RESUMO
The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria-dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod ) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full-length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+ /K+ -ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified.
Assuntos
Catepsina B/metabolismo , Canais Epiteliais de Sódio/metabolismo , Hipertensão/etiologia , Hipertensão/metabolismo , Síndrome Nefrótica/complicações , Síndrome Nefrótica/metabolismo , Amilorida/farmacologia , Animais , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Glomerulosclerose Segmentar e Focal/enzimologia , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/urina , Hipertensão/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Lisossomos/enzimologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Síndrome Nefrótica/genética , Proteinúria/metabolismo , Proteólise , Sódio/metabolismoRESUMO
Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to ß-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography-tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated p-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response.
Assuntos
Catepsina A/farmacologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Proteômica/métodos , Animais , Catepsina A/metabolismo , Cromatografia Líquida , Humanos , Espectrometria de Massas , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Proteólise , Proteoma/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Normal epithelial cells and carcinoma cells can acquire invasiveness by epithelial-to-mesenchymal transition (EMT), a process of considerable cellular remodeling. The endosomal/lysosomal compartment is a principal site of intracellular protein degradation. Lysosomal cathepsin proteases are secreted during cancer progression. The established pro-metastatic role of specific cysteine cathepsins has until now been ascribed to their contribution to extracellular matrix remodeling. We hypothesized that cysteine cathepsins affect transforming growth factor ß-1 (TGFß-1)-induced EMT of normal and malignant mammary epithelial cells. METHODS: The role of lysosomal proteolysis in TGFß-1-induced EMT and invasion was investigated in a normal and a novel malignant murine mammary epithelial cell line. The contribution of cysteine cathepsins was determined by addition of the general cysteine cathepsin inhibitor E64d. Hallmarks of EMT were analyzed by molecular- and cell-biologic analyses including real-time cell migration/invasion assays. A quantitative proteome comparison using stable isotopic labeling with amino acids in culture (SILAC) showed the effect of E64d on TGFß-1 induced proteome changes. Lysosomal patterning and junctional adhesion molecule A (Jam-a) localization and abundance were analyzed by immunofluorescence. RESULTS: We found increased lysosome activity during EMT of malignant mammary epithelial cells. Cysteine cathepsin inhibition had no effect on the induction of the TGFß-1-induced EMT program on transcriptional level. Protease inhibition did not affect invasion of TGFß-1 treated normal mammary epithelial cells, but reduced the invasion of murine breast cancer cells. Remarkably, reduced invasion was also evident if E64d was removed 24 h before the invasion assay in order to allow for recovery of cathepsin activity. Proteome analyses revealed a high abundance of lysosomal enzymes and lysosome-associated proteins in cancer cells treated with TGFß-1 and E64d. An accumulation of those proteins and of lysosomal vesicles was further confirmed by independent methods. Interestingly, E64d caused lysosomal accumulation of Jam-a, a tight junction component facilitating epithelial cell-cell adhesion. CONCLUSION: Our results demonstrate an important role of lysosomal proteolysis in cellular remodeling during EMT and a pivotal contribution of lysosomal cysteine cathepsins to TGFß-1 induced acquisition of breast cancer cell invasiveness. These findings provide an additional rationale to use cathepsin inhibitors to stall tumor metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Neoplasias da Mama/patologia , Catepsinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Camundongos , Proteólise , Proteoma/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
DJ-1 is an abundant and ubiquitous component of cellular proteomes. DJ-1 supposedly exerts a wide variety of molecular functions, ranging from enzymatic activities as a deglycase, protease, and esterase to chaperone functions. However, a consensus perspective on its molecular function in the cellular context has not yet been reached. Structurally, the C-terminal helix 8 of DJ-1 has been proposed to constitute a propeptide whose proteolytic removal transforms a DJ-1 zymogen to an active hydrolase with potential proteolytic activity. To better understand the cell-contextual functionality of DJ-1 and the role of helix 8, we employed post-mitotically differentiated, neuron-like SH-SY5Y neuroblastoma cells with stable over-expression of full length DJ-1 or DJ-1 lacking helix 8 (ΔH8), either with a native catalytically active site (C106) or an inactive site (C106A active site mutation). Global proteome comparison of cells over-expressing DJ-1 ΔH8 with native or mutated active site cysteine indicated a strong impact on mitochondrial biology. N-terminomic profiling however did not highlight direct protease substrate candidates for DJ-1 ΔH8, but linked DJ-1 to elevated levels of activated lysosomal proteases, albeit presumably in an indirect manner. Finally, we show that DJ-1 ΔH8 loses the deglycation activity of full length DJ-1. Our study further establishes DJ-1 as deglycation enzyme. Helix 8 is essential for the deglycation activity but dispensable for the impact on lysosomal and mitochondrial biology; further illustrating the pleiotropic nature of DJ-1.
Assuntos
Cisteína/metabolismo , Neurônios/metabolismo , Proteína Desglicase DJ-1/metabolismo , Proteoma/metabolismo , Humanos , Lisossomos/metabolismo , Mutação/genética , Estresse Oxidativo/fisiologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Peptídeo Hidrolases/metabolismo , Proteína Desglicase DJ-1/genética , Proteoma/genéticaRESUMO
Longitudinal axons provide connectivity between remote areas of the nervous system. Although the molecular determinants driving commissural pathway formation have been well characterized, mechanisms specifying the formation of longitudinal axon tracts in the vertebrate nervous system are largely unknown. Here, we study axon guidance mechanisms of the longitudinal dopaminergic (DA) diencephalospinal tract. This tract is established by DA neurons located in the ventral diencephalon and is thought to be involved in modulating locomotor activity. Using mutant analysis as well as gain of function and loss of function experiments, we demonstrate that longitudinal DA axons navigate by integrating long-range signaling of midline-derived cues. Repulsive Robo2/Slit signaling keeps longitudinal DA axons away from the midline. In the absence of repulsive Robo2/Slit function, DA axons are attracted toward the midline by DCC (deleted in colorectal cancer)/Netrin1 signaling. Thus, Slit-based repulsion counteracts Netrin-mediated attraction to specify lateral positions of the DA diencephalospinal tract. We further identified heparan sulfate proteglycans as essential modulators of DA diencephalospinal guidance mechanisms. Our findings provide insight into the complexity of positioning far-projecting longitudinal axons and allow us to provide a model for DA diencephalospinal pathfinding. Simultaneous integrations of repulsive and attractive long-range cues from the midline act in a concerted manner to define lateral positions of DA longitudinal axon tracts.
Assuntos
Axônios/metabolismo , Diencéfalo/citologia , Dopamina/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Neurônios/citologia , Transdução de Sinais/fisiologia , Medula Espinal/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/genética , Mutação/genética , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Netrina-1 , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed. METHODOLOGY/PRINCIPAL FINDINGS: Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh(-/-) mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh(-/-) mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh(+/+) and Ctsh(-/-) mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh(-/-) mice. CONCLUSIONS/SIGNIFICANCE: We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.