Recombinant Human Uncharacterized protein C9orf4 (C9orf4)

What is Recombinant Human Uncharacterized protein C9orf4?

Recombinant Human Uncharacterized protein C9orf4 is a protein originally identified through genomic analysis as chromosome 9 open reading frame 4. It has several aliases including FRRS1L, CG6, and CG-6, with more recent literature favoring the FRRS1L designation . The protein contains a DOMON (dopamine beta-monooxygenase N-terminal) domain and is classified as ferric-chelate reductase 1-like protein, suggesting potential roles in redox reactions .

The recombinant form is produced in various expression systems including E. coli, yeast, baculovirus, or mammalian cells, with typical purity levels of ≥85% as determined by SDS-PAGE analysis . This recombinant protein serves as a valuable research tool for investigating the protein's structure, function, and interactions, particularly in neurological contexts.

Methodological considerations:

• Selection of expression system should be based on experimental requirements (bacterial systems for simple structural studies; mammalian systems for functional studies requiring post-translational modifications)

• Validate recombinant protein function before experimental use

• Consider using partial recombinant proteins when investigating specific domains

How is C9orf4/FRRS1L expressed in the human body?

C9orf4/FRRS1L is notably expressed in brain tissue, earning it the alternative name "Brain protein CG-6" . Its expression pattern aligns with its functional association with AMPA receptors, which are predominantly found in the central nervous system .

When studying expression patterns, researchers typically employ:

• Western blot analysis using specific antibodies that show reactivity across multiple species (rat, pig, dog, cow, human)

• Immunohistochemistry on both paraffin-embedded and frozen brain tissue sections

• Immunofluorescence and immunocytochemistry for cellular localization studies

These methods reveal that FRRS1L is primarily expressed in neurons, consistent with its role in modulating glutamatergic neurotransmission. While the protein's full expression profile across development and in pathological conditions remains under investigation, its predominant neuronal expression underscores its importance in brain function.

What detection methods are most appropriate for C9orf4/FRRS1L research?

Multiple approaches can be employed for detecting and quantifying C9orf4/FRRS1L, each with specific applications:

When selecting a detection method, consider:

• The nature of your biological sample (tissue sections, cell lysates, body fluids)

• Required sensitivity and specificity

• Need for quantitative versus qualitative data

• Co-detection requirements with other proteins

How can CRISPR/Cas9 technology be applied to study C9orf4/FRRS1L in neurons?

CRISPR/Cas9 genome editing offers powerful approaches for investigating C9orf4/FRRS1L function in neurons. The ORANGE (Open Resource for the Application of Neuronal Genome Editing) system provides an efficient platform for tagging endogenous neuronal proteins .

Implementing this approach for C9orf4/FRRS1L research involves:

• Designing a single CRISPR/Cas9 knock-in template vector containing:

  • A U6-driven expression cassette for the guide RNA targeting the C9orf4/FRRS1L locus

  • A donor sequence containing the desired tag (typically fluorescent)

  • A Cas9 expression cassette driven by a β-actin promoter

• Careful selection of the genomic target sequence and gRNA design to ensure specificity and efficiency

• Electroporation of DIV0 neurons in suspension for expression in a large population of cells

• Verification of successful integration through genomic DNA extraction, PCR amplification of the target locus, and sequencing

This approach enables visualization of endogenous C9orf4/FRRS1L localization, trafficking, and interaction dynamics in live neurons, providing insights not achievable with conventional overexpression approaches.

How does C9orf4/FRRS1L associate with AMPA receptors in the brain?

Proteomic studies have identified C9orf4/FRRS1L as one of several previously uncharacterized proteins associated with endogenous AMPA receptors (AMPARs) in the brain . These receptors are critical mediators of fast excitatory synaptic transmission in the central nervous system.

Experimental evidence indicates that in HEK cells, FRRS1L can reduce non-saturating 100 μM glutamate-evoked, GluA1 homomer-mediated calcium responses, suggesting a modulatory role in AMPAR function . This modulation appears to be physiologically significant, as loss-of-function mutations in FRRS1L lead to severe neurological phenotypes including epilepsy and cognitive impairment .

Research methodologies to investigate this association include:

• Co-immunoprecipitation with AMPAR subunits

• Calcium imaging in heterologous expression systems

• Electrophysiological recordings in neurons with manipulated FRRS1L levels

• Super-resolution microscopy to visualize co-localization at synapses

Understanding FRRS1L's precise mechanism of AMPAR regulation remains an active area of research with significant implications for excitatory synaptic transmission.

What are the functional consequences of C9orf4/FRRS1L mutations?

Human genetic studies have established that loss-of-function mutations in FRRS1L lead to a constellation of severe neurological symptoms, including:

• Epilepsy, often presenting as early infantile epileptic encephalopathy (EIEE37)

• Prominent choreoathetosis (involuntary movements)

• Severe impairment of cognitive functions

These findings highlight the critical importance of FRRS1L in normal brain development and function. The severity of symptoms suggests that FRRS1L plays a non-redundant role in regulating excitatory neurotransmission that cannot be compensated by other proteins when its function is lost.

Methodological approaches to study these mutations include:

• Generating equivalent mutations in model systems (cell lines, rodent models)

• Electrophysiological characterization of AMPAR function in the presence of mutant FRRS1L

• Imaging studies to assess protein localization and trafficking

• Behavioral assessment in animal models expressing FRRS1L mutations

Investigation of these mutations provides a valuable window into both the normal function of FRRS1L and the pathophysiology of associated neurological disorders.

What are the current methodological challenges in studying C9orf4/FRRS1L?

Despite significant progress, several technical challenges remain in C9orf4/FRRS1L research:

How can researchers differentiate the function of C9orf4/FRRS1L from other AMPAR-associated proteins?

AMPARs associate with numerous auxiliary proteins that modulate their trafficking, gating, and signaling properties. Distinguishing the specific contribution of FRRS1L requires:

• Sequential knockdown/knockout approaches: Systematically removing individual AMPAR-associated proteins to identify non-redundant versus synergistic functions.

• Domain-specific mutations: Creating targeted mutations in functional domains of FRRS1L to disrupt specific protein-protein interactions while preserving others.

• Temporal manipulation: Using inducible expression or degradation systems to determine acute versus developmental roles.

• Cell-type specific studies: Investigating FRRS1L function in different neuronal populations that may exhibit distinct AMPAR regulatory mechanisms.

• Reconstitution assays: Building simplified systems with defined components to determine minimal requirements for FRRS1L function.

The challenge remains to integrate these diverse approaches into a coherent understanding of FRRS1L's unique contribution to AMPAR regulation in the context of the complex protein networks at excitatory synapses.

What is the relationship between the DOMON domain of C9orf4/FRRS1L and its neuronal function?

The DOMON domain found in FRRS1L typically participates in electron transfer functions in other proteins, suggesting potential redox-related activities . Current research questions include:

• Does the DOMON domain directly interact with AMPAR subunits?

• Could FRRS1L regulate AMPAR function through redox modulation of the receptor or associated proteins?

• What are the structural determinants within the DOMON domain that contribute to FRRS1L's neuronal functions?

Methodological approaches to address these questions include:

• Site-directed mutagenesis of conserved residues within the DOMON domain

• In vitro binding assays with purified proteins

• Redox-sensitive imaging probes to assess local redox environments at synapses

• Electrophysiological assessment of AMPAR function under different redox conditions

Understanding this relationship may provide mechanistic insights into how FRRS1L modulates glutamatergic transmission and why its dysfunction leads to neurological disorders.

How might therapeutic strategies target C9orf4/FRRS1L-related disorders?

Given the association of FRRS1L mutations with severe neurological disorders, potential therapeutic strategies include:

• Gene therapy approaches to restore FRRS1L expression in affected neurons

• Small molecules that compensate for FRRS1L loss by modulating AMPAR function through alternative mechanisms

• AMPAR antagonists titrated to normalize excessive excitatory transmission in the absence of FRRS1L

• Peptide-based approaches mimicking functional domains of FRRS1L

The development of these strategies requires:

• High-throughput screening platforms to identify compounds that modulate AMPAR function

• Patient-derived iPSC models to test interventions in human neurons carrying FRRS1L mutations

• Animal models that recapitulate key aspects of FRRS1L-associated disorders

• Biomarkers to monitor treatment efficacy in clinical settings