Activity-based protein profiling guided identification of urine proteinase 3 activity in subclinical rejection after renal transplantation.

BACKGROUND: The pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection. METHODS: Serine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine. RESULTS: In-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group. CONCLUSIONS: There appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

Mental retardation-related protease, motopsin (prss12), binds to the BRICHOS domain of the integral membrane protein 2a.

Motopsin (prss12), a mosaic serine protease secreted by neuronal cells, is believed to be important for cognitive function, as the loss of its function causes severe nonsyndromic mental retardation. To understand the molecular role of motopsin, we identified the integral membrane protein 2a (Itm2a) as a motopsin-interacting protein using a yeast two-hybrid system. A pull-down assay showed that the BRICHOS domain of Itm2a was essential for this interaction. Motopsin and Itm2a co-localized in COS cells and in cultured neurons when transiently expressed in these cells. Both proteins were co-immunoprecipitated from lysates of these transfected COS cells. Itm2a was strongly detected in a brain lysate prepared between postnatal day 0 and 10, during which period motopsin protein was also enriched in the brain. Immunohistochemistry detected Itm2a as patchy spots along endothelial cells of brain capillaries (which also expressed myosin II regulatory light chain [RLC]), and on glial fibrillary acidic protein (GFAP)-positive processes in the developing cerebral cortex. The data raise the possibility that secreted motopsin interacts with endothelial cells in the developing brain.

Overexpression of Motopsin, an Extracellular Serine Protease Related to Intellectual Disability, Promotes Adult Neurogenesis and Neuronal Responsiveness in the Dentate Gyrus.

Motopsin, a serine protease encoded by PRSS12, is secreted by neuronal cells into the synaptic clefts in an activity-dependent manner, where it induces synaptogenesis by modulating Na+/K+-ATPase activity. In humans, motopsin deficiency leads to severe intellectual disability and, in mice, it disturbs spatial memory and social behavior. In this study, we investigated mice that overexpressed motopsin in the forebrain using the Tet-Off system (DTG-OE mice). The elevated agrin cleavage or the reduced Na+/K+-ATPase activity was not detected. However, motopsin overexpression led to a reduction in spine density in hippocampal CA1 basal dendrites. While motopsin overexpression decreased the ratio of mature mushroom spines in the DG, it increased the ratio of immature thin spines in CA1 apical dendrites. Female DTG-OE mice showed elevated locomotor activity in their home cages. DTG-OE mice showed aberrant behaviors, such as delayed latency to the target hole in the Barnes maze test and prolonged duration of sniffing objects in the novel object recognition test (NOR), although they retained memory comparable to that of TRE-motopsin littermates, which normally express motopsin. After NOR, c-Fos-positive cells increased in the dentate gyrus (DG) of DTG-OE mice compared with that of DTG-SO littermates, in which motopsin overexpression was suppressed by the administration of doxycycline, and TRE-motopsin littermates. Notably, the numbers of doublecortin- and 5-bromo-2'-deoxyuridine-labeled cells significantly increased in the DG of DTG-OE mice, suggesting increased adult neurogenesis. Importantly, our results revealed a new function in addition to modulating neuronal responsiveness and spine morphology in the DG: the regulation of neurogenesis.

Effects of Cardiac Glycoside Digoxin on Dendritic Spines and Motor Learning Performance in Mice.

Synapse formation following the generation of postsynaptic dendritic spines is essential for motor learning and functional recovery after brain injury. The C-terminal fragment of agrin cleaved by neurotrypsin induces dendritic spine formation in the adult hippocampus. Since the α3 subunit of sodium-potassium ATPase (Na/K ATPase) is a neuronal receptor for agrin in the central nervous system, cardiac glycosides might facilitate dendritic spine formation and subsequent improvements in learning. This study investigated the effects of cardiac glycoside digoxin on dendritic spine turnover and learning performance in mice. Golgi-Cox staining revealed that intraperitoneal injection of digoxin less than its IC50 in the brain significantly increased the density of long spines (≥2 µm) in the cerebral cortex in wild-type mice and neurotrypsin-knockout (NT-KO) mice showing impairment of activity-dependent spine formation. Although the motor learning performance of NT-KO mice was significantly lower than control wild-type mice under the control condition, low doses of digoxin enhanced performance to a similar degree in both strains. In NT-KO mice, lower digoxin doses equivalent to clinical doses also significantly improved motor learning performance. These data suggest that lower doses of digoxin could modify dendritic spine formation or recycling and facilitate motor learning in compensation for the disruption of neurotrypsin-agrin pathway.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6.

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology.

Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability.

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called "NT-mini" and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT's heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

Molecular investigation of mental retardation locus gene PRSS12 by linkage analysis.

The present study was carried out to determine the prevalence of families having mental retardation in Pakistani population. We enrolled seven mentally retarded (MR) families with two or more affected individuals. Family history was taken to minimize the chances of other abnormalities. Pedigrees were drawn using the Cyrillic software (version 2.1). The structure of pedigrees shows that all the marriages are consanguineous and the families have recessive mode of inheritance. All the families were studied by linkage analysis to mental retardation locus (MRT1)/gene PRSS12. Three STR markers (D4S191, D4S2392, and D4S3024) in vicinity of mental retardation (MR) locus (MRT1)/gene PRSS12 were amplified on all the sample of each family by PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). The Haplotype were constructed to determine the pattern of inheritance and also to determine that a family was linked or unlinked to gene PRSS12. One out of the seven families was potentially linked to gene PRSS12, while the other six families remain unlinked.

Novel Peptide NT/K-CRS Derived from Kringle Structure of Neurotrypsin Inhibits Neovascularization In Vitro and In Vivo.

Purpose: To assess the anti-neovascularization effect of a novel peptide NT/K-CRS derived from the kringle domain of neurotrypsin in vitro and in vivo. Methods: Primary human umbilical vein endothelial cells (HUVECs) were treated with vascular endothelial growth factor (VEGF) in advance. Cell migration, lumen formation, and cell proliferation assays were performed to determine the anti-neovascularization effect of NT/K-CRS in HUVECs. TUNEL and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium tests were conducted to evaluate cell viability. Chick chorioallantoic membrane and oxygen-induced retinopathy model were established to assess the anti-angiogenic role of NT/K-CRS in vivo. Results: The in vitro results showed that NT/K-CRS effectively decreased VEGF-induced cell migration and endothelial tube formation, with no significant effect on cell proliferation and cell viability. In addition, NT/K-CRS showed great efficacy in angiogenesis inhibition in chicken embryos. The cytokine release syndrome (CRS) peptide also inhibited retinal neovascularization and improved retinal blood perfusion in oxygen-treated mouse pups through intravitreal injection. Conclusions: NT/K-CRS peptide derived from the kringle domain of neurotrypsin can strongly inhibit neovascularization in vitro and vivo. This novel peptide may become a promising therapeutic agent for neovascular-related ocular diseases.

Novel Peptide NT/K-CFY Derived from Kringle Structure of Neurotrypsin Inhibits Neovascularization.

Purpose: To assess the anti-neovascularization effect of a novel peptide NT/K-CFY derived from the kringle domain of neurotrypsin.Materials and Methods: Cell migration, lumen formation and cell proliferation assays were performed to determine the anti-neovascularization effect of NT/K-CFY in primary human umbilical vein endothelial cells (HUVECs). Chick chorioallantoic membrane (CAM) and oxygen-induced retinopathy (OIR) models were established to assess the anti-angiogenic role of NT/K-CFY in vivo. The retinal expression of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) was examined by western blot and real-time PCR in OIR model.Results: The in vitro results showed that NT/K-CFY effectively and safely decreased VEGF-induced cell migration, cell proliferation and tube formation in HUVECs. In addition, NT/K-CFY showed certain efficacy in angiogenesis inhibition in chicken embryos and oxygen-treated mouse pups. Moreover, the CFY peptide also improved retinal blood perfusion and reversed the abnormal expression of VEGF and PEDF in OIR mouse model.Conclusion: NT/K-CFY peptide strongly inhibits neovascularization in vitro and vivo. This novel peptide may become a promising therapeutic agent for ocular angiogenesis-related diseases.

Motopsin deficiency imparts partial insensitivity to doxorubicin-induced hippocampal impairments in adult mice.

Motopsin is a serine protease that plays a crucial role in synaptic functions. Loss of motopsin function causes severe intellectual disability in humans. In this study, we evaluated the role of motopsin in the neuropathological development of cognitive impairments following chemotherapy, also known as chemobrain. Motopsin knockout (KO) and wild-type (WT) mice were intravenously injected with doxorubicin (Dox) or saline four times every 8 days and were evaluated for open field, novel object recognition, and passive avoidance tests. Parvalbumin-positive neurons in the hippocampus were immunohistochemically analyzed. Dox administration significantly decreased the total distance in the open field test in both WT and motopsin KO mice without affecting the duration spent in the center square. A significant interaction between the genotype and drug treatment was detected in the recognition index (the rate to investigate a novel object) in the novel object recognition test, although Dox treatment did not affect the total investigation time. Additionally, Dox treatment significantly decreased the recognition index in WT mice, whereas it tended to increase the recognition index in motopsin KO mice. Dox treatment did not affect the latency to enter a dark compartment in either WT or motopsin KO mice in the passive avoidance test. Interestingly, Dox treatment increased the parvalbumin-positive neurons in the stratum oriens of the hippocampus CA1 region of only WT mice, not motopsin KO mice. Our data suggest that motopsin deficiency imparted partial insensitivity to Dox-induced hippocampal impairments. Alternatively, motopsin may contribute to the neuropathology of chemobrain.

Structural characterization of the third scavenger receptor cysteine-rich domain of murine neurotrypsin.

Neurotrypsin (NT) is a multi-domain serine protease of the nervous system with only one known substrate: the large proteoglycan Agrin. NT has seen to be involved in the maintenance/turnover of neuromuscular junctions and in processes of synaptic plasticity in the central nervous system. Roles which have been tied to its enzymatic activity, localized in the C-terminal serine-protease (SP) domain. However the purpose of NT's remaining 3-4 scavenger receptor cysteine-rich (SRCR) domains is still unclear. We have determined the crystal structure of the third SRCR domain of murine NT (mmNT-SRCR3), immediately preceding the SP domain and performed a comparative structural analysis using homologous SRCR structures. Our data and the elevated degree of structural conservation with homologous domains highlight possible functional roles for NT SRCRs. Computational and experimental analyses suggest the identification of a putative binding region for Ca2+ ions, known to regulate NT enzymatic activity. Furthermore, sequence and structure comparisons allow to single out regions of interest that, in future studies, might be implicated in Agrin recognition/binding or in interactions with as of yet undiscovered NT partners.

Dissecting the Extracellular Complexity of Neuromuscular Junction Organizers.

Synapse formation is a very elaborate process dependent upon accurate coordination of pre and post-synaptic specialization, requiring multiple steps and a variety of receptors and signaling molecules. Due to its relative structural simplicity and the ease in manipulation and observation, the neuromuscular synapse or neuromuscular junction (NMJ)-the connection between motor neurons and skeletal muscle-represents the archetype junction system for studying synapse formation and conservation. This junction is essential for survival, as it controls our ability to move and breath. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the formation of a highly specialized post-synaptic architecture and a highly differentiated nerve terminal. Furthermore, to ensure a fast and reliable synaptic transmission following neurotransmitter release, ligand-gated channels (acetylcholine receptors, AChRs) are clustered on the post-synaptic muscle cell at high concentrations in sites opposite the presynaptic active zone, supporting a direct role for nerves in the organization of the post-synaptic membrane architecture. This organized clustering process, essential for NMJ formation and for life, relies on key signaling molecules and receptors and is regulated by soluble extracellular molecules localized within the synaptic cleft. Notably, several mutations as well as auto-antibodies against components of these signaling complexes have been related to neuromuscular disorders. The recent years have witnessed strong progress in the understanding of molecular identities, architectures, and functions of NMJ macromolecules. Among these, prominent roles have been proposed for neural variants of the proteoglycan agrin, its receptor at NMJs composed of the lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific kinase (MuSK), as well as the regulatory soluble synapse-specific protease Neurotrypsin. In this review we summarize the current state of the art regarding molecular structures and (agrin-dependent) canonical, as well as (agrin-independent) non-canonical, MuSK signaling mechanisms that underscore the formation of neuromuscular junctions, with the aim of providing a broad perspective to further stimulate molecular, cellular and tissue biology investigations on this fundamental intercellular contact.

Vulnerability or resilience of motopsin knockout mice to maternal separation stress depending on adulthood behaviors.

BACKGROUND: Both environmental and genetic conditions contribute to the robust development of neuronal circuits and adulthood behaviors. Loss of motopsin gene function causes severe intellectual disability in humans and enhanced social behavior in mice. Furthermore, childhood maltreatment is a risk factor for some psychiatric disorders, and children with disabilities have a higher risk of abuse than healthy children. MATERIALS AND METHODS: In this study, we investigated the effects of maternal separation (MS) on adulthood behaviors of motopsin knockout (KO) and wild-type (WT) mice. RESULTS: The MS paradigm decreased the duration that WT mice stayed in the center area of an open field, but not for motopsin KO mice; however, it decreased the novel object recognition index in both genotypes. In the marble burying test, motopsin KO mice buried fewer marbles than WT mice, regardless of the rearing conditions. The MS paradigm slightly increased and reduced open arm entry in the elevated plus maze by WT and motopsin KO mice, respectively. In the three-chamber test, the rate of sniffing the animal cage was increased by the MS paradigm only for motopsin KO mice. After the three-chamber test, motopsin KO mice had fewer cFos-positive cells in the prelimbic cortex, which is involved in emotional response, than WT mice. In the infralimbic cortex, the MS paradigm decreased the number of cFos-positive cells in motopsin KO mice. CONCLUSION: Our results suggest that motopsin deficiency and childhood adversity independently affect some behaviors, but they may interfere with each other for other behaviors. Defective neuronal circuits in the prefrontal cortex may add to this complexity.

Tequila Regulates Insulin-Like Signaling and Extends Life Span in Drosophila melanogaster.

The aging process is a universal phenomenon shared by all living organisms. The identification of longevity genes is important in that the study of these genes is likely to yield significant insights into human senescence. In this study, we have identified Tequila as a novel candidate gene involved in the regulation of longevity in Drosophila melanogaster. We have found that a hypomorphic mutation of Tequila (Teq(f01792)), as well as cell-specific downregulation of Tequila in insulin-producing neurons of the fly, significantly extends life span. Tequila deficiency-induced life-span extension is likely to be associated with reduced insulin-like signaling, because Tequila mutant flies display several common phenotypes of insulin dysregulation, including reduced circulating Drosophila insulin-like peptide 2 (Dilp2), reduced Akt phosphorylation, reduced body size, and altered glucose homeostasis. These observations suggest that Tequila may confer life-span extension by acting as a modulator of Drosophila insulin-like signaling.