Marit Stirnberg

Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

Mutations in transmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2, are responsible for the familial anemia disorder iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have inappropriately elevated levels of the iron regulatory hormone hepcidin, suggesting that TMPRSS6 is involved in negatively regulating hepcidin expression. Hepcidin is positively regulated by iron via the bone morphogenetic protein (BMP)-SMAD signaling pathway. In this study, we investigated whether BMP6 and iron also regulate TMPRSS6 expression. Here we demonstrate that, in vitro, treatment with BMP6 stimulates TMPRSS6 expression at the mRNA and protein levels and leads to an increase in matriptase-2 activity. Moreover, we identify that inhibitor of DNA binding 1 is the key element of the BMP-SMAD pathway to regulate TMPRSS6 expression in response to BMP6 treatment. Finally, we show that, in mice, Tmprss6 mRNA expression is stimulated by chronic iron treatment or BMP6 injection and is blocked by injection of neutralizing antibody against BMP6. Our results indicate that BMP6 and iron not only induce hepcidin expression but also induce TMPRSS6, a negative regulator of hepcidin expression. Modulation of TMPRSS6 expression could serve as a negative feedback inhibitor to avoid excessive hepcidin increases by iron to help maintain tight homeostatic balance of systemic iron levels.

Identification of the first low-molecular-weight inhibitors of matriptase-2.

As recently discovered, matriptase-2, a type II transmembrane serine protease, plays a crucial role in body iron homeostasis by down-regulating hepcidin expression, which results in increased iron levels. Thus, matriptase-2 represents a novel target for the development of enzyme inhibitors potentially useful for the treatment of systemic iron overload (hemochromatosis). A comparative three-dimensional model of the catalytic domain of matriptase-2 was generated and utilized for structure-based virtual screening in combination with similarity searching and knowledge-based compound design. Two N-protected dipeptide amides containing a 4-amidinobenzylamide as P1 residue (compounds 1 and 3) were identified as the first small molecule inhibitors of matriptase-2 with K(i) values of 170 and 460 nM, respectively. An inhibitor of the closely related protease matriptase (compound 2, K(i) = 220 nM), with more than 50-fold selectivity over matriptase-2, was also identified.

Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

Matriptase-2 (TMPRSS6) is directly up-regulated by hypoxia inducible factor-1: identification of a hypoxia-responsive element in the TMPRSS6 promoter region.

Abstract The type II transmembrane serine protease matriptase-2 (TMPRSS6) down-regulates the expression of hepcidin, the main regulator of systemic iron homeostasis, and increases in this way iron plasma levels. Matriptase-2 is up-regulated under hypoxic conditions, providing a new link between hypoxia signaling and iron homeostasis. In this study, we have characterized the TMPRSS6 promoter region and identified a functional hypoxia-responsive element (HRE). Mutations of the hypoxia inducible factor (HIF)-binding site located within the HRE abrogate HIF-1α-dependent induction of TMPRSS6 expression. The action of HIF-1α on TMPRSS6 promoter activity reveals a new regulative element for the suppression of hepcidin synthesis.

Inhibitors of Matriptase-2 Based on the Trypsin Inhibitor SFTI-1

A series of 17 new analogues of trypsin inhibitor SFTI‐1 were designed and synthesized to obtain matriptase‐2 inhibitors. A number of the modified bicyclic peptides displayed much higher affinity towards matriptase‐2 than towards the highly homologous matriptase‐1. Replacement of Lys5 by Arg in the wild‐type SFTI‐1 led to an 11‐fold increase in the matriptase‐2 inhibitory activity. Replacement of Arg2 by its enantiomer (D‐arginine) slightly lowered the inhibition of matriptase‐2, but almost completely abolished the affinity towards matriptase‐1, thus yielding the most selective matriptase‐2 inhibitor. This is the first report describing inhibitors of the recently discovered matriptase‐2 based on the SFTI‐1 structure. The results showed that SFTI‐1 is a promising scaffold for the design of potent and selective inhibitors of this enzyme.

Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore

An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) modulates hepcidin expression by inhibiting the cell surface protease matriptase-2.

Matriptase-2, a recently identified cell surface protease, is the key enzyme of iron homoeostasis modulating the expression of the liver peptide hormone hepcidin. HAI (hepatocyte growth factor activator inhibitor) types 1 and 2 (HAI-1 and HAI-2 respectively) have been shown to inhibit the close homologue, i.e. matriptase. By co-expressing matriptase-2 and the inhibitor HAI-2 we have identified HAI-2 displaying high inhibitory potential against matriptase-2 at the cell surface as well as in conditioned medium. Accordingly, complex formation between matriptase-2 and HAI-2 was demonstrated by isolation of the complex via immobilizing either HAI-2 or matriptase-2 from lysates and conditioned medium of co-expressing cells. Furthermore, HAI-2 indirectly influences the expression of the hepcidin-encoding gene HAMP. The inhibitor abrogates the matriptase-2-mediated suppression of HAMP expression, presumably by inhibiting the supposed potential of matriptase-2 to cleave membrane-bound HJV (haemojuvelin). Taken together, the results of the present study have characterized HAI-2 as an inhibitor of matriptase-2 that modulates the synthesis of hepcidin and provides new insights into the regulatory mechanism of iron homoeostasis, with clinical importance for a treatment of iron overload diseases.

Active site mapping of trypsin, thrombin and matriptase-2 by sulfamoyl benzamidines

The benzamidine moiety, a well-known arginine mimetic, has been introduced in a variety of ligands, including peptidomimetic inhibitors of trypsin-like serine proteases. According to their primary substrate specificity, the benzamidine residue interacts with the negatively charged aspartate at the bottom of the S1 pocket of such enzymes. Six series of benzamidine derivatives (1-73) were synthesized and evaluated as inhibitors of two prototype serine proteases, that is, bovine trypsin and human thrombin. As a further target, human matriptase-2, a recently discovered type II transmembrane serine protease, was investigated. Matriptase-2 represents an important regulatory protease in iron homeostasis by down-regulation of the hepcidin expression. Compounds 1-73 were designed to contain a fixed sulfamoyl benzamidine moiety as arginine mimetic and a linker-connected additional substructure, such as a tert-butyl ester, carboxylate or second benzamidine functionality. A systematic mapping approach was performed with these inhibitors to scan the active site of the three target proteases. In particular, bisbenzamidines, able to interact with both the S1 and S3/S4 binding sites, showed notable affinity. In branched bisbenzamidines 66-73 containing a third hydrophobic residue, opposite effects of the stereochemistry on trypsin and thrombin inhibition were observed.

Matriptase-2, a regulatory protease of iron homeostasis: possible substrates, cleavage sites and inhibitors.

Matriptase-2 is a cell surface serine protease with a modular structure. The exploration of its function in iron homeostasis was of significance for the understanding of the regulation of hepcidin expression, the master protein in iron control. Mutations in matriptase- 2 cause iron-refractory iron deficiency anemia (IRIDA), an iron deficiency disorder where the level of hepcidin is inappropriately high. Matriptase-2 controls hepcidin expression through the suppression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic homologue protein (SMAD) signaling, probably by cleaving the BMP co-receptor hemojuvelin. Since prospective studies revealed that genetic inactivation of matriptase-2 reduces iron loading in different mouse models, matriptase-2 becomes highly attractive as a novel target for the design of low-molecular weight inhibitors. The first synthetic peptidomimetic matriptase-2 inhibitors have been reported. A computational model of the active site of matriptase-2 based on the X-ray crystal structure of the close homologue matriptase was generated and mutational studies were performed in order to identify critical amino acids specifying the preferred recognition site of matriptase-2. So far, the only known putative natural substrates of matriptase-2 are hemojuvelin and matriptase-2 itself, as this protease undergoes complex auto-processing during zymogen activation. Cleavage sites within both natural substrates were identified.

Substrate specificity of human matriptase-2

Human matriptase-2 is an enzyme that belongs to the family of type II transmembrane serine proteases. So far there is a limited knowledge regarding its specificity and protein substrate(s). One of the identified natural substrates is hemojuvelin, a protein involved in the control of iron homeostasis. In this work, we describe the synthesis and evaluation of internal quenched substrates using a combinatorial approach. The iterative deconvolution of two libraries to define the specificity of matriptase-2 yielded to the identification of the substrate ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 with a k(cat)/K(m) value of 4.5 × 10(5) M(-1) × s(-1), i.e. the highest specificity constant reported so far for matriptase-2.