RAC2 Human, 189a.a

What is the molecular structure of RAC2 Human, 189a.a?

RAC2 Human is a single, non-glycosylated polypeptide chain containing 189 amino acids. When produced as a recombinant protein, it is typically fused with a 20 amino acid His tag at the N-terminus, resulting in a total length of 209 amino acids and a molecular mass of 23.3kDa . RAC2 belongs to the RAS superfamily of GTPases and cycles between inactive GDP-bound and active GTP-bound states to regulate hematopoietic cell signaling in the immune system . The protein's structure includes regions critical for GTP binding, effector binding, and membrane localization, all essential for its proper signaling function.

How does RAC2 differ structurally and functionally from other RAC family members?

Despite high sequence homology with RAC1 (92% identical), RAC2 exhibits hematopoietic-specific expression patterns and non-redundant functions. The key structural differences lie in the C-terminal region, which affects subcellular localization and interaction with regulatory proteins. While RAC1 is ubiquitously expressed, RAC2 expression is largely restricted to hematopoietic cells, indicating tissue-specific functions . Research methodologies to distinguish between RAC proteins should include isoform-specific antibodies and gene knockout approaches. Functional studies in neutrophils from RAC2-deficient models have demonstrated that RAC2, but not RAC1, is critical for certain aspects of neutrophil migration and superoxide production .

What are the recommended handling and storage conditions for RAC2 recombinant protein?

For optimal stability and activity of RAC2 Human recombinant protein:

Following these storage guidelines is crucial for maintaining protein activity for experimental applications. When working with the protein, always keep it on ice during experimental setup and avoid prolonged exposure to room temperature .

How does RAC2 regulate neutrophil function at the molecular level?

RAC2 serves as a molecular switch in neutrophils, regulating multiple critical functions including:

• Chemotaxis: RAC2 coordinates actin cytoskeleton remodeling necessary for directional migration. Patients with RAC2 mutations show impaired neutrophil migration .

• Phagocytosis: RAC2 mediates actin rearrangements required for pathogen engulfment.

• Superoxide production: RAC2 is an essential component of the NADPH oxidase complex, which generates reactive oxygen species for pathogen destruction. Most RAC2 mutants produce elevated superoxide, though some mutations unable to support superoxide formation are associated with increased bacterial infections .

• Neutrophil granule dynamics: Electron microscopy studies of neutrophils from patients with RAC2 mutations reveal reduced numbers of neutrophil granules and morphological changes in secondary granules .

Research methodologies for investigating these functions should include chemotaxis assays, respiratory burst assays, and advanced imaging of granule mobilization.

What role does RAC2 play in lymphocyte development and function?

RAC2 is critical for proper T and B lymphocyte development and function. Studies of patients with RAC2 mutations reveal:

• T-cell development: RAC2 mutations can lead to reduced T-cell receptor excision circles (TRECs), indicating decreased recent thymic emigrants .

• B-cell development: Reduced kappa-deleting recombination excision circles (KRECs) in peripheral blood suggest impaired B-cell development .

• Progressive antibody deficiency: RAC2 deficiency can cause progressive hypogammaglobulinemia, with varying levels of different immunoglobulin classes .

To study these aspects, researchers should employ flow cytometry-based immunophenotyping, TREC/KREC analysis, and functional assays measuring T-cell proliferation and B-cell antibody production.

What spectrum of immunodeficiencies is associated with different RAC2 mutations?

RAC2 mutations cause a spectrum of immune dysfunction that correlates with the functional impact on RAC2 activity:

The recently identified E62K mutation in RAC2 promotes immune dysfunction through RAC2 hyperactivation, altering guanine nucleotide exchange factor (GEF) specificity, and impairing GTPase-activating protein (GAP) function .

What are the laboratory findings and clinical manifestations in patients with RAC2 mutations?

Patients with RAC2 mutations exhibit a range of laboratory abnormalities and clinical manifestations:

Laboratory findings:

• Significant T- and B-lymphopenia

• Low immunoglobulin levels (variable patterns)

• Neutropenia

• Altered oxidative burst

• Impaired neutrophil migration

• Visible neutrophil macropinosomes on microscopy

Clinical manifestations:

• Recurrent upper and lower respiratory infections

• Increased susceptibility to viral infections

• Autoimmune manifestations (including glomerulonephritis)

• Multiple hormone deficiencies (potentially autoimmune basis)

• Coagulopathy

• Abnormal wound healing

Research methodologies should include comprehensive immunological workup, genetic testing, and functional assays of neutrophil and lymphocyte function.

How do homozygous loss-of-function RAC2 mutations differ clinically from other RAC2 mutations?

Homozygous loss-of-function RAC2 mutations present with distinctive features compared to dominant mutations. In the first reported cases of homozygous RAC2 loss-of-function mutations, patients presented with:

• Features of common variable immunodeficiency (CVID)

• Glomerulonephritis

• Coagulopathy

• Multiple hormone deficiencies (potentially on an autoimmune basis)

• Abnormalities of neutrophil granules

Unlike dominant-negative mutations that cause severe neonatal presentations, these patients did not exhibit severe clinical abnormalities in the neonatal period. The antibody deficiency was progressive, with the first manifestations appearing at 6 months of age with recurrent pneumonia . This progressive nature of the disease could not be evaluated in patients with dominant mutations, as they typically received hematopoietic cell transplantation at an early age.

What methodologies are most effective for studying RAC2 activation and downstream signaling?

To effectively study RAC2 activation and signaling, researchers should employ multiple complementary approaches:

• GTPase activation assays: Pull-down assays using the binding domains of RAC effector proteins (e.g., PAK-PBD) to isolate active GTP-bound RAC2.

• Effector binding assays: Assess interactions with downstream effectors like p67phox (for NADPH oxidase) and PAK (for cytoskeletal regulation) .

• Superoxide production measurement: Nitroblue tetrazolium (NBT) or dihydrorhodamine (DHR) assays to measure respiratory burst activity.

• Confocal microscopy: To visualize altered actin assembly, membrane ruffling, and macropinosome formation .

• Heterologous expression systems: To assess downstream effector functions including AKT activation and protein stability .

Recent research indicates that no single assay is sufficient to determine the functional consequence of RAC2 mutations, necessitating multiple methodological approaches .

How can researchers effectively study the role of RAC2 in macrophage differentiation and tumor microenvironment?

Studies investigating RAC2's role in macrophage differentiation and tumor microenvironment should incorporate:

• Genetic approaches: Utilize Rac2-/- mouse models combined with syngeneic and orthotopic tumor models to assess tumor growth, angiogenesis, and metastasis .

• Transcriptomic analysis: Microarray or RNA-seq on bone marrow-derived macrophages to identify RAC2's role in M1-M2 macrophage differentiation .

• Metabolomic analysis: To detect metabolic shifts associated with macrophage polarization states .

• Signaling pathway analysis: Focus on signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor that lead to the activation of RAC2 .

• Systems biology approaches: Develop hierarchical protein-protein interaction networks to predict additional mechanisms by which RAC2 controls macrophage differentiation .

Research has shown that Rac2-/- mice display marked defects in tumor growth, angiogenesis, and metastasis, highlighting the importance of these methodological approaches in understanding RAC2's role in cancer biology .

What is the significance of RAC2's interaction with specific GEFs and GAPs in different pathological contexts?

RAC2 activity is precisely regulated by guanine nucleotide exchange factors (GEFs) that promote activation and GTPase-activating proteins (GAPs) that facilitate inactivation. Research indicates that:

• The E62K mutation in RAC2 alters GEF specificity: This mutation completely abolishes TIAM1 (a DH domain GEF) stimulation while retaining DOCK2 activity, representing a shift in RAC2 GEF specificity .

• The E62K mutation impairs GAP-mediated inactivation: This contributes to RAC2 hyperactivation and associated immunopathology .

• Different GEF-GAP interactions may explain tissue-specific manifestations: Despite RAC2 being hematopoietic-specific, patients with RAC2 mutations can develop extra-hematopoietic manifestations like hormone deficiencies and kidney disease .

For studying these interactions, researchers should employ biochemical assays measuring nucleotide exchange and GTPase activity, coupled with structural biology approaches to understand the molecular basis of altered interactions.

How might therapeutic approaches targeting RAC2 be developed for immunodeficiencies?

Developing therapeutic strategies for RAC2-related immunodeficiencies requires consideration of:

• Mutation-specific approaches: Different mutations cause different disease mechanisms (hyperactivation vs. loss-of-function) .

• Cell-specific delivery: Given RAC2's hematopoietic-specific expression, cell-targeted therapies might minimize off-target effects.

• Timing considerations: For progressive defects like antibody deficiencies in homozygous loss-of-function mutations, early intervention may be critical .

• Hematopoietic cell transplantation (HCT): Currently used for severe RAC2 mutations, with patients receiving HCT as early as 3-10 months of age for dominant negative mutations .

• Small molecule modulators: Potential development of molecules that could restore normal RAC2 cycling between active and inactive states.

Research should focus on animal models that recapitulate the specific mutations found in humans to develop and test targeted therapeutic approaches before clinical translation.