Recombinant Pongo abelii Ras-related protein Ral-B (RALB)

What is Ras-related protein Ral-B and what is its role in cellular signaling pathways?

Ras-related protein Ral-B (RALB) is a small GTPase that functions as a critical downstream effector in the Ras signaling pathway. RALB acts as a molecular switch that cycles between active (GTP-bound) and inactive (GDP-bound) states to regulate numerous cellular processes.

RALB plays a distinct role in supporting the invasiveness of transformed cells. While RalA and RalB share approximately 85% sequence identity and collaborate in supporting cancer cell proliferation, they have markedly different effects: RalA is important in mediating proliferation, while depletion of RalB results in transformed cells undergoing apoptosis .

The primary signaling pathway involves:

• Activation of Ras (often through oncogenic mutations)

• Ras activates RalGEFs (particularly RGL1 and RGL2)

• RalGEFs activate RALB by promoting GDP-GTP exchange

• Activated RALB binds to effector proteins including the exocyst complex

• The exocyst complex recruits the Wave Regulatory Complex (WRC)

• WRC stimulates actin polymerization leading to protrusion formation, motility and invasion

How does the structure of RALB affect its function in signaling pathways?

Structural analysis has revealed four regions of disorder in RALB:

• The P-loop

• Switch I

• Switch II

• The loop comprising residues 116-121, which has a single residue insertion compared to RalA

What innovative approaches have been developed to study RALB activation and its downstream effects?

Recent advances in RALB research have employed optogenetics, a powerful approach that allows spatiotemporal control of protein activity using light. This method enables precise activation of RALB at the plasma membrane using a laser that produces blue light .

Key methodological components:

• Light-controlled RALB activation system

• Real-time visualization of cellular responses

• Ability to dissect molecular mechanisms involved in RALB-mediated processes

Using this optogenetic approach, researchers demonstrated that:

• Light-controlled activation of Ral at the plasma membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst

• This recruitment leads to induction of protrusions and cellular invasion

• Active Ras signals to RALB via two RalGEFs (RGL1 and RGL2) to foster invasiveness

• RALB's contribution appears to be more important than that of MAPK and PI3K pathways

What experimental techniques are most effective for studying RALB's role in radiation resistance of cancer cells?

To investigate RALB's role in radiation resistance, researchers have developed several effective experimental approaches:

Key experimental techniques:

• Stable shRNA knockdown: RalA, RalB, and their major effectors (RalBP1 and Sec5) can be knocked down by stable expression of short hairpin RNAs in K-Ras-dependent cancer cell lines

• Clonogenic survival assays: To measure reproductive survival post-irradiation

• γH2AX expression analysis: For quantifying double-strand DNA breaks (DSBs)

• PARP cleavage assays: To detect apoptosis

Research findings:

• Knockdown of K-Ras, RalA, or RalB reduced colony-forming ability post-IR

• Knockdown of either Ral isoform decreased the rate of DSB repair post-IR

• Knockdown of RalB, but not RalA, increased cell death

• Surprisingly, neither RalBP1 nor Sec5, the two best-characterized Ral effectors, affected colony formation post-IR

• This suggests Ral-mediated radioresistance may utilize an atypical or novel effector pathway

How does RALB contribute to cancer cell invasion and metastasis mechanisms?

RALB plays a specialized role in cancer cell invasion and metastasis that is distinct from its family member RalA. Multiple lines of evidence demonstrate RALB's specific contribution to invasive phenotypes:

RALB-specific invasion mechanisms:

• In UMUC3 human bladder cancer cells (K-Ras mutated), RalA antagonizes the pro-migratory function of RalB, highlighting their distinct roles

• In 7 out of 9 K-Ras mutated human pancreatic cancer cell lines, RalB knockdown inhibits invasion in Transwell assays

• RalB, but not RalA, plays a crucial role in invadopodia formation in pancreatic cancer cell lines

• In lung cancer cells (A549, K-Ras mutated), RalB specifically drives contractility-dependent invasion

• In vivo metastasis assays in mice (tail vein injection) and hamsters (subcutaneous injection) confirm RALB's importance in metastasis formation

Molecular mechanism of RALB-driven invasion:
RALB activation leads to exocyst-dependent recruitment of the Wave Regulatory Complex (WRC) at the leading edge of cells. This WRC activation promotes actin cytoskeleton formation through interaction with the Arp2/3 complex, unexpectedly independent of the small GTPase Rac1 (a well-established WRC activator) .

What is the clinical relevance of RALB expression in human cancers?

Clinical investigations have revealed significant correlations between RALB expression and cancer progression:

Breast cancer findings:
RALB protein expression levels increase in a manner consistent with progression toward metastasis. Samples of breast cancer cells that had metastasized to other body sites showed abnormally high levels of RALB protein, suggesting RALB as a potential biomarker for metastatic progression .

Experimental evidence in transformation models:
In the genetically controlled cell model of HEK-HT and HEK-HT-H-RasV12 cells:

• The silencing of RALB impaired Transwell invasion by approximately 60%

• RalA silencing had no effect

• Combined silencing of both RALB and RalA reached up to 90% invasion inhibition

• This indicates that Ras-dependent RALB activation substantially contributes to the invasive phenotype, while RalA is dispensable but may partially compensate when RALB is absent

These findings highlight the Ras-RGL1/2-RALB-exocyst-WRC axis as an appealing target for novel anticancer strategies.

How does the RALB-exocyst-WRC signaling axis function in cell invasion processes?

The RALB-exocyst-WRC signaling axis represents a critical pathway in controlling cell invasion through coordination of cytoskeletal dynamics and membrane remodeling:

Signaling pathway components:

• RALB activation: Oncogenic Ras activates RALB through RGL1 and RGL2 (RalGEFs)

• Exocyst recruitment: Activated RALB binds to the exocyst complex, promoting its assembly and recruitment to the leading edge of cells

• WRC mobilization: The exocyst, through direct association with the WRC complex, drives WRC to the leading edge

• Actin polymerization: At the leading edge, WRC stimulates actin polymerization through Arp2/3 complex, leading to protrusion formation, motility and invasion

Experimental evidence:

• Knockdown of RGL1 and RGL2 substantially reduces invasion in HEK-HT-RasV12 cells

• Immunofluorescence staining shows increased recruitment of endogenous RGL2 and RALB at cell edges in HEK-HTRasV12 cells compared to normal HEK-HT cells

• The RALB pathway appears to have greater significance than MAPK and PI3K pathways in driving Ras-dependent invasion

How do RALB and RalA differentially contribute to radiation resistance in cancer cells?

Despite their high sequence similarity, RALB and RalA have distinct functional roles in cancer cell radioresistance:

Differential contributions to radioresistance:

Key findings:

• Both RALB and RalA contribute to K-Ras-dependent IR resistance in MIA PaCa-2 pancreatic cancer cells

• Sensitization due to suppressed Ral expression likely results from decreased efficiency of DNA repair (both RALB and RalA) and increased susceptibility to apoptosis (RALB only)

• RALB-mediated radioresistance does not depend on either the RalBP1 or the exocyst complex, suggesting an alternative mechanism

What expression systems are optimal for producing functional recombinant RALB protein?

Several expression systems have been employed for recombinant Ral protein production, though specific data for Pongo abelii RALB is limited. Based on related recombinant protein production from Pongo abelii and similar GTPases:

Common expression systems for recombinant proteins:

Key production considerations:

• For functional small GTPases like RALB, proper folding and nucleotide binding are critical

• Addition of 5-50% glycerol in the final preparation helps maintain stability during storage

• Storage at -20°C/-80°C is recommended, with liquid preparations having a typical shelf life of 6 months and lyophilized forms 12 months

What purification strategies ensure high activity of recombinant RALB for functional studies?

To ensure high activity of recombinant RALB for functional studies, specific purification strategies should be implemented:

Recommended purification protocol:

• Initial capture: Affinity chromatography using His-tag (N-terminal 6xHis or 10xHis tags are common)

• Intermediate purification: Ion exchange chromatography to remove contaminants

• Polishing step: Size exclusion chromatography to ensure monomeric state and remove aggregates

• Quality control: SDS-PAGE analysis to confirm >85% purity

Handling recommendations for maintaining activity:

• Avoid repeated freezing and thawing (store working aliquots at 4°C for up to one week)

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) before long-term storage

• For GTPases like RALB, include appropriate nucleotides (GTP, GDP, or non-hydrolyzable analogs like GMPPNP) depending on the desired activation state