Recombinant Human FERM domain-containing protein 5 (FRMD5)

What is the basic structure of human FRMD5 protein?

Human FRMD5 is a 570 amino acid protein containing a FERM (four-point-one, ezrin, radixin, and moesin) domain. The protein's structure includes the FERM domain, which is involved in plasma membrane association, and a functionally important FA (FERM adjacent) domain. The FA domain is particularly significant as it mediates interactions with other proteins such as ROCK1. According to AlphaFold Protein Structure Database predictions, several disease-associated variants cluster in a loop within the FA domain, though these variants do not show obvious structural differences compared to the reference protein .

What are the primary cellular functions of FRMD5?

FRMD5 primarily localizes at cell adherens junctions where it stabilizes cell-cell contacts. It interacts with ROCK1 via its FA domain and inhibits ROCK1 kinase activity, thereby regulating actin-based cytoskeletal remodeling . This function is critical for maintaining proper cellular architecture and communication. In neurons, FRMD5 appears to play essential roles in development and function, as variants in this protein are associated with neurological disorders, including developmental delay and seizures .

How is FRMD5 expression regulated in different tissues?

In Drosophila models, the FRMD5 ortholog (dFrmd) is expressed in the larval and adult central nervous systems, specifically in neurons but not in glial cells . In humans, FRMD5 expression patterns vary across tissues, with significant expression noted in neural tissues. Research in papillary thyroid carcinoma has shown that FRMD5 expression can influence cellular migration, invasion, and adhesion properties, suggesting tissue-specific roles in normal and pathological conditions .

What expression systems are most effective for producing recombinant human FRMD5?

E. coli expression systems have been successfully used to produce recombinant full-length human FRMD5 protein (amino acids 1-570) with N-terminal His-tags . This prokaryotic expression system allows for substantial protein yield and facilitates purification through affinity chromatography. When expressing FRMD5, researchers should consider using codon-optimized sequences for E. coli to enhance expression efficiency, as the protein contains 570 amino acids and may present challenges for bacterial expression systems .

What purification strategies yield the highest purity for recombinant FRMD5?

For His-tagged recombinant FRMD5, immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices provides effective initial purification. Further purification can be achieved through size exclusion chromatography to remove aggregates and other contaminants. Commercially available recombinant FRMD5 is typically greater than 90% pure as determined by SDS-PAGE . When working with purified FRMD5, it's recommended to store the protein in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 to maintain stability .

How can FRMD5 function be effectively assessed in cellular models?

Functional assessment of FRMD5 can be accomplished through multiple complementary approaches:

• RNAi-mediated knockdown using siRNA targeting FRMD5 (approximately 75% knockdown efficiency has been reported)

• Wound-healing assays to assess effects on cell migration

• Transwell migration and invasion assays to quantify metastatic potential

• Scaffold-free cell spheroid models to mimic tumor growth

• Expression analysis of multidrug resistance genes following FRMD5 manipulation

Different cell types may respond differently to FRMD5 manipulation; for example, in thyroid cancer models, FRMD5 silencing in TPC1 (BRAF-wt) cells reduces migration, while in BCPAP (BRAF-V600E) cells, it increases migration .

What neurological phenotypes are associated with FRMD5 variants?

Heterozygous missense variants in FRMD5 have been associated with a spectrum of neurological symptoms including:

• Developmental delay and motor delay

• Intellectual disability

• Ataxia (observed in 7 of 8 reported probands)

• Abnormalities of eye movement (including nystagmus, opsoclonus, strabismus)

• Seizures (including refractory seizures in some cases)

• Abnormal brain MRI findings (including pachygyria in bilateral temporal lobes)

These neurological manifestations appear to be caused by de novo variants in most cases, with evidence suggesting FRMD5 is intolerant to loss of function .

How do animal models help understand FRMD5-related neurological disorders?

Drosophila models have proven invaluable for understanding FRMD5-related disorders. Flies with loss-of-function mutations in dFrmd (the fly ortholog of FRMD5) remain viable but demonstrate extreme sensitivity to heat shock, which induces severe seizures. These mutants also exhibit defective responses to light . Importantly, these phenotypes can be rescued by expressing human FRMD5 reference cDNA, confirming functional conservation across species. This model system allows for testing whether human variants are pathogenic—all tested human FRMD5 variants (c.340T>C, c.1051A>G, c.1053C>G, c.1054T>C, c.1045A>C, and c.1637A>G) behaved as partial loss-of-function variants in the fly model .

What molecular mechanisms underlie FRMD5-associated developmental disorders?

FRMD5 variants associated with neurological disorders appear to function through both loss-of-function and dominant-negative mechanisms. When human FRMD5 reference and variant proteins are co-expressed in dFrmd loss-of-function flies, the variants impair the rescue ability of the reference protein, indicating dominant-negative effects . At the molecular level, disruption of FRMD5's interaction with ROCK1 may alter cytoskeletal dynamics essential for proper neuronal development and function. This places FRMD5 within a broader context of FERM domain-containing proteins implicated in neurological disorders, such as FRMD4A, which is associated with microcephaly and intellectual disability when mutated .

How does FRMD5 influence cancer cell metastatic potential?

FRMD5 appears to significantly influence the metastatic potential of cancer cells, though its effects may vary depending on the genetic background of the cells. In papillary thyroid carcinoma (PTC) models, FRMD5 depletion results in divergent effects between BRAF-wild-type and BRAF-mutated cells:

• In BRAF-wild-type PTC cells (TPC1), FRMD5 knockdown reduces migration and invasion by up to 3-fold and 2-fold, respectively

• In BRAF-V600E mutated PTC cells (BCPAP), FRMD5 knockdown increases migration and invasion up to 2-fold

These findings suggest that FRMD5's role in cancer cell motility is context-dependent and influenced by the underlying genetic alterations in the cancer cells .

What is the relationship between FRMD5 and multidrug resistance in cancer?

Research indicates that FRMD5 can significantly influence multidrug resistance gene expression in cancer cells. Knockdown of FRMD5 has been shown to alter the expression of multidrug resistance genes, suggesting a potential role in therapeutic resistance mechanisms . This connection positions FRMD5 as a potential target for overcoming treatment resistance in certain cancers, though more research is needed to fully characterize the specific resistance pathways modulated by FRMD5 and how these might be therapeutically exploited.

How can 3D cell culture models enhance FRMD5 research in cancer biology?

Scaffold-free cell spheroid suspension models offer significant advantages for studying FRMD5's role in cancer biology as they better mimic the growth patterns of naturally occurring tumors compared to traditional 2D cultures. These 3D models have been used to investigate how FRMD5 deficiency affects the formation and growth characteristics of cancer cell spheroids . Researchers can assess parameters such as spheroid formation efficiency, size, compactness, and growth rates to understand how FRMD5 influences tumor-like growth. Additionally, these models allow for studying cell-cell interactions and adhesion properties that are particularly relevant to FRMD5's function at adherens junctions.

How do dominant-negative FRMD5 variants differ functionally from simple loss-of-function variants?

Dominant-negative FRMD5 variants represent a distinct mechanism of pathogenicity compared to simple loss-of-function variants. In experimental models, co-expression of human FRMD5 reference protein with variant proteins (particularly c.1051A>G or c.1054T>C) results in intermediate phenotypes between wild-type and complete loss-of-function, indicating that these variants actively interfere with normal protein function . This interference likely occurs through disruption of protein complexes or signaling pathways that require properly functioning FRMD5. Understanding these mechanistic differences is crucial for developing potential therapeutic approaches, as dominant-negative effects may require different intervention strategies than simple haploinsufficiency.

What techniques can resolve contradictory findings in FRMD5 functional studies?

When faced with contradictory findings, such as the opposing effects of FRMD5 depletion in different thyroid cancer cell lines, researchers should consider:

• Employing multiple cell lines representing diverse genetic backgrounds

• Using both in vitro and in vivo models to validate findings

• Implementing complementary gene manipulation techniques (siRNA, CRISPR-Cas9, overexpression)

• Conducting comprehensive signaling pathway analyses to identify context-dependent factors

• Performing rescue experiments with wild-type and mutant proteins

Additionally, researchers should thoroughly investigate the genetic and epigenetic context of experimental models, as the contradictory results observed in PTC cells with different BRAF mutation status highlight how genetic background can fundamentally alter FRMD5 function .

How might FRMD5 interact with other FERM domain-containing proteins in cellular networks?

The human genome encodes approximately 50 FERM domain-containing proteins (FDCPs), including eight specifically named "FRMD" proteins (FRMD1, 3, 4A, 4B, 5, 6, 7, and 8) . Research into potential interactions and functional redundancy among these related proteins remains an important frontier. Like FRMD5, other FRMD proteins have been implicated in human diseases—FRMD4A mutations are associated with microcephaly and intellectual disability, while FRMD7 is also linked to neurological conditions . Investigating potential interactions, compensatory mechanisms, and shared signaling pathways among FRMD family proteins could reveal important insights into both physiological functions and pathological mechanisms. Techniques such as co-immunoprecipitation, proximity labeling, and interactome analyses would be valuable for mapping these protein-protein interaction networks.

What are the optimal storage conditions for maintaining recombinant FRMD5 stability?

Recombinant FRMD5 protein stability is best maintained through proper storage protocols:

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

• After reconstitution in deionized sterile water (to a concentration of 0.1-1.0 mg/mL), add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• For short-term storage, working aliquots can be kept at 4°C for up to one week

• Store buffer-reconstituted protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0

These storage recommendations help preserve protein structure and function, ensuring reliable experimental results.

What quality control measures should be implemented when working with recombinant FRMD5?

When working with recombinant FRMD5, researchers should implement several quality control measures:

• Verify protein purity through SDS-PAGE (>90% purity is typically achievable)

• Confirm protein identity via western blotting with specific antibodies

• Assess batch-to-batch consistency through functional assays

• Validate protein activity using known interaction partners (e.g., ROCK1 binding assays)

• Monitor protein stability over time and after freeze-thaw cycles

Implementing these quality control measures ensures experimental reproducibility and reliable results.