TMPRSS13 zymogen activation, surface localization, and shedding is regulated by proteolytic cleavage within the non-catalytic stem region.

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Here we characterize a novel post-translational mechanism important for TMPRSS13 function: proteolytic cleavage within the extracellular TMPRSS13 stem region located between the transmembrane domain and the first site of N-linked glycosylation at asparagine (N)-250 in the scavenger receptor cysteine rich (SRCR) domain. Importantly, the catalytic competence of TMPRSS13 is essential for stem region cleavage, suggesting an autonomous mechanism of action. Site-directed mutagenesis of the 10 basic amino acids (four arginine and six lysine residues) in this region abrogated zymogen activation and catalytic activity of TMPRSS13, as well as phosphorylation, cell surface expression, and shedding. Mutation analysis of individual arginine residues identified R223, a residue located between the low-density lipoprotein receptor class A domain and the SRCR domain, as important for stem region cleavage. Mutation of R223 causes a reduction in the aforementioned functional processing steps of TMPRSS13. These data provide further insight into the roles of different post-translational modifications as regulators of the function and localization of TMPRSS13. Additionally, the data suggest the presence of complex interconnected regulatory mechanisms that may serve to ensure the proper levels of cell-surface and pericellular TMPRSS13-mediated proteolysis under homeostatic conditions.

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation, proteolytic activity, and cell surface localization.

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.

Mechanism of Deoxyguanosine Diphosphate Insertion by Human DNA Polymerase ß.

DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) ß, we examine how Pol ß incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol ß to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (kpol), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.

The cell-surface anchored serine protease TMPRSS13 promotes breast cancer progression and resistance to chemotherapy.

Breast cancer progression is accompanied by increased expression of extracellular and cell-surface proteases capable of degrading the extracellular matrix as well as cleaving and activating downstream targets. The type II transmembrane serine proteases (TTSPs) are a family of cell-surface proteases that play critical roles in numerous types of cancers. Therefore, the aim of this study was to identify novel and uncharacterized TTSPs with differential expression in breast cancer and to determine their potential roles in progression. Systematic in silico data analysis followed by immunohistochemical validation identified increased expression of the TTSP family member, TMPRSS13 (transmembrane protease, serine 13), in invasive ductal carcinoma patient tissue samples compared to normal breast tissue. To test whether loss of TMPRSS13 impacts tumor progression, TMPRSS13 was genetically ablated in the oncogene-induced transgenic MMTV-PymT tumor model. TMPRSS13 deficiency resulted in a significant decrease in overall tumor burden and growth rate, as well as a delayed formation of detectable mammary tumors, thus suggesting a causal relationship between TMPRSS13 expression and the progression of breast cancer. Complementary studies using human breast cancer cell culture models revealed that siRNA-mediated silencing of TMPRSS13 expression decreases proliferation, induces apoptosis, and attenuates invasion. Importantly, targeting TMPRSS13 expression renders aggressive triple-negative breast cancer cell lines highly responsive to chemotherapy. At the molecular level, knockdown of TMPRSS13 in breast cancer cells led to increased protein levels of the tumor-suppressive protease prostasin. TMPRSS13/prostasin co-immunoprecipitation and prostasin zymogen activation experiments identified prostasin as a potential novel target for TMPRSS13. Regulation of prostasin levels may be a mechanism that contributes to the pro-oncogenic properties of TMPRSS13 in breast cancer. TMPRSS13 represents a novel candidate for targeted therapy in combination with standard of care chemotherapy agents in patients with hormone receptor-negative breast cancer or in patients with tumors refractory to endocrine therapy.

TMPRSS13 promotes cell survival, invasion, and resistance to drug-induced apoptosis in colorectal cancer.

Cancer progression is often accompanied by increased levels of extracellular proteases capable of remodeling the extracellular matrix and promoting pro-cancerous signaling pathways by activating growth factors and receptors. The type II transmembrane serine protease (TTSP) family encompasses several proteases that play critical roles in cancer progression; however, the expression or function of the TTSP TMPRSS13 in carcinogenesis has not been examined. In the present study, we found TMPRSS13 to be differentially expressed at both the transcript and protein levels in human colorectal cancer (CRC). Immunohistochemical analyses revealed consistent high expression of TMPRSS13 protein on the cancer cell surface in CRC patient samples; in contrast, the majority of normal colon samples displayed no detectable expression. On a functional level, TMPRSS13 silencing in CRC cell lines increased apoptosis and impaired invasive potential. Importantly, transgenic overexpression of TMPRSS13 in CRC cell lines increased tolerance to apoptosis-inducing agents, including paclitaxel and HA14-1. Conversely, TMPRSS13 silencing rendered CRC cells more sensitive to these agents. Together, our findings suggest that TMPRSS13 plays an important role in CRC cell survival and in promoting resistance to drug-induced apoptosis; we also identify TMPRSS13 as a potential new target for monotherapy or combination therapy with established chemotherapeutics to improve treatment outcomes in CRC patients.

Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cell-surface localization.

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Although various TTSPs have been characterized in detail biochemically and functionally, the basic properties of TMPRSS13 remain unclear. Here, we investigate the activation, inhibition, post-translational modification, and localization of TMPRSS13. We show that TMPRSS13 is a glycosylated, active protease and that its own proteolytic activity mediates zymogen cleavage. Full-length, active TMPRSS13 exhibits impaired cell-surface expression in the absence of the cognate Kunitz-type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 or HAI-2. Concomitant presence of TMPRSS13 with either HAI-1 or -2 mediates phosphorylation of residues in the intracellular domain of the protease, and it coincides with efficient transport of the protease to the cell surface and its subsequent shedding. Cell-surface labeling experiments indicate that the dominant form of TMPRSS13 on the cell surface is phosphorylated, whereas intracellular TMPRSS13 is predominantly non-phosphorylated. These data provide novel insight into the cellular properties of TMPRSS13 and highlight phosphorylation of TMPRSS13 as a novel post-translational modification of this TTSP family member and potentially other members of this family of proteases.

Type II transmembrane serine proteases as potential targets for cancer therapy.

Carcinogenesis is accompanied by increased protein and activity levels of extracellular cell-surface proteases that are capable of modifying the tumor microenvironment by directly cleaving the extracellular matrix, as well as activating growth factors and proinflammatory mediators involved in proliferation and invasion of cancer cells, and recruitment of inflammatory cells. These complex processes ultimately potentiate neoplastic progression leading to local tumor cell invasion, entry into the vasculature, and metastasis to distal sites. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression. In this review the knowledge collected over the past two decades about the molecular mechanisms underlying the pro-cancerous properties of selected TTSPs will be summarized. Furthermore, we will discuss how these insights may facilitate the translation into clinical settings in the future by specifically targeting TTSPs as part of novel cancer treatment regimens.

Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis.

Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro-cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin-like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non-proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas.

Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling.

Matriptase is an epithelia-specific membrane-anchored serine protease that has received considerable attention in recent years because of its consistent dysregulation in human epithelial tumours, including breast cancer. Mice with reduced levels of matriptase display a significant delay in oncogene-induced mammary tumour formation and blunted tumour growth. The abated tumour growth is associated with a decrease in cancer cell proliferation. Here we demonstrate by genetic deletion and silencing that the proliferation impairment in matriptase-deficient breast cancer cells is caused by their inability to initiate activation of the c-Met signalling pathway in response to fibroblast-secreted pro-HGF. Similarly, inhibition of matriptase catalytic activity using a selective small-molecule inhibitor abrogates the activation of c-Met, Gab1 and AKT, in response to pro-HGF, which functionally leads to attenuated proliferation in breast carcinoma cells. We conclude that matriptase is critically involved in breast cancer progression and represents a potential therapeutic target in breast cancer.

Repeated aripiprazole treatment causes dopamine D2 receptor up-regulation and dopamine supersensitivity in young rats.

Aripiprazole is a second-generation antipsychotic that is increasingly being prescribed to children and adolescents. Despite this trend, little preclinical research has been done on the neural and behavioral actions of aripiprazole during early development. In the present study, young male and female Sprague-Dawley rats were pretreated with vehicle, haloperidol (1 mg/kg), or aripiprazole (10 mg/kg) once daily on postnatal days (PD) 10-20. After 1, 4, or 8 days (i.e. on PD 21, PD 24, or PD 28), amphetamine-induced locomotor activity and stereotypy, as well as dorsal striatal D2 receptor levels, were measured in separate groups of rats. Pretreating young rats with aripiprazole or haloperidol increased D2 binding sites in the dorsal striatum. Consistent with these results, dopamine supersensitivity was apparent when aripiprazole- and haloperidol-pretreated rats were given a test day injection of amphetamine (2 or 4 mg/kg). Increased D2 receptor levels and altered behavioral responding persisted for at least 8 days after conclusion of the pretreatment regimen. Contrary to what has been reported in adults, repeated aripiprazole treatment caused D2 receptor up-regulation and persistent alterations of amphetamine-induced behavior in young rats. These findings are consistent with human clinical studies showing that children and adolescents are more prone than adults to aripiprazole-induced side effects, including extrapyramidal symptoms.

Novelty-induced conditioned place preference, sucrose preference, and elevated plus maze behavior in adult rats after repeated exposure to methylphenidate during the preweanling period.

Early treatment with methylphenidate has a persistent effect on the affective (i.e., anxiety- and depressive-like) behaviors of adult rats and mice. Interestingly, age at methylphenidate exposure appears to be a critical determinant influencing the expression of affective behaviors. In the present study, we exposed rats to methylphenidate during the preweanling period (i.e., PD 11-PD 20) because this ontogenetic period is analogous to early childhood in humans (an age associated with increasing methylphenidate usage). Rats were injected with methylphenidate (0, 2, or 5mg/kg) from PD 11 to PD 20 and reactivity to rewarding and aversive stimuli were measured in early adulthood. Specifically, novelty-induced CPP, sucrose preference, and elevated plus maze behavior were assessed on PD 60. Early treatment with 2 or 5mg/kg methylphenidate increased total time spent in the white compartment of the CPP chamber. This methylphenidate-induced effect occurred regardless of exposure condition. Performance on the elevated plus maze was also impacted by early methylphenidate exposure, because rats treated with 5mg/kg methylphenidate spent more time in the closed compartment of the elevated plus maze than vehicle controls. Early methylphenidate exposure did not alter sucrose preference. These data indicate that exposing rats to methylphenidate during the preweanling period differentially affects anxiety-like behavior depending on the type of anxiety-provoking stimulus. Specifically, early methylphenidate exposure decreased aversion to a bright white room when measured on a novelty-induced CPP task, whereas methylphenidate caused a long-term increase in anxiety when measured on the elevated plus maze.

Postnatal manganese exposure alters the expression of D2L and D2S receptor isoforms: relationship to PKA activity and Akt levels.

Postnatal manganese chloride (Mn) exposure causes persistent changes in presynaptic dopamine (DA) functioning (e.g., Mn reduces DA transporter levels and DA uptake), but evidence that Mn affects postsynaptic DA receptors and their associated second messenger systems is equivocal. Therefore, a goal of the present study was to determine whether exposing rats to Mn on postnatal days (PD) 1-21 would cause long-term alterations in D2 long (D2L) and D2 short (D2S) receptors that were detectible in adulthood (i.e., on PD 90). Signaling systems associated with D2 receptors were also assessed. Specifically, we measured protein kinase A (PKA) activity in the dorsal striatum and prefrontal cortex (PFC), whereas immunoblotting was used to quantify phosphorylated Akt (p-Akt) and phosphorylated ERK. Results showed that early Mn exposure caused a persistent elevation of D2L and D2S protein expression in the dorsal striatum, as well as an increase in the number of D2 binding sites. Conversely, Mn reduced D2 specific binding in the PFC on PD 90. PKA activity of Mn-treated rats was enhanced in both the dorsal striatum and PFC, whereas p-Akt levels were elevated in the dorsal striatum. When considered together, these results suggest that postnatal Mn exposure either directly or indirectly alters the functioning of postsynaptic DA receptors. One possibility is that early Mn exposure depresses presynaptic dopaminergic functioning and reduces DA levels, thereby causing an up-regulation of D2 receptors and a dysregulation of DA-associated signaling pathways. An alternative explanation is that early Mn exposure affects D2 receptors and PKA/p-Akt levels via independent mechanisms.

Effects of repeated and acute aripiprazole or haloperidol treatment on dopamine synthesis in the dorsal striatum of young rats: comparison to adult rats.

The purpose of the present study was to determine whether repeated treatment with the D2 partial agonist aripiprazole or the D2 antagonist haloperidol alters dopamine (DA) synthesis characteristics in the dorsal striatum of young rats. To this end, rats received a daily pretreatment regimen of aripiprazole or haloperidol on postnatal days (PD) 10-20 and were tested 24 or 72 h later after an acute injection of vehicle, aripiprazole, haloperidol, or quinpirole (a D2 agonist). For comparison purposes, adult rats were pretreated with an 11-day regimen of saline or haloperidol on PD 70-80 and DA synthesis was measured after acute drug treatment on PD 83. Dorsal striatal DA synthesis was determined by measuring L-dihydroxyphenylalanine accumulation after NSD-1015 treatment. In a separate experiment, the ability of repeated drug treatment to up-regulate dorsal striatal D2 receptors was assessed in young and adult rats 72 h after drug discontinuation. The major findings of this study were that: (a) acute treatment with haloperidol and aripiprazole increased DA synthesis while quinpirole reduced it; (b) pretreatment with haloperidol and aripiprazole blunted the synthesis-modulating effects of acutely administered dopaminergic drugs; and (c) DA synthesis of young and adult rats was affected in a qualitatively similar manner by DA agonist, antagonist, and partial agonist drugs. In conclusion, results from the present study suggest that synthesis-modulating autoreceptors in the dorsal striatum are functionally mature by the end of the preweanling period and DA synthesis declines to near basal levels during the course of repeated aripiprazole treatment.