RELM b Human

What is the molecular structure of human RELM-β and how does it compare to other RELM family members?

Human RELM-β is a 19.0 kDa protein consisting of two identical 89 amino acid polypeptide chains linked by a single disulfide bond. It belongs to the resistin-like molecule (RELM) family, characterized by conserved cysteine residues in their C-terminus . The RELM family includes Resistin (also called FIZZ3), RELM-α (FIZZ1), RELM-β (FIZZ2), and RELM-γ. Notably, only Resistin and RELM-β are found in humans, whereas all four family members are present in rodents .

Human RELM-β shares approximately 59% sequence homology with rodent RELM-β and 50% with rodent RELM-α, making it more closely related to its direct ortholog . This difference in sequence homology has important implications for translational research and the applicability of rodent models to human studies.

Table 1: RELM Family Distribution Across Species

What structural features of RELM-β contribute to its antimicrobial function?

The bactericidal activity of RELM-β is primarily attributed to its ability to form size-selective pores in bacterial membranes, particularly in Gram-negative bacteria . The protein's cysteine-rich C-terminal domain is crucial for this function, as it facilitates the formation of the proper tertiary structure through disulfide bonding.

When investigating structure-function relationships, researchers should consider employing site-directed mutagenesis of key cysteine residues, followed by functional assays measuring membrane permeabilization, such as fluorescent dye leakage assays or bacterial viability testing. Current research demonstrates that both mouse and human RELM-β selectively kill Gram-negative bacteria through this pore-forming mechanism, suggesting conservation of this critical function across species .

What factors regulate RELM-β expression in human tissues?

RELM-β expression is regulated by multiple factors, with Th2 cytokines playing a particularly important role. Both rodent and human lung epithelial cells show induced RELM-β expression in response to Th2 cytokines, with this induction mediated through STAT6 signaling pathways . This regulatory mechanism links RELM-β expression to type 2 immune responses, which are prominent in allergic and parasitic conditions.

For optimal experimental design when studying RELM-β regulation, researchers should:

• Include appropriate pathway inhibitors (e.g., STAT6 inhibitors) to establish causality

• Employ quantitative PCR for mRNA analysis

• Use immunohistochemistry and western blotting for protein detection

• Consider the tissue-specific nature of RELM-β expression when designing in vitro models

How does RELM-β expression differ between healthy tissues and disease states?

RELM-β expression is significantly elevated in multiple disease states compared to healthy tissues. In asthma patients, RELM-β expression is markedly increased in the bronchial submucosa compared to healthy controls . This increased expression positively correlates with the expression of fibronectin and α-smooth muscle actin, suggesting a role in tissue remodeling .

In pulmonary fibrosis, RELM-β is highly induced in both rodent models with bleomycin-induced fibrosis and human patients with idiopathic pulmonary fibrosis . Similar upregulation occurs in inflammatory bowel conditions .

Beyond epithelial cells, which are the primary source of RELM-β in healthy tissues, disease states show expanded RELM-β expression in macrophages, fibroblasts, and vascular endothelial cells in the submucosa . This altered cellular expression pattern may contribute to RELM-β's pathological effects in chronic inflammatory conditions.

What is the mechanism by which RELM-β kills Gram-negative bacteria?

RELM-β functions as a bactericidal protein that selectively targets and kills Gram-negative bacteria through a direct membrane-disrupting mechanism. Studies have demonstrated that both mouse and human RELM-β form size-selective pores that permeabilize bacterial membranes, compromising cellular integrity and leading to bacterial death . This mechanism is similar to other pore-forming antimicrobial peptides but with specificity for Gram-negative species.

To effectively study this mechanism, researchers should employ:

• Membrane permeability assays using fluorescent dyes such as propidium iodide or SYTOX Green that penetrate compromised membranes

• Advanced imaging techniques including atomic force microscopy or electron microscopy to visualize pore formation

• Comparative bacterial viability assays across different bacterial species to confirm selectivity

• Concentration-dependent killing assays to determine minimum inhibitory concentrations

How does RELM-β contribute to intestinal homeostasis and microbial compartmentalization?

RELM-β plays a crucial role in maintaining spatial segregation between intestinal bacteria and the epithelial surface. As part of the intestine's antimicrobial arsenal, RELM-β is secreted from intestinal epithelial cells into the mucus layer where it helps prevent bacterial penetration into host tissues .

The significance of this function is evidenced in studies of RELM-β-deficient mice, which show Proteobacteria (Gram-negative bacteria) present in the inner mucus layer and invading mucosal tissues . This breakdown in compartmentalization can disrupt the mutualistic relationship between the host and intestinal microbiota, potentially leading to inflammatory responses.

Table 2: Effects of RELM-β Deficiency on Intestinal Homeostasis

What role does RELM-β play in airway remodeling and fibrosis?

RELM-β has emerged as a significant mediator of tissue remodeling in respiratory diseases, particularly asthma and pulmonary fibrosis. Research demonstrates that RELM-β expression is significantly increased in the bronchial submucosa of asthmatic patients compared to controls, with its expression positively correlating with markers of tissue remodeling such as fibronectin and α-smooth muscle actin .

Mechanistically, RELM-β directly affects lung fibroblast function by:

• Increasing expression of TGF-β1 and TGF-β2, potent pro-fibrotic mediators

• Promoting production of extracellular matrix proteins including collagen I, fibronectin, laminin α1, and hyaluronan and proteoglycan link protein 1 (Hapl1)

• Enhancing fibroblast proliferation

• Activating ERK1/2 signaling pathways that drive fibrotic responses

These effects collectively contribute to airway remodeling processes that characterize chronic respiratory diseases.

Table 3: RELM-β Effects on Human Lung Fibroblasts

How can researchers effectively measure RELM-β's contribution to inflammatory disease models?

To comprehensively assess RELM-β's role in inflammatory diseases, researchers should employ multiple complementary approaches:

• Genetic Models: RELM-β knockout mice provide valuable insights into protein function, as demonstrated by studies showing altered susceptibility to colonic inflammation in these animals . For respiratory studies, conditional knockouts with lung-specific deletion can isolate tissue-specific effects.

• Expression Analysis: Quantitative PCR, immunohistochemistry, and western blotting should be used to measure RELM-β levels in tissues of interest. Single-cell RNA sequencing can further delineate cell type-specific expression patterns.

• Functional Readouts: For intestinal studies, ex vivo permeability assays using chambers with fluorescent tracers can quantify barrier integrity. In respiratory models, airway hyperresponsiveness measurements, bronchoalveolar lavage fluid analysis, and histopathological scoring provide functional insights.

• Molecular Pathway Analysis: Phosphorylation status of ERK1/2 and other signaling molecules should be assessed to understand the molecular mechanisms of RELM-β action .

• Translational Validation: Findings in animal models should be validated in human samples whenever possible, comparing tissue expression patterns between healthy controls and patients with the relevant disease.

What are the optimal protocols for expressing and purifying active recombinant human RELM-β?

For successful expression and purification of biologically active human RELM-β, researchers should follow these evidence-based protocols:

• Expression System: E. coli has been successfully used to produce non-glycosylated, disulfide-linked homodimeric RELM-β .

• Purification Process: The protein should be sterile filtered and lyophilized from 0.1% TFA to ensure stability .

• Storage Conditions: The lyophilized product is very stable at -20°C. For reconstituted protein, aliquoting and freezing at -20°C is recommended .

• Reconstitution Protocol: Prior to opening, centrifuge the vial, then gently pipet and wash down the sides to ensure full recovery. Reconstitute with sterile water at 0.1 mg/ml concentration, which can be further diluted as needed .

• Long-term Storage: Addition of a carrier protein (0.1% HSA or BSA) is recommended for long-term storage .

• Quality Control: Verify purity and structural integrity using:

  • UV spectroscopy at 280 nm

  • RP-HPLC calibrated against known standards

  • SDS-PAGE under reducing and non-reducing conditions

  • Endotoxin testing (levels should be <0.01ng/μg or <0.1EU/μg)

What experimental approaches best elucidate RELM-β's interactions with bacteria and host cells?

To thoroughly investigate RELM-β's interactions with bacteria and host cells, researchers should employ these methodological approaches:

• Direct Bacterial Interaction Studies:

  • Membrane permeabilization assays using fluorescent dyes

  • Electron microscopy to visualize bacterial membrane disruption

  • Bacterial killing assays comparing susceptibility across various species

• Host-Microbe Interface Analysis:

  • Fluorescence in situ hybridization (FISH) with bacterial 16S rRNA probes combined with immunofluorescence for RELM-β to visualize spatial relationships

  • Live imaging of fluorescently-labeled bacteria in the presence of RELM-β

  • Co-culture systems with intestinal epithelial cells, RELM-β, and bacterial strains

• Genetic Approaches:

  • RELM-β knockout models compared with wild-type in infection models

  • Bacterial transcriptomics before and after RELM-β exposure to identify stress responses

  • CRISPR/Cas9 modification of bacterial genes to identify targets critical for RELM-β susceptibility

• Structural Biology:

  • X-ray crystallography or cryo-electron microscopy of RELM-β-membrane interactions

  • NMR studies to identify protein domains involved in bacterial binding

  • Protein-lipid interaction assays to characterize membrane binding specificity

What are the most promising therapeutic applications of RELM-β?

Based on RELM-β's established biological functions, several therapeutic applications show particular promise:

• Antimicrobial Applications: RELM-β's selective bactericidal activity against Gram-negative bacteria positions it as a potential novel antimicrobial agent, particularly for intestinal infections resistant to conventional antibiotics . Its specificity could allow for targeted therapy with potentially fewer disruptions to beneficial microbiota.

• Gut Barrier Enhancement: For conditions characterized by compromised intestinal barrier function, such as inflammatory bowel disease, RELM-β supplementation might help restore proper bacterial compartmentalization and reduce bacterial translocation .

• Fibrosis Modulation: Conversely, in airway diseases like asthma where RELM-β contributes to pathological remodeling, inhibiting its activity might have therapeutic benefits . Small molecule inhibitors or neutralizing antibodies targeting RELM-β could potentially reduce fibrotic processes.

What challenges must be addressed in developing RELM-β-targeted therapies?

Developing RELM-β-targeted therapeutics presents several significant challenges:

• Context-Dependent Functions: RELM-β displays dual roles—protective in maintaining gut barrier function but potentially harmful in promoting airway remodeling . This context-dependent activity necessitates tissue-specific targeting approaches.

• Microbiome Implications: Since RELM-β affects microbial communities, therapies targeting it must carefully monitor potential microbiome alterations to avoid dysbiosis.

• Delivery Challenges: As a protein that acts primarily in mucosal environments, achieving effective drug concentrations at the site of action presents delivery challenges.

• Species Differences: Given the differences between human and rodent RELM family complexity, findings from animal models require careful validation in human systems .

• Biomarker Development: Identifying reliable biomarkers that reflect RELM-β activity in different tissues would be essential for patient stratification and monitoring therapeutic responses.