Structure and function of invertebrate Kunitz serine protease inhibitors

Ranasinghe, Shiwanthi L.; McManus, Donald P.

doi:10.1016/j.dci.2012.10.005

Cited by 115 publications

(109 citation statements)

References 79 publications

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“…The inhibitory mechanism is characterized by an essentially non-hydrolyzable scissile bond and exceptionally slow rates of dissociation, resulting in effectively irreversible inhibition of the protease (for a review, see Ref. 43). The Kunitz scaffold has received considerable pharmaceutical interest due to the relative ease of engineering protease inhibitors with high affinity and specificity and the applicability to directed evolution strategies (for a review, see Ref.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

Hong

Meulemeester

Jacobi

et al. 2016

Journal of Biological Chemistry

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Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knockout mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain).

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Section: Discussionmentioning

confidence: 99%

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

Hong

Meulemeester

Jacobi

et al. 2016

Journal of Biological Chemistry

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“…Endogenous proteases are found in all organisms from microbes to mammals and participate in various physiological and pathological processes including coagulation, immune responses, and cancer metastasis (Ranasinghe and McManus, 2013). In the central nervous system (CNS), a subfamily of proteases, serine proteases, and their inhibitors have been identified in both neurons and glia (Choi et al, 1990, Weinstein et al, 1995, Vivien and Buisson, 2000).…”

Section: Introductionmentioning

confidence: 99%

Expression and localization of Inter-alpha Inhibitors in rodent brain

et al. 2016

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Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day-18 mice and postnatal (P) day-10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIPs proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS.

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“…They contain six cysteine residues that form three conserved disulfide bonds in a 1–6, 2–4, and 3–5 arrangement that maintains structural integrity of the inhibitor and allows presentation of a protease-binding loop at its surface (see Fig. 2) (11–13). A highly exposed P1 active site residue at position 15, which inserts into the S1 site of the cognate protease, is located at the peak of the binding loop and is of prime importance in determining the specificity of serine protease inhibition (14).…”

Section: Introductionmentioning

confidence: 99%

Unexpected Activity of a Novel Kunitz-type Inhibitor

Smith¹,

Tikhonova

Jewhurst³

et al. 2016

Journal of Biological Chemistry

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Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host.

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Structure and function of invertebrate Kunitz serine protease inhibitors

Cited by 115 publications

References 79 publications

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

Expression and localization of Inter-alpha Inhibitors in rodent brain

Unexpected Activity of a Novel Kunitz-type Inhibitor

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