Chinemerem P. Ogbu

Purpose of Review Apolipoprotein A-I (APOA1) is the primary protein and structural scaffold on high density lipoproteins (HDL), playing key roles in HDL biogenesis, reverse cholesterol transport (RCT), regulation of systemic lipid metabolism, and cardiovascular protection. This review aims to trace the lifecycle of APOA1, from its cellular synthesis to its contributions in HDL formation, remodeling and eventual clearance or recycling through the body. By highlighting APOA1’s structural versatility, we focus on how its transitional states enable functional adaptation at various stages of its metabolic journey. Recent Findings Advancements in structural biology highlight how APOA1’s conformations drive its interactions with lipid transporters, enzymes, and cellular receptors. These transitions regulate HDL biogenesis and remodeling, with evidence suggesting APOA1’s early lipid-free state may serve as a critical determinant of its role on mature HDL particles. New studies also explore underexplored pathways, such as APOA1’s potential contributions to immune modulation and journey in the lymphatics system. Summary With its dynamic structural properties, APOA1 remains essential for HDL metabolic adaptation, systemic lipid transport, and inflammatory protection. Despite recent years of progress, key gaps remain, including elucidating structural conformations on lipid surfaces, their role in particle remodeling, and their relevance to metabolic fate. Addressing these areas through advanced imaging techniques and structural workflows in human-relevant models could provide therapeutic opportunities to enhance APOA1 function and combat cardiovascular and other metabolic diseases.

Claudins are a 27-member protein family that form and fortify specialized cell contacts in endothelium and epithelium called tight junctions. Tight junctions restrict paracellular transport across tissues by forming molecular barriers between cells. Claudin-binding molecules thus hold promise for modulating tight junction permeability to deliver drugs or as therapeutics to treat tight junction-linked disease. The development of claudin-binding molecules, however, is hindered by their intractability and small targetable surfaces. Here, we determine that a synthetic antibody fragment (sFab) we developed binds directly to 10 claudin subtypes with nanomolar affinity by targeting claudin’s paracellular-exposed surface. Application of this sFab to cells that model intestinal epithelium show that it opens the paracellular barrier comparable to a known, but application limited, tight junction modulator. This novel pan-claudin-binding molecule can probe claudin or tight junction structure and holds potential as a broad modulator of tight junction permeability for basic or translational applications.

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

Claudins are a family of ∼25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. Humans have 27 subtypes, which homo- and hetero-oligomerize to impart distinct properties and physiological functions to tissues and organs. As the structural and functional backbone of tight junctions, claudins are attractive targets for therapeutics capable of modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. We have developed a synthetic antibody fragment (sFab) that binds human claudin-4 and used it to resolve structures of its complex with Clostridium perfringens enterotoxin (CpE) using cryogenic electron microscopy (cryo-EM). The resolution of the structures reveals the architectures of 22 kDa claudin-4, the 14 kDa C-terminal domain of CpE, and the mechanism by which this sFab binds claudins. Further, we elucidate the biochemical and biophysical bases of sFab binding and demonstrate that this molecule exhibits subtype-selectivity by assaying homologous claudins. Our results provide a framework for developing sFabs against hard-to-target claudins and establishes the utility of sFabs as fiducial markers for determining cryo-EM structures of this small membrane protein family at resolutions that surpass X-ray crystallography. Taken together, this work highlights the ability of sFabs to elucidate claudin structure and function and posits their potential as therapeutics for modulating tight junctions by targeting specific claudin subtypes.

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.

Due to the COVID-19 pandemic and multiple devastating forest fires, the 2020 meeting of the Rocky Mountain Virology Association was held virtually. The three-day gathering featured talks describing recent advances in virology and prion research. The keynote presentation described the measles virus paradox of immune suppression and life-long immunity. Special invited speakers presented information concerning visualizing antiviral effector cell biology in mucosal tissues, uncovering the T-cell tropism of Epstein-Barr virus type 2, a history and current survey of coronavirus spike proteins, a summary of Zika virus vaccination and immunity, the innate immune response to flavivirus infections, a discussion concerning prion disease as it relates to multiple system atrophy, and clues for discussing virology with the non-virologist. On behalf of the Rocky Mountain Virology Association, this report summarizes selected presentations.