Recombinant Mouse Uncharacterized protein C18orf25 homolog

What is C18orf25 and what are its main structural features?

C18orf25 is a protein homologous to Arkadia (RNF111), which is known as an E3 ubiquitin ligase with SUMO-interaction motifs (SIMs). The critical structural distinction is that C18orf25 lacks the entire C-terminal RING domain that RNF111 possesses, which is required for ubiquitin binding. This structural difference suggests that C18orf25 lacks ubiquitination activity and instead likely functions as an adaptor or signaling scaffold in cellular processes .

For experimental characterization of C18orf25's structure, researchers typically use a combination of:

• X-ray crystallography to determine three-dimensional structure

• Domain mapping through truncation analyses

• Comparative modeling with RNF111

• Mass spectrometry to confirm post-translational modifications

When designing experiments to investigate C18orf25 structure-function relationships, researchers should consider both wild-type proteins and strategic deletion constructs targeting specific domains to assess their functional importance.

What phenotypes are observed in C18orf25 knockout mouse models?

C18orf25 knockout mice exhibit several distinct phenotypes that provide insights into the protein's physiological functions:

When designing experiments with these models, researchers should include appropriate controls and employ multiple measurement techniques to fully characterize the phenotypes. Age and sex-matched controls are essential, as metabolic and muscle phenotypes can vary significantly with these parameters .

How should recombinant C18orf25 protein be prepared for functional studies?

For optimal preparation of recombinant C18orf25 protein:

• Expression system selection: Mammalian expression systems are preferred for proper post-translational modifications, particularly for studying phosphorylation events. HEK293 cells are commonly used.

• Purification strategy:

  • Affinity tags (His-tag, FLAG-tag) facilitate purification while minimizing interference with protein function

  • For carrier-free preparations, avoid BSA additions that might interfere with downstream applications

  • Consider size-exclusion chromatography as a final purification step to ensure homogeneity

• Quality control methods:

  • SDS-PAGE to verify size and purity

  • Western blotting to confirm identity

  • Mass spectrometry to verify sequence integrity and post-translational modifications

  • Circular dichroism to assess proper folding

• Storage recommendations:

  • Store in appropriate buffer (typically containing MES and NaCl)

  • Use 0.2 μm filtered solutions to ensure sterility

  • Aliquot to avoid repeated freeze-thaw cycles

  • Store at -80°C for long-term stability

When selecting between carrier-free versus BSA-containing preparations, consider that carrier-free versions are preferable for applications where BSA might interfere, while BSA-containing preparations offer enhanced stability for cell culture applications or ELISA standards .

What is the significance of S67 phosphorylation on C18orf25 function?

The phosphorylation of serine 67 (S67) on C18orf25 represents a critical regulatory mechanism for this protein's function:

AMPK directly catalyzes the phosphorylation of S67 on C18orf25, and this post-translational modification is exercise-regulated. Experimental evidence demonstrates that this phosphorylation is functionally crucial - re-expression of a phospho-mimetic S66/67D mutant in C18orf25 knockout mice successfully rescues skeletal muscle contractile defects, whereas a phospho-dead S66/67A mutant fails to restore normal function .

For investigating S67 phosphorylation experimentally:

• Phospho-specific antibodies can detect native phosphorylation states

• Site-directed mutagenesis to generate:

  • Phospho-mimetic mutants (S67D or S66/67D)

  • Phospho-dead mutants (S67A or S66/67A)

• In vitro kinase assays with purified AMPK to confirm direct phosphorylation

• Mass spectrometry to quantify phosphorylation stoichiometry

• Functional rescue experiments using phospho-mutants in knockout backgrounds

The phosphorylation state analysis should be performed under various physiological conditions, particularly comparing resting versus post-exercise states, to understand the dynamic regulation of this modification.

What methodologies are optimal for studying C18orf25 protein-protein interactions?

Several complementary methodologies should be employed to comprehensively map the protein-protein interaction landscape of C18orf25:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • This is the gold standard for unbiased identification of protein interactions

  • Tagged C18orf25 (FLAG, HA, or GFP) is expressed in relevant cell types

  • Protein complexes are isolated via the tag and identified by mass spectrometry

  • Include appropriate controls (empty vector, unrelated protein) to filter non-specific interactions

  • Comparative analysis between wild-type and phospho-mutants can reveal phosphorylation-dependent interactions

• Proximity-dependent labeling methods:

  • BioID or TurboID fusion proteins to identify proximal proteins in living cells

  • APEX2 for temporally controlled labeling

  • These methods capture transient interactions that may be missed by AP-MS

• Co-immunoprecipitation followed by Western blotting:

  • For validation of specific interactions identified by high-throughput methods

  • Can be performed with endogenous proteins using specific antibodies

• Yeast two-hybrid or mammalian two-hybrid:

  • For direct binary interaction testing

  • Useful for domain mapping to identify interaction interfaces

• Fluorescence-based interaction assays:

  • FRET or BiFC to visualize interactions in living cells

  • Allows for spatial and temporal resolution of interactions

When analyzing interaction data, researchers should employ quantitative proteomics approaches and statistical filtering to distinguish true interactors from background. Crosslinking mass spectrometry can provide additional structural insights into interaction interfaces .

How can researchers investigate the molecular mechanisms linking C18orf25 to calcium handling in skeletal muscle?

To investigate the molecular mechanisms by which C18orf25 regulates calcium handling in skeletal muscle:

• Calcium imaging methodologies:

  • Use fluorescent calcium indicators (Fura-2, Fluo-4) in isolated muscle fibers

  • Measure SR calcium release and reuptake kinetics

  • Compare fast-twitch versus slow-twitch fibers, as C18orf25 KO specifically affects fast-twitch fiber calcium cycling

  • Employ electrical stimulation protocols that mimic physiological activation patterns

• Molecular characterization approaches:

  • Phosphoproteomic analysis of calcium handling proteins (RyR1, SERCA, calsequestrin) in wild-type versus KO tissue

  • Co-immunoprecipitation to detect physical interactions between C18orf25 and calcium regulatory proteins

  • In vitro reconstitution assays with purified components to test direct effects

• Functional rescue experiments:

  • Re-expression of wild-type C18orf25 versus phospho-mutants in KO background

  • Targeted expression in specific fiber types using appropriate promoters

  • Temporal control of expression using inducible systems to distinguish developmental versus acute effects

• Analysis of PKA signaling:

  • Measure PKA activity using FRET-based reporters in living fibers

  • Assess PKA-dependent phosphorylation of calcium handling proteins

  • Test whether PKA activators can bypass the requirement for C18orf25

When designing these experiments, researchers should consider fiber type-specific effects and employ both biochemical and physiological readouts to establish causative relationships rather than correlations .

What experimental approaches can differentiate the adaptor/scaffold function of C18orf25 from potential enzymatic activities?

To definitively characterize C18orf25's function as an adaptor/scaffold versus potential enzymatic activities:

• In vitro activity assays:

  • Test for ubiquitination activity using purified components (E1, E2, ubiquitin, potential substrates)

  • Assess potential deubiquitinating activity

  • Examine for other enzymatic functions (phosphatase, kinase activity)

  • Include appropriate positive controls (RNF111 for ubiquitination)

• Structure-function analysis:

  • Systematic domain deletion/mutation constructs

  • Focus on regions conserved with RNF111 outside the RING domain

  • Complementation assays in knockout cells with various constructs

  • Protein interaction mapping with each construct

• Scaffolding activity characterization:

  • Co-expression of interaction partners with/without C18orf25

  • Analysis of complex formation dynamics by size exclusion chromatography

  • Visualization of protein complexes by single-particle cryo-EM

  • In vitro reconstitution of multiprotein complexes using purified components

• Substrate trapping approaches:

  • If enzymatic activity is suspected, use catalytically dead mutants to trap substrates

  • Chemical crosslinking combined with mass spectrometry to identify transient interactions

• Evolutionary analysis:

  • Compare C18orf25 sequence and function across species to identify conserved features

  • Assess selective pressure on different domains to infer functional importance

When interpreting results, researchers should consider that adaptor/scaffold functions can be as specific and regulated as enzymatic activities, with equally important physiological consequences .

What are the optimal methods for validating C18orf25 antibodies for research applications?

Comprehensive antibody validation is critical for obtaining reliable results when studying C18orf25:

• Specificity testing:

  • Western blot analysis in wild-type versus knockout tissues

  • Preabsorption with recombinant antigen to confirm specific binding

  • Testing in multiple tissues with known expression patterns

  • Comparison of multiple antibodies targeting different epitopes

• Application-specific validation:

  • For immunoprecipitation: verify pull-down efficiency and specificity

  • For immunofluorescence: compare staining patterns with GFP-tagged expressed protein

  • For flow cytometry: include isotype controls and blocking peptides

  • For chromatin immunoprecipitation: include IgG controls and test in knockout cells

• Phospho-specific antibody validation:

  • Test antibody recognition using phosphatase-treated samples

  • Validate with phospho-mimetic and phospho-dead mutants

  • Confirm phosphorylation-state specificity using in vitro kinase assays

• Quantitative assessment:

  • Determine linear dynamic range for quantitative applications

  • Assess lot-to-lot variability

  • Document optimal working concentrations for each application

A systematic validation matrix should be established and documented, with antibody performance assessed across all intended applications. Researchers should be particularly cautious when using antibodies for detecting post-translational modifications, as these require rigorous validation protocols.

How should researchers design experiments to study the impact of exercise on C18orf25 phosphorylation?

To effectively study exercise-regulated phosphorylation of C18orf25:

• Exercise protocol design:

  • Compare acute versus chronic exercise interventions

  • Include both endurance and resistance exercise protocols

  • Establish appropriate time points for tissue collection (immediately post-exercise, 1h, 3h, 24h)

  • Control for circadian variations in phosphorylation

• Tissue collection and processing:

  • Rapid tissue harvesting and snap-freezing to preserve phosphorylation status

  • Consider in vivo phosphorylation labeling with metabolic tracers

  • Include phosphatase inhibitors in all extraction buffers

  • Process all samples simultaneously to minimize technical variation

• Analytical approaches:

  • Phospho-specific western blotting for targeted analysis

  • Mass spectrometry-based phosphoproteomics for unbiased profiling

  • Quantitative analysis using stable isotope labeling or label-free approaches

  • Single-fiber analysis to account for fiber type heterogeneity

• Functional correlation studies:

  • Parallel assessment of AMPK activation status

  • Correlation with physiological parameters (exercise performance, metabolic measurements)

  • Ex vivo contractile measurements on the same cohort

• Mechanistic validation:

  • Pharmacological AMPK activation versus inhibition

  • Genetic models with altered AMPK activity

  • In vitro kinase assays with muscle lysates from exercised animals

Statistical analysis should account for individual variability in exercise response and include appropriate corrections for multiple comparisons when performing phosphoproteomic analyses .

What approach should be taken to investigate potential tissue-specific roles of C18orf25?

To systematically investigate tissue-specific roles of C18orf25:

• Expression profiling strategy:

  • Quantitative PCR panel across tissues to establish baseline expression patterns

  • Western blot analysis to confirm protein expression correlates with mRNA

  • Single-cell RNA sequencing to identify cell type-specific expression within heterogeneous tissues

  • Developmental time course to identify temporal regulation

• Tissue-specific knockout approaches:

  • Cre-loxP system with tissue-specific promoters

  • Temporal control using inducible Cre systems to distinguish developmental versus adult roles

  • Careful validation of knockout efficiency in target tissues

  • Assessment of potential compensatory mechanisms (related family members)

• Phenotypic characterization:

  • Standardized phenotyping pipeline across physiological systems

  • Focused analysis on tissues with high C18orf25 expression

  • Comparison between tissue-specific and global knockout models

  • Stress challenges to reveal phenotypes not apparent under baseline conditions

• Molecular characterization by tissue:

  • Tissue-specific interactome mapping using AP-MS

  • Phosphoproteomic analysis to identify tissue-specific phosphorylation events

  • Transcriptomic analysis to identify tissue-specific gene expression changes

  • Metabolomic profiling to identify tissue-specific metabolic alterations

• Rescue experiments:

  • Re-expression of C18orf25 in specific tissues of global knockout

  • Testing phospho-mutants for tissue-specific functional requirements

  • Cross-tissue expression to test for functional conservation

When designing these experiments, researchers should prioritize tissues based on expression levels and phenotypic observations from global knockout models, with particular attention to skeletal muscle given the established phenotypes in this tissue .

What are the current limitations in C18orf25 research and future directions?

Current limitations in C18orf25 research include incomplete structural characterization, limited understanding of its regulation beyond S67 phosphorylation, and gaps in knowledge regarding its tissue-specific functions outside skeletal muscle. Advanced structural biology approaches, comprehensive interactome mapping across tissues, and development of small molecule modulators represent promising future directions to address these knowledge gaps.