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Study of a naturally occurring inhibitor of essential enzymes with latent disease-causing properties

A research team based in Denmark has obtained results that may be a step towards the fight against protease-related diseases such as influenza, skin diseases and cancer by taking advantage of a naturally occurring protease inhibitor.

A research team based in Denmark has obtained results that may be a step towards the fight against protease-related diseases such as influenza, skin diseases and cancer by taking advantage of a naturally occurring protease inhibitor. Figure: Jan K. Jensen

Proteases are enzymes that cleave other proteins, which are then either activated or inactivated. Defects in the regulation of specific proteases may lead to serious diseases. By understanding and thereby potentially exploit naturally occurring protease inhibitors, it may be possible to develop effective therapies against diseases caused by unregulated activity of certain proteases.

More specifically, the research team have studied a protease inhibitor known as HAI-1 (hepatocyte growth factor activator inhibitor-1), which is very important for normal development. It has been shown that HAI-1 is an essential protein, as genetically modified mice lacking the HAI-1 die in the utero due to placental defects. This is due to a faulty regulation of extracellular proteolysis is seen as mouse embryos were born when the protein expression of the target protease matriptase was simultaneously removed. Especially matriptase is known to play an important role in several serious skin diseases, the amplification of influenza virus in the body, as well as cancer. Changes in the natural balance between matriptase and HAI-1 in favour of the protease activity lead to cancer in mice. This imbalance is also observed in many human tumours. Hence, the proteolytic activity of matriptase is a promising target for drug therapy.

In addition to the N-terminal MANEC domain, which were recently characterised with NMR in the laboratory of Jan K Jensen, a second non-characterised domain has been proposed to exist in HAI-1’s extracellular part, the so-called "internal" domain. In addition, two so-called Kunitz-type inhibitor domains (Kunitz) are found in HAI-1, of which Kunitz-1 contains HAI-1’s protease inhibitory activity. The Kunitz domain is commonly described as a separate and functionally independent unit.

By solving the X-ray protein crystal structure of a HAI-1 fragment, which covers the unknown internal domain and Kunitz-1, it was revealed that the internal domain belongs to the relative unknown family of polycystic kidney disease (PKD) domains. Surprisingly, the structure also proposed how the internal domain form a complex with the neighbouring Kunitz-1. Using a combination of the liquid phase technique small-angle X-ray scattering, mutagenesis of selected amino acids and protein function-based experiments, the researchers succeeded in demonstrating how these inter-domain interactions not only stabilises the internal domain but also how the inhibitory activity of the Kunitz-1 is stimulated.

By completing the structural characterisation of HAI-1's until now unknown N-terminal region, the researchers have provided new knowledge about the close links between the tertiary structure and the overall activity of a multidomain protease inhibitor. An unknown mechanism has been presented where the binding of an adjacent domain stimulate the inhibitory activity of the Kunitz inhibitor. The new structure represents the first described intra-molecular binding between a Kunitz domain and another domain. Based on this new knowledge, the researchers hope to develop new ways for pharmacological intervention of protease-related diseases by exploiting naturally occurring protease inhibitors.


The results are published in the international journal Journal of Biological Chemistry (JBC) med titlen: Crystal Structure of a Two-Domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: Functional Interactions between the Kunitz-Type Inhibitor Domain-1 and the Neighboring Polycystic Kidney Disease-like Domain


For further information, please contact

Associate Professor Jan K. Jensen
Department of Molecular Biology and Genetics
Aarhus University, Denmark
jkj@mbg.au.dk - +45 87155542