Mutagenesis of Claudins to Probe in vivo Interactions and Assemblies

Paracellular transport of solutes and the control of the flow of molecules through the intracellular space in vertebrate epithelia is directed by tight junctions (TJs). Claudins form paracellular barriers and pores that determine tight junction permeability. This investigation attempts to explain the molecular bases for destruction and reconstruction of tight junctions within epithelial cells, occurring via both natural and disease-causing mechanisms. This interdisciplinary research is important in the advancement of our understanding of human biology and health, as different disruptions to epithelial tight junctions are hallmarks of many human diseases. The overall objective was to take previously made Claudin containing vectors (pEGFP-N3) containing a Green Fluorescent Protein (GFP) sequence and utilize site specific mutations to change the sequences to express Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP). Proteins tested in this experiment were Human Claudins 1, 9, 19, Tricellulin, and Occludin. Once the specified claudins and TAMPs are tagged with CFP and YFP, FRET microscopy techniques can be utilized to determine generic interactions within the formation of TJs, forming a baseline for future research

Disruption of Claudin-Made Tight Junction Barriers by Clostridium perfringens Enterotoxin: Insights from Structural Biology

Claudins are a family of integral membrane proteins that enable epithelial cell/cell interactions by localizing to and driving the formation of tight junctions. Via claudin self-assembly within the membranes of adjoining cells, their extracellular domains interact, forming barriers to the paracellular transport of small molecules and ions. The bacterium Clostridium perfringens causes prevalent gastrointestinal disorders in mammals by employing an enterotoxin (CpE) that targets claudins. CpE binds to claudins at or near tight junctions in the gut and disrupts their barrier function, potentially by disabling their assembly or via cell signaling means—the mechanism(s) remain unclear. CpE ultimately destroys claudin-expressing cells through the formation of a cytotoxic membrane-penetrating β-barrel pore. Structures obtained by X-ray crystallography of CpE, claudins, and claudins in complex with CpE fragments have provided the structural bases of claudin and CpE functions, revealing potential mechanisms for the CpE-mediated disruption of claudin-made tight junctions. This review highlights current progress in this space—what has been discovered and what remains unknown—toward efforts to elucidate the molecular mechanism of CpE disruption of tight junction barriers. It further underscores the key insights obtained through structure that are being applied to develop CpE-based therapeutics that combat claudin-overexpressing cancers or modulate tight junction barriers

Development, structure, and mechanism of synthetic antibodies that target claudin and \u3ci\u3eClostridium perfringens\u3c/i\u3e enterotoxin complexes

Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE’s C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membranepenetrating β-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via β-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin–CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs’ mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4–cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab’s targeting mechanism. From these insights, we generated a model for CpE’s claudin-bound β-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases

Early Change in Urine Protein as a Surrogate End Point in Studies of IgA Nephropathy: An Individual-Patient Meta-analysis

Background The role of change in proteinuria as a surrogate end point for randomized trials in immunoglobulin A nephropathy (IgAN) has previously not been thoroughly evaluated. Study Design Individual patient–level meta-analysis. Setting & Population Individual-patient data for 830 patients from 11 randomized trials evaluating 4 intervention types (renin-angiotensin system [RAS] blockade, fish oil, immunosuppression, and steroids) examining associations between changes in urine protein and clinical end points at the individual and trial levels. Selection Criteria for Studies Randomized controlled trials of IgAN with measurements of proteinuria at baseline and a median of 9 (range, 5-12) months follow-up, with at least 1 further year of follow-up for the clinical outcome. Predictor 9-month change in proteinuria. Outcome Doubling of serum creatinine level, end-stage renal disease, or death. Results Early decline in proteinuria at 9 months was associated with lower risk for the clinical outcome (HR per 50% reduction in proteinuria, 0.40; 95% CI, 0.32-0.48) and was consistent across studies. Proportions of treatment effect on the clinical outcome explained by early decline in proteinuria were estimated at 11% (95% CI, −19% to 41%) for RAS blockade and 29% (95% CI, 6% to 53%) for steroid therapy. The direction of the pooled treatment effect on early change in proteinuria was in accord with the direction of the treatment effect on the clinical outcome for steroids and RAS blockade. Trial-level analyses estimated that the slope for the regression line for the association of treatment effects on the clinical end points and for the treatment effect on proteinuria was 2.15 (95% Bayesian credible interval, 0.10-4.32). Limitations Study population restricted to 11 trials, all having fewer than 200 patients each with a limited number of clinical events. Conclusions Results of this analysis offer novel evidence supporting the use of an early reduction in proteinuria as a surrogate end point for clinical end points in IgAN in selected settings

Mutagenesis of Claudins to Probe in vivo Interactions and Assemblies

Paracellular transport of solutes and the control of the flow of molecules through the intracellular space in vertebrate epithelia is directed by tight junctions (TJs). Claudins form paracellular barriers and pores that determine tight junction permeability. This investigation attempts to explain the molecular bases for destruction and reconstruction of tight junctions within epithelial cells, occurring via both natural and disease-causing mechanisms. This interdisciplinary research is important in the advancement of our understanding of human biology and health, as different disruptions to epithelial tight junctions are hallmarks of many human diseases. The overall objective was to take previously made Claudin containing vectors (pEGFP-N3) containing a Green Fluorescent Protein (GFP) sequence and utilize site specific mutations to change the sequences to express Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP). Proteins tested in this experiment were Human Claudins 1, 9, 19, Tricellulin, and Occludin. Once the specified claudins and TAMPs are tagged with CFP and YFP, FRET microscopy techniques can be utilized to determine generic interactions within the formation of TJs, forming a baseline for future research

Structural basis for Clostridium perfringens enterotoxin targeting of claudins at tight junctions in mammalian gut

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell–cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, 24 claudins govern TJ barriers—but the basis for CpE’s selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that entero-toxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other sub-types. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin–enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin–CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans

High-Throughput Nano-Scale Characterization of Membrane Proteins Using Fluorescence-Detection Size-Exclusion Chromatography

Structural biology has revealed predicting heterologous expression levels, homogeneity, and stability of a protein from its primary structure are exceedingly difficult. Membrane proteins, in particular, present numerous challenges that make obtaining milligram quantities of quality samples problematic. For structural and functional investigation of these molecules, however, this is what is required. Fluorescence size-exclusion chromatography (F-SEC), a technique where a protein of biological interest is fused to green fluorescent protein (GFP) and monitored, circumvents many bottlenecks inherent to membrane protein structural biology. In vivo expression yields, as well as in vitro homogeneity and stability, can be rapidly evaluated utilizing nanogram quantities of unpurified protein. In this chapter we describe our most current protocols for expression screening and biochemical characterization of membrane proteins using F-SEC, as it pertains to a high-throughput (HTP) crystallographic pipeline. Therein, the methods and workflow were designed and optimized for structure–function elucidation of eukaryotic integral membrane proteins, but may be applied to prokaryotic or water-soluble proteins with minor modifications, thus making it a useful general approach