Biophysics of claudin proteins in tight junction architecture: Three decades of progress.

Tight junctions are cell-cell adhesion complexes that act as gatekeepers of the paracellular space. Formed by several transmembrane proteins, the claudin family performs the primary gate-keeping function. The claudin proteins form charge and size-selective diffusion barriers to maintain homeostasis across endothelial and epithelial tissue. Of the 27 known claudins in mammals, some are known to seal the paracellular space, while others provide selective permeability. The differences in permeability arise due to the varying expression levels of claudins in each tissue. The tight junctions are observed as strands in freeze-fracture electron monographs; however, at the molecular level, tight junction strands form when multiple claudin proteins assemble laterally (cis assembly) within a cell and head-on (trans assembly) with claudins of the adjacent cell in a zipper-like architecture, closing the gap between the neighboring cells. The disruption of tight junctions caused by changing claudin expression levels or mutations can lead to diseases. Therefore, knowledge of the molecular architecture of the tight junctions and how that is tied to tissue-specific function is critical for fighting diseases. Here, we review the current understanding of the tight junctions accrued over the last three decades from experimental and computational biophysics perspectives.

Claudin-23 reshapes epithelial tight junction architecture to regulate barrier function.

Claudin family tight junction proteins form charge- and size-selective paracellular channels that regulate epithelial barrier function. In the gastrointestinal tract, barrier heterogeneity is attributed to differential claudin expression. Here, we show that claudin-23 (CLDN23) is enriched in luminal intestinal epithelial cells where it strengthens the epithelial barrier. Complementary approaches reveal that CLDN23 regulates paracellular ion and macromolecule permeability by associating with CLDN3 and CLDN4 and regulating their distribution in tight junctions. Computational modeling suggests that CLDN23 forms heteromeric and heterotypic complexes with CLDN3 and CLDN4 that have unique pore architecture and overall net charge. These computational simulation analyses further suggest that pore properties are interaction-dependent, since differently organized complexes with the same claudin stoichiometry form pores with unique architecture. Our findings provide insight into tight junction organization and propose a model whereby different claudins combine to form multiple distinct complexes that modify epithelial barrier function by altering tight junction structure.

How can we discover developable antibody-based biotherapeutics?

Antibody-based biotherapeutics have emerged as a successful class of pharmaceuticals despite significant challenges and risks to their discovery and development. This review discusses the most frequently encountered hurdles in the research and development (R&D) of antibody-based biotherapeutics and proposes a conceptual framework called biopharmaceutical informatics. Our vision advocates for the syncretic use of computation and experimentation at every stage of biologic drug discovery, considering developability (manufacturability, safety, efficacy, and pharmacology) of potential drug candidates from the earliest stages of the drug discovery phase. The computational advances in recent years allow for more precise formulation of disease concepts, rapid identification, and validation of targets suitable for therapeutic intervention and discovery of potential biotherapeutics that can agonize or antagonize them. Furthermore, computational methods for de novo and epitope-specific antibody design are increasingly being developed, opening novel computationally driven opportunities for biologic drug discovery. Here, we review the opportunities and limitations of emerging computational approaches for optimizing antigens to generate robust immune responses, in silico generation of antibody sequences, discovery of potential antibody binders through virtual screening, assessment of hits, identification of lead drug candidates and their affinity maturation, and optimization for developability. The adoption of biopharmaceutical informatics across all aspects of drug discovery and development cycles should help bring affordable and effective biotherapeutics to patients more quickly.

Unique structural features of claudin-5 and claudin-15 lead to functionally distinct tight junction strand architecture.

Members of the claudin family impart unique paracellular selectivity to tight junctions. However, the structure-function relationship between claudin's strand architecture and the paracellular charge- and size-selectivity is not well-understood. This work examines the molecular assembly of claudin-5, a barrier-forming protein, and claudin-15, a channel-forming protein, to determine their structural and functional properties. We adopt a bottom-up approach starting from claudin monomers to build the molecular architecture of the tight junction strands. First, we investigated the cis assembly of claudin-5 and -15 dimers using the Protein Association Energy Landscape method. Out of the millions of dimer conformations, we narrowed down key cis claudin-5 and -15 dimer conformations that were thermodynamically and kinetically stable. Second, we performed the trans assembly of dimers to identify the tetrameric building blocks that serve as the repeat units for strand formation. Finally, the strand assembly of the tetrameric repeat units showed fundamentally distinct strand architectures. In claudin-5, the cis and trans interactions seal the paracellular space, while in claudin-15, the gaps in the paracellular space lead to pore formation. This detailed study suggests that each member of the claudin family is unique and requires systematic molecular-level analysis for determining the strand architecture.

Joint Profile Characteristics of Long-Latency Transient Evoked and Distortion Otoacoustic Emissions.

PURPOSE: In clinical practice, otoacoustic emissions (OAEs) are interpreted as either "present" or "absent." However, OAEs have the potential to inform about etiology and severity of hearing loss if analyzed in other dimensions. A proposed method uses the nonlinear component of the distortion product OAEs together with stimulus frequency OAEs to construct a joint reflection-distortion profile. The objective of the current study is to determine if joint reflection-distortion profiles can be created using long-latency (LL) components of transient evoked OAEs (TEOAEs) as the reflection-type emission. METHOD: LL TEOAEs and the nonlinear distortion OAEs were measured from adult ears. Individual input-output (I/O) functions were created, and OAE level was normalized by dividing by the stimulus level yielding individual gain functions. Peak strength, compression threshold, and OAE level at compression threshold were derived from individual gain functions to create joint reflection-distortion profiles. RESULTS: TEOAEs with a poststimulus window starting at 6 ms had I/O functions with compression characteristics similar to LL TEOAE components. The model fit the LL gain functions, which had R 2 > .93, significantly better than the nonlinear distortion OAE gain functions, which had R 2 = .596-.99. Interquartile ranges for joint reflection-distortion profiles were larger for compression threshold and OAE level at compression threshold but smaller for peak strength than those previously published. CONCLUSIONS: The gain function fits LL TEOAEs well. Joint reflection-distortion profiles are a promising method that could enhance diagnosis of hearing loss, and use of the LL TEOAE in the profile for peak strength may be important because of narrow interquartile ranges. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.20323593.

Computational Nanoscopy of Tight Junctions at the Blood-Brain Barrier Interface.

The selectivity of the blood-brain barrier (BBB) is primarily maintained by tight junctions (TJs), which act as gatekeepers of the paracellular space by blocking blood-borne toxins, drugs, and pathogens from entering the brain. The BBB presents a significant challenge in designing neurotherapeutics, so a comprehensive understanding of the TJ architecture can aid in the design of novel therapeutics. Unraveling the intricacies of TJs with conventional experimental techniques alone is challenging, but recently developed computational tools can provide a valuable molecular-level understanding of TJ architecture. We employed the computational methods toolkit to investigate claudin-5, a highly expressed TJ protein at the BBB interface. Our approach started with the prediction of claudin-5 structure, evaluation of stable dimer conformations and nanoscale assemblies, followed by the impact of lipid environments, and posttranslational modifications on these claudin-5 assemblies. These led to the study of TJ pores and barriers and finally understanding of ion and small molecule transport through the TJs. Some of these in silico, molecular-level findings, will need to be corroborated by future experiments. The resulting understanding can be advantageous towards the eventual goal of drug delivery across the BBB. This review provides key insights gleaned from a series of state-of-the-art nanoscale simulations (or computational nanoscopy studies) performed on the TJ architecture.

Obtaining Protein Association Energy Landscape for Integral Membrane Proteins.

Integral membrane proteins are ubiquitous in biological cellular and subcellular membranes. Despite their significance to cell function, isolation of membrane proteins from their hydrophobic lipid environment and further characterization remains a challenge. To obtain insights into membrane proteins, computational approaches such as docking or self-assembly simulations have been used; however, the promise of these approaches has been limited due to the computational cost. Here we present a new approach called Protein AssociatioN Energy Landscape (PANEL) that provides an extensive and converged data set for all possible conformations of membrane protein associations using a combination of stochastic sampling and equilibration simulations. The PANEL method samples the rotational space around both interacting proteins to obtain the comprehensive interaction energy landscape. We demonstrate the versatility of the PANEL method using two distinct applications: (a) dimerization of claudin-5 tight junction proteins in phospholipid bilayer membrane and (b) dimer and trimer formation of the Outer membrane protein F (OmpF) in the lipopolysaccharide-rich bacterial outer membrane. Both applications required only a fraction of simulation cost compared to self-assembly simulations. The method is robust as it can capture changes in protein-protein conformations caused by point mutations. Moreover, the method is versatile and independent of the molecular resolution (atomistic or coarse grain) or the choice of force field employed to compute the pair-interaction energies. The PANEL method is implemented in easy-to-use scripts that are available for download for general use by the scientific community to characterize any pair of interacting integral membrane proteins.

Palmitoylation of Claudin-5 Proteins Influences Their Lipid Domain Affinity and Tight Junction Assembly at the Blood-Brain Barrier Interface.

Post-translational lipid modification of integral membrane proteins is recognized as a key mechanism to modulate protein-protein and membrane-protein associations. Despite numerous reports of lipid-modified proteins, molecular-level understanding of the influence of lipid-modification of key membrane proteins remains elusive. This study focuses on the lipid modification of one such protein-claudin-5, a critical component of the blood-brain barrier tight junctions. Claudin-5 proteins are responsible for regulating the size and charge-selective permeability at the blood-brain interface. Palmitoylation of the claudin family of proteins is implicated in influencing the tight junction permeability in prior experimental studies. Here, we investigate the impact of palmitoylation on claudin-5 self-assembly using multiscale molecular simulations. To elucidate protein-membrane interactions, we used three model membrane compositions (endoplasmic reticulum, cholesterol-enriched endoplasmic reticulum, and plasma membrane) that mimic the complexity of cell organelles encountered by a typical membrane protein in its secretion pathway. The results show that palmitoylation enhances protein's affinity for cholesterol-rich domains in a membrane, and it can elicit a site-specific response based on the location of the palmitoyl chain on the protein. Also, in claudin-5 self-assembly, palmitoylation restricts specific protein-protein conformations. Overall, this study demonstrates the significance of post-translational lipid modification of proteins in cellular and subcellular membranes, and the impact palmitoylation can have on critical cellular functions of the protein.