Mutations of hepatocyte growth factor activator inhibitor (HAI)-2 in humans cause sodium loss in the gastrointestinal (GI) tract in patients with syndromic congenital sodium diarrhea (SCSD). Aberrant ...regulation of HAI-2 target protease(s) was proposed as the cause of the disease. Here functional linkage of HAI-2 with two membrane-associated serine proteases, matriptase and prostasin was analyzed in Caco-2 cells and the human GI tract. Immunodepletion-immunoblot analysis showed that significant proportion of HAI-2 is in complex with activated prostasin but not matriptase. Unexpectedly, prostasin is expressed predominantly in activated forms and was also detected in complex with HAI-1, a Kunitz inhibitor highly related to HAI-2. Immunohistochemistry showed a similar tissue distribution of prostasin and HAI-2 immunoreactivity with the most intense labeling near the brush borders of villus epithelial cells. In contrast, matriptase was detected primarily at the lateral plasma membrane, where HAI-1 was also detected. The tissue distribution profiles of immunoreactivity against these proteins, when paired with the species detected suggests that prostasin is under tight control by both HAI-1 and HAI-2 and matriptase by HAI-1 in human enterocytes. Furthermore, HAI-1 is a general inhibitor of prostasin in a variety of epithelial cells. In contrast, HAI-2 was not found to be a significant inhibitor for prostasin in mammary epithelial cells or keratinocytes. The high levels of constitutive prostasin zymogen activation and the selective prostasin inhibition by HAI-2 in enterocytes suggest that dysregulated prostasin proteolysis may be particularly important in the GI tract when HAI-2 function is lost and/or dysregulated.
Matriptase plays important roles in epithelial integrity and function, which depend on its sorting to the basolateral surface of cells, where matriptase zymogen is converted to an active enzyme in ...order to act on its substrates. After activation, matriptase undergoes HAI-1-mediated inhibition, internalization, transcytosis, and secretion from the apical surface into the lumen. Matriptase is a mosaic protein with several distinct protein domains and motifs, which are a reflection of matriptase's complex cellular itinerary, life cycle, and the tight control of its enzymatic activity. While the molecular determinants for various matriptase regulatory events have been identified, the motif(s) required for translocation of human matriptase to the basolateral plasma membrane is unknown. The motif previously identified in rat matriptase is not conserved between the rodent and the primate. We, here, revisit the question for human matriptase through the use of a fusion protein containing a green fluorescent protein linked to the matriptase N-terminal fragment ending at Gly-149. A conserved seven amino acid motif EEGEVFL, which is similar to the monoleucine C-terminal to an acidic cluster motif involved in the basolateral targeting for some growth factors, has been shown to be required for matriptase translocation to the basolateral plasma membrane of polarized MDCK cells. Furthermore, time-lapse video microscopy showed that the motif appears to be required for entry into the correct transport vesicles, by which matriptase can undergo rapid trafficking and translocate to the plasma membrane. Our study reveals that the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting and serves as the basis for further research on its pathophysiological roles.
The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a ...matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation.
Matriptase is a type II transmembrane serine protease that is widely expressed in normal epithelial cells and epithelial cancers. Studies have shown that regulation of matriptase expression and ...activation becomes deranged in several cancers and is associated with poor disease-free survival. Although the central mechanism of its activation has remained unknown, our lab has previously demonstrated that inflammatory conditions such as intracellular pH decrease strongly induces matriptase activation. In this investigation, we first demonstrate clear matriptase activation following Fulvestrant (ICI) and Tykerb (Lapatinib) treatment in HER2-amplified, estrogen receptor (ER)-positive BT474, MDA-MB-361 and ZR-75-30 or single ER-positive MCF7 cells, respectively. This activation modestly involved Phosphoinositide 3-kinase (PI3K) activation and occurred as quickly as six hours post treatment. We also demonstrate that matriptase activation is not a universal hallmark of stress, with Etoposide treated cells showing a larger degree of matriptase activation than Lapatinib and ICI-treated cells. While etoposide toxicity has been shown to be mediated through reactive oxygen species (ROS) and MAPK/ERK kinase (MEK) activity, MEK activity showed no correlation with matriptase activation. Novelly, we demonstrate that endogenous and exogenous matriptase activation are ROS-mediated in vitro and inhibited by N-acetylcysteine (NAC). Lastly, we demonstrate matriptase-directed NAC treatment results in apoptosis of several breast cancer cell lines either alone or in combination with clinically used therapeutics. These data demonstrate the contribution of ROS-mediated survival, its independence of kinase-mediated survival, and the plausibility of using matriptase activation to indicate the potential success of antioxidant therapy.
As the most common cause of heart failure, dilated cardiomyopathy (DCM) is characterized by dilated ventricles and weakened contractile force. Mutations in the calcium handling protein phospholamban ...(PLN) are known to cause inherited DCM. Here, we introduced a PLN-R9C mutation in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9. The genome-edited iPSC line showed typical pluripotent cell morphology, robust expression of pluripotency markers, normal karyotype, and the capacity to differentiate into all three germ layers in vitro. The PLN-R9C iPSC line provides a valuable resource to dissect the molecular mechanisms underlying PLN mutation-related DCM.
Epidermal differentiation and barrier function require well-controlled matriptase and prostasin proteolysis, in which the Kunitz-type serine protease inhibitor HAI-1 represents the primary enzymatic ...inhibitor for both proteases. HAI-1, however, also functions as a chaperone-like protein necessary for normal matriptase synthesis and intracellular trafficking. Furthermore, other protease inhibitors, such as antithrombin and HAI-2, can also inhibit matriptase and prostasin in solution or in keratinocytes. It remains unclear, therefore, whether aberrant increases in matriptase and prostasin enzymatic activity would be the consequence of targeted deletion of HAI-1 and so subsequently contribute to the epidermal defects observed in HAI-1 knockout mice. The impact of HAI-1 deficiency on matriptase and prostasin proteolysis was, here, investigated in HaCaT human keratinocytes. Our results show that HAI-1 deficiency causes an increase in prostasin proteolysis via increased protein expression and zymogen activation. It remains unclear, however, whether HAI-1 deficiency increases “net” prostasin enzymatic activity because all of the activated prostasin was detected in complexes with HAI-2, suggesting that prostasin enzymatic activity is still under tight control in HAI-1-deficient keratinocytes. Matriptase proteolysis is, however, unexpectedly suppressed by HAI-1 deficiency, as manifested by decreases in zymogen activation, shedding of active matriptase, and matriptase-dependent prostasin zymogen activation. This suppressed proteolysis results mainly from the reduced ability of HAI-1-deficient HaCaT cells to activate matriptase and the rapid inhibition of nascent active matriptase by HAI-2 and other yet-to-be-identified protease inhibitors. Our study provides novel insights with opposite impacts by HAI-1 deficiency on matriptase versus prostasin proteolysis in keratinocytes.
The pathophysiological functions of matriptase, a type 2 transmembrane serine protease, rely primarily on its enzymatic activity, which is under tight control through multiple mechanisms. Among those ...regulatory mechanisms, the control of zymogen activation is arguably the most important. Matriptase zymogen activation not only generates the mature active enzyme but also initiates suppressive mechanisms, such as rapid inhibition by HAI-1, and matriptase shedding. These tightly coupled events allow the potent matriptase tryptic activity to fulfill its biological functions at the same time as limiting undesired hazards. Matriptase is converted to the active enzyme via a process of autoactivation, in which the activational cleavage is thought to rely on the interactions of matriptase zymogen molecules and other as yet identified proteins. Matriptase autoactivation can occur spontaneously and is rapidly followed by the formation and then shedding of matriptase-HAI-1 complexes, resulting in the presence of relatively low levels of the complex on cells. Activation can also be induced by several non-protease factors, such as the exposure of cells to a mildly acidic buffer, which rapidly causes high-level matriptase zymogen activation in almost all cell lines tested. In the current study, the structural requirements for this acid-induced zymogen activation are compared with those required for spontaneous activation through a systematic analysis of the impact of 18 different mutations in various structural domains and motifs on matriptase zymogen activation. Our study reveals that both acid-induced matriptase activation and spontaneous activation depend on the maintenance of the structural integrity of the serine protease domain, non-catalytic domains, and posttranslational modifications. The common requirements of both modes of activation suggest that acid-induced matriptase activation may function as a physiological mechanism to induce pericellular proteolysis by accelerating matriptase autoactivation.
Danon disease (DD) is caused by mutations of the gene encoding lysosomal-associated membrane protein type 2 (
), which lead to impaired autophagy, glycogen accumulation, and cardiac hypertrophy. ...However, it is not well understood why a large portion of DD patients develop arrhythmia and sudden cardiac death. In the current study, we generated
knockout (KO) human iPSC-derived cardiomyocytes (CM), which mimic the LAMP2 dysfunction in DD heart. Morphologic analysis demonstrated the sarcomere disarrangement in
KO CMs. In functional studies,
KO CMs showed near-normal calcium handling at base level. However, treatment of pro-maturation medium (MM) exaggerated the disease phenotype in the KO cells as they exhibited impaired calcium recycling and increased irregular beating events, which recapitulates the pro-arrhythmia phenotypes of DD patients. Further mechanistic study confirmed that MM treatment significantly enhanced the autophagic stress in the
KO CMs, which was accompanied by an increase of both cellular and mitochondrial reactive oxygen species (ROS) levels. Excess ROS accumulation in
KO CMs resulted in the over-activation of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and arrhythmogenesis, which was partially rescued by the treatment of ROS scavenger. In summary, our study has revealed ROS induced CaMKIIδ overactivation as a key mechanism that promotes cardiac arrhythmia in DD patients.
The type 2 transmembrane serine protease matriptase is involved in many pathophysiological processes probably via its enzymatic activity, which depends on the dynamic relationship between zymogen ...activation and protease inhibition. Matriptase shedding can prolong the life of enzymatically active matriptase and increase accessibility to substrates. We show here that matriptase shedding occurs via a de novo proteolytic cleavage at sites located between the SEA domain and the CUB domain. Point or combined mutations at the four positively charged amino acid residues in the region following the SEA domain allowed Arg-186 to be identified as the primary cleavage site responsible for matriptase shedding. Kinetic studies further demonstrate that matriptase shedding is temporally coupled with matriptase zymogen activation. The onset of matriptase shedding lags one minute behind matriptase zymogen activation. Studies with active site triad Ser-805 point mutated matriptase, which no longer undergoes zymogen activation or shedding, further suggests that matriptase shedding depends on matriptase zymogen activation, and that matriptase proteolytic activity may be involved in its own shedding. Our studies uncover an autonomous mechanism coupling matriptase zymogen activation, proteolytic activity, and shedding such that a proportion of newly generated active matriptase escapes HAI-1-mediated rapid inhibition by shedding into the extracellular milieu.
Abstract
Mutations of SPINT2, the gene encoding the integral membrane, Kunitz-type serine inhibitor HAI-2, primarily affect the intestine, while sparing many other HAI-2-expressing tissues, causing ...sodium loss in patients with syndromic congenital sodium diarrhea. The membrane-bound serine protease prostasin was previously identified as a HAI-2 target protease in intestinal tissues but not in the skin. In both tissues, the highly related inhibitor HAI-1 is, however, the default inhibitor for prostasin and the type 2 transmembrane serine protease matriptase. This cell-type selective functional linkage may contribute to the organ-selective damage associated with SPINT 2 mutations. To this end, the impact of HAI-2 deletion on matriptase and prostasin proteolysis was, here, compared using Caco-2 human colorectal adenocarcinoma cells and HaCaT human keratinocytes. Greatly enhanced prostasin proteolytic activity with a prolonged half-life and significant depletion of HAI-1 monomer were observed with HAI-2 loss in Caco-2 cells but not HaCaT cells. The constitutive, high level prostasin zymogen activation observed in Caco-2 cells, but not in HaCaT cells, also contributes to the excessive prostasin proteolytic activity caused by HAI-2 loss. HAI-2 deletion also caused increased matriptase zymogen activation, likely as an indirect result of increased prostasin proteolysis. This increase in activated matriptase, however, only had a negligible role in depletion of HAI-1 monomer. Our study suggests that the constitutive, high level of prostasin zymogen activation and the cell-type selective functional relationship between HAI-2 and prostasin renders Caco-2 cells more susceptible than HaCaT cells to the loss of HAI-2, causing a severe imbalance favoring prostasin proteolysis.
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