Identification of the First Low-Molecular-Weight Inhibitors of Matriptase-2

Sisay, Mihiret T.; Steinmetzer, Torsten; Stirnberg, Marit; Maurer, Eva; Hammami, Muhammad M.; Bajorath, Jürgen; Gütschow, Michael

doi:10.1021/jm100183e

Cited by 63 publications

(86 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These assays are based on the measurements of: 1) the luciferase activity driven by a HAMP promoter after overexpression of wild-type or mutated MT-2 in cells in the presence of HJV; 68 2) the release of the serine protease domain in the culture media of transfected cells to assess the proteolytic activity of MT-2; 43,49,[70][71][72][73] 3) the release of the chromogenic molecule p-nitroaniline based on the ability of the serine protease domain to cleave the artificial substrate N-(tert-butoxycarbonyl)-Gln-Ala-Arg-pnitroanilide (chromogenic assay developed by Sisay and collaborators). 34,73,74 Therefore, a MT-2 variant may be considered as pathogenic on the basis of the following findings: 1) a lack of release of the MT-2 S/P domain in the culture media of transfected cells; 2) a defective ability to repress the HAMP promoter, using the luciferase assay; or 3) no detection of membrane-derived HJV fragments, using cells co-transfected with HJV and MT-2.…”

Section: Functional Studies Of the Normal And Mutated Mt-2 Proteinsmentioning

confidence: 99%

Iron refractory iron deficiency anemia

et al. 2013

View full text Add to dashboard Cite

Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding Matriptase-2. This protein is a transmembrane serine protease that plays an essential role in down-regulating hepcidin, the key regulator of iron homeostasis. Hallmarks of this disease are microcytic hypochromic anemia, low transferrin saturation and normal/high serum hepcidin values. The anemia appears in the post-natal period, although in some cases it is only diagnosed in adulthood. The disease is refractory to oral iron treatment but shows a slow response to intravenous iron injections and partial correction of the anemia. To date, 40 different Matriptase-2 mutations have been reported, affecting all the functional domains of the large ectodomain of the protein. In vitro experiments on transfected cells suggest that Matriptase-2 cleaves Hemojuvelin, a major regulator of hepcidin expression and that this function is altered in this genetic form of anemia. In contrast to the low/undetectable hepcidin levels observed in acquired iron deficiency, in patients with Matriptase-2 deficiency, serum hepcidin is inappropriately high for the low iron status and accounts for the absent/delayed response to oral iron treatment. A challenge for the clinicians and pediatricians is the recognition of the disorder among iron deficiency and other microcytic anemias commonly found in pediatric patients. The current treatment of iron refractory iron deficiency anemia is based on parenteral iron administration; in the future, manipulation of the hepcidin pathway with the aim of suppressing it might become an alternative therapeutic approach

show abstract

Section: Functional Studies Of the Normal And Mutated Mt-2 Proteinsmentioning

confidence: 99%

Iron refractory iron deficiency anemia

et al. 2013

View full text Add to dashboard Cite

show abstract

“…As previously described, 38 in brief, activity was assayed in Tris/ saline buffer (50mM Tris, pH 8.0, and 150mM NaCl) at 37°C by monitoring the release of p-nitroaniline from the chromogenic substrate N-(tert-butoxycarbonyl)-Gln-Ala-Arg-p-nitroanilide (666M; Bachem) at a wavelength of 405 nm for up to 20 minutes.…”

Section: Measurement Of Mtp-2 Activitymentioning

confidence: 99%

“…Under our experimental conditions, detection of cleaved soluble HJV protein by Western blot analysis in the conditioned media of Hep3B cells treated with BMP6 ligand was not a sensitive assay for assessing changes in MTP-2 activity (supplemental Figure 3). To measure MTP-2 protein activity, we determined the hydrolysis rate of N-(tert-butoxycarbonyl)-Gln-Ala-Arg-p-nitroanilide, a specific chromogenic substrate for trypsin-like proteases, 38,39 in conditioned media derived from Hep3B cells treated with BMP6 and transfected with either control siRNA or TMPRSS6 siRNA. Treatment with BMP6 induced an increase by 3.6-fold of protease activity in the conditioned media ( Figure 1D).…”

Section: Tmprss6 Expression Is Increased By Bmp6mentioning

confidence: 99%

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

Meynard

Vaja

Sun

et al. 2011

Blood

Self Cite

106

104

View full text Add to dashboard Cite

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 TM-PRSS6 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 TM-PRSS6 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. (Blood. 2011;118(3): 747-756) IntroductionTransmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2 (MTP-2), a transmembrane serine protease produced by the liver, 1,2 was recently identified as a critical gene for iron homeostasis. 3,4 In both humans and mice, mutations in the TMPRSS6 gene lead to a strong increase in hepcidin expression, resulting in a dramatic decrease in ferroportin expression, and severe iron deficiency anemia that is unresponsive to oral iron treatment but partially responsive to parenteral iron therapy (IRIDA). [3][4][5][6] Moreover, genome-wide association studies identified common TMPRSS6 variants associated with hematologic parameters 7-9 and serum iron concentration, 8,10 highlighting that TMPRSS6 is important in the control of iron homeostasis and normal erythropoiesis. It was recently proposed that the mechanism by which TMPRSS6 inhibits hepcidin expression is by down-regulation of the bone morphogenetic protein (BMP)-SMAD signaling pathway via proteolytic cleavage of the BMP coreceptor hemojuvelin. 11,12 Hepcidin is an iron-regulated hepatic peptide hormone that controls iron absorption at the intestinal level and iron release from macrophages and hepatocytes. Hepcidin binds to the plasma membrane iron exporter ferroportin and induces its endocytosis and proteolysis, preventing release of iron into plasma. 13 Iron 14 and inflammatory cytokines (eg, interleukin-6) 15 stimulate hepcidin expression, leading to reduced plasma iron levels. In contrast, hypoxia, high erythropoietic activity, and iron deficiency inhibit hepcidin expression. 16 The role of the BMP-SMAD signaling pathway in regulating hepcidin...

show abstract

“…To assess the protease activity resulting from the shedding of MT2 into the culture medium of transfected Hela cells, proteolytic activity in concentrated medium from transfected cells were assayed, using a chromogenic substrate previously used in two different studies [Meynard et al, 2011;Sisay et al, 2010]. None of the mutants tested showed any detectable protease activity, whereas absorbance increased linearly with incubation time in the WT MT2 (Fig.…”

Section: Proteolytic Activity In the Media Of Cells Transfected With mentioning

confidence: 99%

Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity

Guillem¹,

Kannengiesser²,

Oudin³

et al. 2012

Hum. Mutat.

View full text Add to dashboard Cite

ABSTRACT:Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.

show abstract

Identification of the First Low-Molecular-Weight Inhibitors of Matriptase-2

Cited by 63 publications

References 61 publications

Iron refractory iron deficiency anemia

Iron refractory iron deficiency anemia

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

Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity

Contact Info

Product

Resources

About