PRELP secreted from mural cells protects the function of blood brain barrier through regulation of endothelial cell-cell integrity.

Introduction: Proline/arginine-rich end leucine-rich repeat protein (PRELP), is a small secreted proteoglycan expressed by pericytes and vascular smooth muscle cells surrounding the brain vasculature of adult mouse. Methods: We utilised a Prelp knockout (Prelp -/-) mouse model to interrogate vasculature integrity in the brain alongside performing in vitro assays to characterise PRELP application to endothelial cells lines. Our findings were supplemented with RNA expression profiling to elucidate the mechanism of how PRELP maintains neurovasculature function. Results: Prelp -/- mice presented with neuroinflammation and reducedneurovasculature integrity, resulting in IgG and dextran leakage in the cerebellum and cortex. Histological analysis of Prelp -/- mice revealed reducedcell-cell integrity of the blood brain barrier, capillary attachment of pericytes andastrocyte end-feet. RNA-sequencing analysis found that cell-cell adhesion andinflammation are affected in Prelp -/- mice and gene ontology analysis as well as gene set enrichment analysis demonstrated that inflammation related processes and adhesion related processes such as epithelial-mesenchymal transition and apical junctions were significantly affected, suggesting PRELP is a regulator of cell-cell adhesion. Immunofluorescence analysis showed that adhesion junction protein expression levels of cadherin, claudin-5, and ZO-1, was suppressed in Prelp -/- mice neurovasculature. Additionally, in vitro studies revealed that PRELP application to endothelial cells enhances cell-cell integrity, induces mesenchymal-endothelial transition and inhibits TGF-ß mediated damage to cell-cell adhesion. Discussion: Our study indicates that PRELP is a novel endogenous secreted regulator of neurovasculature integrity and that PRELP application may be a potential treatment for diseases associated with neurovascular damage.

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain.

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al. developed a rational design of peptide binders targeting intrinsically disordered proteins based on contact energies between residue pairs. In this study, we designed 18 producible peptide binder candidates to TDP-43 LCD by using this method. Fluorescence anisotropy titration and surface plasmon resonance assays demonstrated that one of the designed peptides bound to TDP-43 LCD at 30 µM. Thioflavin-T fluorescence and sedimentation assays showed that the peptide binder suppressed the aggregation of TDP-43. In summary, this study highlights the potential applicability of peptide binder design for aggregation prone proteins.

Regulation of cadherin dimerization by chemical fragments as a trigger to inhibit cell adhesion.

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the process of X dimerization. Our findings will impact on a strategy for regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules.

Molecular basis for governing the morphology of type-I collagen fibrils by Osteomodulin.

Small leucine-rich repeat proteoglycan (SLRP) proteins have an important role in the organization of the extracellular matrix, especially in the formation of collagen fibrils. However, the mechanism governing the shape of collagen fibrils is poorly understood. Here, we report that the protein Osteomodulin (OMD) of the SLRP family is a monomeric protein in solution that interacts with type-I collagen. This interaction is dominated by weak electrostatic forces employing negatively charged residues of OMD, in particular Glu284 and Glu303, and controlled by entropic factors. The protein OMD establishes a fast-binding equilibrium with collagen, where OMD may engage not only with individual collagen molecules, but also with the growing fibrils. This weak electrostatic interaction is carefully balanced so it modulates the shape of the fibrils without compromising their viability.

Inhibition of homophilic dimerization and disruption of cell adhesion by P-cadherin-specific small molecules from SPR-based assays.

The inhibitor for the homophilic dimerization of P-cadherin was discovered by SPR-based screening using fragment compounds. Our SPR assays identified a specific P-cadherin binder, which was able to inhibit the cell adhesion of living CHO cells that expressed P-cadherin.

Osteomodulin regulates diameter and alters shape of collagen fibrils.

Osteomodulin (OMD) is a member of the small leucine-rich repeat proteoglycan family, which is involved in the organization of the extracellular matrix. OMD is located in bone tissue and is reportedly important for bone mineralization. However, the details of OMD function in bone formation are poorly understood. Using the baculovirus expression system, we produced recombinant human OMD and analyzed its interaction with type I collagen, which is abundant in bone. In this result, OMD directly interacted with purified immobilized collagen and OMD suppressed collagen fibril formation in a turbidity assay. Morphological analysis of collagen in the presence or absence of OMD demonstrated that OMD reduces the diameter and changes the shape of collagen fibrils. We conclude that OMD regulates the extracellular matrix during bone formation.