Modulating the selectivity of matriptase-2 inhibitors with unnatural amino acids (original) (raw)
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Cell Metabolism, 2008
The liver peptide hepcidin regulates body iron, is upregulated in iron overload and inflammation and downregulated in iron deficiency/hypoxia. The transmembrane serine protease matriptase-2 (TMPRSS6) inhibits the hepcidin response and its mutational inactivation causes iron-deficient anemia in mice and humans. Here we confirm the inhibitory effect of matriptase-2 on hepcidin promoter; we show that matriptase-2 lacking the serine protease domain, identified in the anemic Mask mouse (matriptase-2 MASK ), is fully inactive and that mutant R774C found in patients with genetic iron deficiency has decreased inhibitory activity. Matriptase-2 cleaves hemojuvelin (HJV), a regulator of hepcidin, on plasma membrane; matriptase-2 MASK shows no and the human mutant only partial cleavage capacity. Matriptase-2 interacts with HJV through the ectodomain since the interaction is conserved in matriptase-2 MASK . The expression of matriptase-2 mutants in zebrafish results in anemia, confirming the matriptase-2 role in iron metabolism and its interaction with HJV.
Low intracellular iron increases the stability of matriptase-2
The Journal of biological chemistry, 2014
Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin (HJV) into an inactive form. HJV is a bone morphogenetic protein (BMP) co-receptor. Here we report that MT2 is upregulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of MT2 gene, TMPRSS6, incubation with apo-transferrin or membrane impermeable iron chelator, desferroxamine, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane permeable iron chelator, SIH, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron-depletion. Neither acute nor chronic iron d...
Journal of Biological Chemistry
Edited by F. Peter Guengerich Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an ironregulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2 ؊/؊ mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.
Blood, 2011
Recent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative real-time reverse transcription polymerase chain reaction analysis revealed no signifi...
Journal of Biological Chemistry
Edited by Amanda J. Fosang Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv ؊/؊ mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2 ؊/؊ mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway. Systemic iron homeostasis is maintained by coordinately regulating the iron absorption in the duodenum, iron recycling from senescent erythrocytes, and iron mobilization from the liver storage (1). In this regulation, hepatic hormone hepcidin plays an essential role (2). Hepcidin inhibits iron efflux into the circulation by targeting the plasma membrane iron exporter, ferroportin, for degradation (3). Lack of hepcidin causes juvenile hemochromatosis, a severe form of iron overload disorder (4). In contrast, inappropriately high levels of hepcidin cause iron-deficiency anemia (5-7). Under physiological conditions, hepcidin expression is regulated positively by body iron content to maintain iron homeostasis. Hepatic hepcidin expression is induced via the bone morphogenetic protein (BMP) 2 signaling pathway (8-10). BMPs are cytokines that are a subset of the transforming growth factor- (TGF-) superfamily (11). BMP signaling is initiated upon the binding of BMP ligands to type I and type II BMP receptors on the cell surface. Upon BMP binding, the type II receptors phosphorylate the type I receptors, leading to the phosphorylation of SMAD1/5/8 in the cytoplasm. The phosphorylated SMADs form heteromeric complexes with the co-SMAD, SMAD4, and then translocate into the nucleus where they induce the transcription of target genes. The BMP ligands that are responsible for hepcidin induction in hepatocytes are derived from adjacent sinusoidal endothelial cells (12, 13). Depletion of Bmp6 or Bmp2 in the liver sinusoidal endothelial cells markedly reduces hepcidin expression and causes iron overload in mice to a similar extent as seen in the Bmp6 global knockout mice (13-16). There are multiple type I BMP receptors (ALK1, ALK2, ALK3, and ALK6) and type II BMP receptors (BMPR2, ActR2a, and ActR2b). Type I BMP receptors exist as dimers, as do type II BMP receptors. Upon ligand binding, they form heterotetramers. Liver-specific deletion of ALK3, to a lesser extent ALK2, or both BMPR2 and ActRIIA ablates BMP signaling and hepcidin expression in mice (17, 18). Additionally, liver-specific depletion of Smad1/5 or Smad4 also reduces hepcidin expression and causes iron overload (10, 19). These observations indicate that hepatocytes utilize a selective set of BMP ligands, BMP receptors, and SMADs to induce hepcidin expression. A normal range of hepcidin expression also requires the involvement of other plasma membrane proteins, including
PloS one, 2016
Matriptase-2 (TMPRSS6) is an important negative regulator of hepcidin expression; however, the effects of iron overload or accelerated erythropoiesis on liver TMPRSS6 protein content in vivo are largely unknown. We determined TMPRSS6 protein content in plasma membrane-enriched fractions of liver homogenates by immunoblotting, using a commercial antibody raised against the catalytic domain of TMPRSS6. Plasma membrane-enriched fractions were obtained by centrifugation at 3000 g and washing. TMPRSS6 was detected in the 3000 g fraction as a 120 kDa full-length protein in both mice and rats. Feeding of iron-deficient diet as well as erythropoietin treatment increased TMPRSS6 protein content in rats and mice by a posttranscriptional mechanism; the increase in TMPRSS6 protein by erythropoietin was also observed in Bmp6-mutant mice. Administration of high doses of iron to mice (200, 350 and 700 mg/kg) decreased TMPRSS6 protein content. Hemojuvelin was detected in the plasma membrane-enriche...
Blood, 2009
Matriptase-2 is a transmembrane serine protease that negatively regulates hepcidin expression by cleaving membrane-bound hemojuvelin. Matriptase-2 has a complex ectodomain, including a C-terminal serine protease domain and its activation requires an autocatalytic cleavage. Matriptase-2 mutations have been reported in several patients with iron-refractory iron deficiency anemia. Here we describe a patient with 2 missense mutations in the second class A low-density lipoprotein receptor (LDLRA) domain. Functional studies of these 2 mutations and of a previously reported mutation in the second C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1 (CUB) domain were performed. Transfection of mutant cDNAs showed that membrane targeting of the 2 LDLRA mutants was impaired, with Golgi retention of the variants. The activating cleavage was absent for the LDLRA mutants and reduced for the CUB mutant. All 3 mutated proteins were still able to physically interact with hemojuv...