between-subjects design: Randomly assign animals to experimental and control groups to minimize bias .

  • Randomized block design: Group subjects by relevant factors (e.g., age, weight) before random assignment to treatment conditions .

  • Time-course controls: Include measurements at multiple time points during fasting to capture dynamic responses .

• Methodological controls:

  • Vehicle controls: For famsin administration studies, include proper vehicle-only injections .

  • Sham procedures: For surgical interventions, include sham-operated controls.

  • Antibody specificity controls: Validate antibodies using knockout tissues or blocking peptides.

• Validation approach:

  • Independent methods: Verify findings using complementary techniques (e.g., both qPCR and immunoblotting) .

  • Dose-response relationships: Test multiple concentrations of recombinant famsin to establish physiological relevance .

  • Temporal controls: Compare fasting-induced versus fed states to establish physiological context .

These controls help ensure that observed effects are specifically attributable to Gm11437/famsin rather than experimental artifacts or confounding factors.

What are the best experimental approaches to identify physiological receptors and signaling pathways for famsin?

Identifying the receptors and signaling pathways for famsin requires a systematic experimental approach:

• Receptor identification strategies:

  • Affinity purification coupled with mass spectrometry:

    • Generate tagged recombinant famsin (e.g., His-tagged or Fc-fusion).

    • Incubate with membrane fractions from target tissues.

    • Perform cross-linking to stabilize interactions.

    • Purify complexes and identify binding partners by mass spectrometry.

  • Genetic screening approaches:

    • CRISPR-Cas9 screens in cell lines responsive to famsin.

    • Transcriptome analysis of famsin-responsive versus non-responsive cells.

• Signaling pathway analysis:

  • Phosphoproteomic analysis:

    • Treat cells with purified famsin for various time points (e.g., 5, 15, 30 minutes).

    • Perform global phosphoproteomic analysis to identify rapidly activated pathways.

    • Validate key phosphorylation events by Western blotting.

  • Transcriptional profiling:

    • RNA-seq analysis of tissues from wild-type versus Gm11437 knockout mice during fasting.

    • Identify differentially expressed genes associated with famsin signaling.

• Functional validation experiments:

  • Pathway inhibition studies:

    • Use specific inhibitors of candidate pathways to block famsin effects.

    • Test whether famsin's effects on gluconeogenesis and ketogenesis are abrogated.

  • Receptor knockout validation:

    • Generate knockouts of candidate receptors in cellular models.

    • Test famsin responsiveness in these receptor-deficient models.

• Experimental design considerations:

  • Use quasi-experimental designs when full randomization is not possible .

  • Include appropriate positive controls (e.g., known hormones that stimulate similar pathways).

  • Use time-course experiments to distinguish between primary and secondary signaling events.

The above methodological approach provides a comprehensive framework for delineating the molecular mechanisms of famsin action in metabolic regulation.

How should researchers design experiments to study the regulation of Gm11437 expression and famsin secretion?

Designing experiments to investigate the regulation of Gm11437 expression and famsin secretion requires attention to multiple aspects:

• Studying transcriptional regulation:

  • Promoter analysis:

    • Clone the Gm11437 promoter region upstream of reporter genes (luciferase, GFP).

    • Test response to fasting-related stimuli and transcription factors.

    • Create deletion constructs to identify key regulatory elements.

  • ChIP-seq analysis:

    • Identify transcription factors that bind the Gm11437 promoter during fed versus fasted states.

    • Validate binding using chromatin immunoprecipitation followed by qPCR.

• Investigating hormonal and nutritional regulation:

  • In vivo models:

    • Subject mice to various fasting durations (6h, 12h, 24h, 48h).

    • Measure Gm11437 mRNA expression and circulating famsin levels.

    • Test effects of insulin, glucagon, and other fasting-related hormones on expression.

  • Ex vivo models:

    • Intestinal organoids cultured under various nutrient conditions.

    • Primary enteroendocrine cells exposed to different nutrients and hormones.

• Studying post-translational regulation and secretion:

  • Pulse-chase experiments:

    • Use metabolic labeling to track Gm11437 processing and famsin secretion.

    • Determine half-life and secretion kinetics under different conditions.

  • Live-cell imaging:

    • Generate Gm11437-fluorescent protein fusions.

    • Track trafficking and secretion in real-time using confocal microscopy.

• Experimental design structure:

  • Independent variables: Fasting duration, hormone treatments, nutrient availability.

  • Dependent variables: Gm11437 mRNA levels, protein expression, famsin secretion.

  • Controls: Time-matched fed controls, vehicle treatments, irrelevant hormones.

• Methodological considerations:

  • True experimental designs with randomization are preferable when studying in vivo regulation .

  • For some mechanistic studies, quasi-experimental designs may be necessary when full randomization is not feasible .

  • Include both molecular (mRNA, protein) and functional (secretion) readouts for comprehensive analysis.

This structured approach enables researchers to systematically investigate the complex regulation of Gm11437 expression and famsin secretion under physiologically relevant conditions.