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Hepatocyte growth factor activator inhibitor‐1 (HAI‐1, also known as SPINT1) is an inhibitor of matriptase, a type‐2 transmembrane protease widely expressed in epithelial cells. HAI‐1 also functions as a chaperone to maintain the processing and localization of matriptase required for epithelial integrity. However, mechanisms underpinning the chaperone function remain to be elucidated. Here, we show that the first Kunitz domain (KD1) and the adjacent polycystic kidney disease (PKD) domain‐like internal domain of HAI‐1 are essential for the chaperone function. In HEK293T cells, which do not express endogenous HAI‐1 or matriptase, forced matriptase overexpression was unsuccessful unless sufficient HAI‐1 was co‐expressed. Among mutant HAI‐1 constructs, HAI‐1 with inactivation mutation in KD1 (HAI‐1mKD1) or HAI‐1 lacking the PKD domain (HAI‐1dPKD) was unable to support matriptase expression, and neither mutant formed a complex with activated matriptase. Matriptase did not localize to the cell surface when co‐expressed with HAI‐1dPKD. Moreover, HAI‐1dPKD accumulated in the cytoplasm of HEK293T and HaCaT cells rather than localizing to the cell surface, presumably due to misfolding as judged by altered antibody recognition. On the other hand, activationlocked and activity‐incompetent matriptase were stable and readily overexpressed and localized to the cell surface without HAI‐1. Therefore, the observed matriptase instability was caused by its own catalytic activity in the absence of inhibitory HAI‐1. The matriptase chaperone function of HAI‐1 is thus mediated primarily by the inhibition of undesired intracellular matriptase activity, and the PKD domain is essential for the proper folding and trafficking of inhibitory HAI‐1 and its chaperone function. [ABSTRACT FROM AUTHOR]

Abstract: Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre-pro-protein that undergoes several N- and C-terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755–838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca2+-binding site located near linker regions between domains. Conformational changes in the Ca2+-free and Ca2+-bound forms suggest that Ca2+-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein. [Copyright &y& Elsevier]

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry. [ABSTRACT FROM AUTHOR]