ROBLD3 Human

What is ROBLD3 and what are its alternative nomenclatures?

ROBLD3 (Roadblock domain-containing protein 3) is also known by several alternative names including LAMTOR2 (Late endosomal/lysosomal adaptor and MAPK and MTOR activator 2), Endosomal adaptor protein p14, Late endosomal/lysosomal Mp1-interacting protein, and Mitogen-activated protein-binding protein-interacting protein. This adapter protein is a member of the GAMAD family and plays crucial roles in cellular signaling pathways, particularly in enhancing MAP kinase cascade efficiency and facilitating MAPK2 activation .

What are the structural characteristics of the human ROBLD3 protein?

Human ROBLD3 is a relatively small protein with a molecular mass of approximately 14 kDa as detected by Western blotting . When produced as a recombinant protein with a His tag at the N-terminus, it forms a single, non-glycosylated polypeptide chain containing 149 amino acids (with the native portion comprising 125 amino acids) and has a molecular mass of 16 kDa . The protein maintains its stability in specific buffer conditions containing Tris-HCl, NaCl, DTT, and glycerol .

What cellular compartments contain ROBLD3 and what is its primary function?

ROBLD3 primarily localizes to late endosomes where it forms a stable heterodimeric complex with MP1. This interaction is critical for proper ERK activation on endosomal membranes . The protein functions as an adapter that enhances the efficiency of the MAP kinase cascade and compels the recruitment of MP1 to late endosomes . Its presence in this compartment suggests a significant role in endosomal biogenesis and signal transduction from endosomal platforms.

What are the recommended methods for detecting endogenous ROBLD3 in cellular samples?

Endogenous ROBLD3/LAMTOR2 can be effectively detected using specific antibodies such as the LAMTOR2/ROBLD3 (D7C10) Rabbit mAb, which has demonstrated reactivity across human, mouse, rat, and monkey samples . Western blotting represents the primary method for detection, with the protein appearing at approximately 14 kDa. Immunoprecipitation can also be employed for isolation and subsequent analysis . For optimal results, researchers should select antibodies raised against conserved epitopes, such as those surrounding Leu31 of the human protein, to ensure consistent detection across experimental models .

How should recombinant ROBLD3 protein be stored to maintain stability for experimental use?

For optimal stability of recombinant ROBLD3, researchers should follow these evidence-based storage protocols: (1) For short-term use (2-4 weeks), store at 4°C in the original formulation; (2) For longer storage periods, maintain the protein at -20°C; (3) For extended preservation, add a carrier protein such as 0.1% HSA or BSA to prevent degradation; (4) Minimize freeze-thaw cycles as these significantly impact protein integrity . The protein is typically supplied in a buffer containing 20mM Tris-HCl (pH8.0), 0.2M NaCl, 2mM DTT, and 10% glycerol at a concentration of 1 mg/ml, which helps maintain its stability .

What experimental design considerations are important when studying ROBLD3 interactions with other proteins?

When designing experiments to study ROBLD3 interactions, researchers should implement a structured experimental pipeline that allows for reproducible results. This includes: (1) Clear documentation of experimental intent and hypothesis; (2) Consistent metadata collection throughout the experimental process; (3) Automated tracking of samples and conditions to reduce human error; (4) Implementation of controlled validation steps to verify interactions . The Round Trip (RT) experimental design approach, which connects inception to analysis with automated metadata tracking, has been shown to reduce experimental cycle duration from weeks to hours (excluding laboratory execution time) .

How does ROBLD3 contribute to the Ragulator complex and mTORC1 signaling?

ROBLD3, as LAMTOR2, functions as a critical component of the Ragulator complex that regulates mTORC1 signaling at the lysosomal surface. In this context, ROBLD3/LAMTOR2 contributes to amino acid sensing and subsequent mTORC1 activation through the recruitment and anchoring of Rag GTPases to lysosomal membranes . The protein's role extends beyond simple structural support, as it participates in the dynamic regulation of this signaling hub. Mutations affecting ROBLD3 can disrupt this signaling axis, potentially explaining the link between ROBLD3 mutations and immunodeficiency syndromes through altered metabolic regulation in immune cells .

What are the methodological approaches for investigating ROBLD3's role in endosomal biogenesis?

Investigating ROBLD3's role in endosomal biogenesis requires multiple complementary approaches: (1) Live-cell imaging using fluorescently tagged ROBLD3 to track its dynamics during endosomal maturation; (2) Proximity labeling techniques such as BioID or APEX to identify the spatial interactome of ROBLD3 at endosomal membranes; (3) CRISPR/Cas9-mediated gene editing to generate knockout or knockin models for functional studies; (4) High-content screening to assess endosomal morphology and dynamics in ROBLD3-perturbed cells . Implementation of automated experimental pipelines can significantly enhance the reproducibility and throughput of these studies, allowing for more comprehensive analysis of ROBLD3's functional impact .

How do mutations in ROBLD3 contribute to primary immunodeficiency syndromes at the molecular level?

Mutations in the ROBLD3 gene have been associated with primary immunodeficiency syndromes through disruption of endosomal biogenesis and altered cellular signaling . At the molecular level, these mutations likely impair the formation of the ROBLD3-MP1 heterodimeric complex, which is essential for proper ERK activation on endosomes. This disruption affects downstream signaling cascades critical for immune cell development, proliferation, and function . Research methodologies to investigate these molecular mechanisms should include patient-derived cell models, phosphoproteomic analysis of affected signaling networks, and high-resolution imaging of endosomal structures in mutation-carrying cells.

What approaches can resolve experimental variability in ROBLD3 functional studies?

Addressing experimental variability in ROBLD3 functional studies requires systematic methodological approaches: (1) Implementation of structured metadata tracking to identify sources of variation; (2) Time-course experiments with appropriate intervals, as experimental evidence shows greater variation in cellular responses at certain time points (e.g., 12 to 24 hours compared to later time points) ; (3) Standardization of strain identifiers and experimental protocols across collaborating teams; (4) Reduction of manual data entry points to minimize human error . The Round Trip experimental management approach has demonstrated significant improvements in this area, reducing the personnel requirements from approximately ten persons to three through automation and standardization .

How should researchers approach contradictory data regarding ROBLD3 function?

When confronting contradictory data regarding ROBLD3 function, researchers should: (1) Systematically evaluate experimental conditions, including cell types, experimental timeframes, and detection methods; (2) Consider the potential impact of protein interaction partners that may vary between experimental systems; (3) Implement statistical analysis methods that can identify variables contributing to observed differences; (4) Design validation experiments specifically targeting the discrepant findings with controlled variations in experimental parameters . Automated experimental planning and analysis tools can help systematically explore these contradictions by facilitating replicate experiments with precise control over variables .

What high-throughput screening approaches are most effective for studying ROBLD3-dependent pathways?

For high-throughput screening of ROBLD3-dependent pathways, researchers should consider: (1) Automated liquid handling systems integrated with standardized experimental protocols; (2) Machine learning approaches for experimental design optimization; (3) Containerized analysis tools that ensure reproducibility across different research settings; (4) Versioned data sets that maintain the integrity of experimental conditions and results . The implementation of automation in both experimental execution and data analysis has been shown to reduce inception-to-analysis latency from several weeks to approximately 8-10 hours (not including laboratory execution time) .

How can researchers effectively plan ROBLD3 experiments that integrate with AI-driven discovery platforms?

To effectively integrate ROBLD3 experiments with AI-driven discovery platforms, researchers should: (1) Establish standardized metadata frameworks that capture all relevant experimental parameters; (2) Implement automated experimental planning tools that translate scientific intent into executable protocols; (3) Ensure data collection includes sufficient contextual information to be "AI-ready"; (4) Utilize containerized analysis tools that can be consistently applied across datasets . This approach has been successfully applied in programs involving approximately one hundred researchers across multiple universities, companies, and government laboratories, demonstrating its effectiveness in complex collaborative research environments .

What are the emerging research frontiers for ROBLD3 beyond its established roles?

Beyond its established roles in endosomal function and MAP kinase signaling, emerging research frontiers for ROBLD3 include: (1) Its potential involvement in metabolic regulation through mTORC1 signaling modulation; (2) Possible contributions to cellular stress responses and adaptation mechanisms; (3) Unexplored roles in specialized cell types, particularly in the immune system where mutations show phenotypic effects; (4) Potential therapeutic targeting in conditions characterized by dysregulated endosomal trafficking or signaling. Future research should leverage high-throughput experimental designs with comprehensive metadata tracking to systematically explore these potential functions .