RESUMEN

Pulmonary arterial hypertension (PAH) is characterised by pulmonary vascular remodelling causing premature death from right heart failure. Established DNA variants influence PAH risk, but susceptibility from epigenetic changes is unknown. We addressed this through epigenome-wide association study (EWAS), testing 865,848 CpG sites for association with PAH in 429 individuals with PAH and 1226 controls. Three loci, at Cathepsin Z (CTSZ, cg04917472), Conserved oligomeric Golgi complex 6 (COG6, cg27396197), and Zinc Finger Protein 678 (ZNF678, cg03144189), reached epigenome-wide significance (p < 10-7) and are hypermethylated in PAH, including in individuals with PAH at 1-year follow-up. Of 16 established PAH genes, only cg10976975 in BMP10 shows hypermethylation in PAH. Hypermethylation at CTSZ is associated with decreased blood cathepsin Z mRNA levels. Knockdown of CTSZ expression in human pulmonary artery endothelial cells increases caspase-3/7 activity (p < 10-4). DNA methylation profiles are altered in PAH, exemplified by the pulmonary endothelial function modifier CTSZ, encoding protease cathepsin Z.

Asunto(s)

Hipertensión Arterial Pulmonar , Humanos , Proteínas Morfogenéticas Óseas , Catepsina Z , Metilación de ADN/genética , Células Endoteliales , Hipertensión Pulmonar Primaria Familiar

Asunto(s)

Queratodermia Palmoplantar , Neoplasias Cutáneas , Humanos , Catepsinas/metabolismo , Catepsina Z , Inflamación , Receptores ErbB

RESUMEN

BACKGROUND: The abnormal function of epidermal growth factor receptor (EGFR) has recently been shown to underlie various disorders of cornification. OBJECTIVES: To delineate the genetic basis of a novel dominant form of palmoplantar keratoderma (PPK). METHODS: Whole-exome (WES) and direct sequencing, quantitative real-time polymerase chain reaction, protein modelling, confocal immunofluorescence microscopy, immunoblotting, three-dimensional skin equivalents and an enzyme activity assay were used to delineate the genetic basis of a novel dominant form of PPK. RESULTS: WES revealed heterozygous variants (c.274T > C and c.305C > T) in CTSZ (encoding cathepsin Z) in four individuals (belonging to three unrelated families) with focal PPK. Bioinformatics and protein modelling predicted the variants to be pathogenic. Previous studies have suggested that EGFR expression may be subject to cathepsin regulation. Immunofluorescence revealed reduced cathepsin Z expression in the upper epidermal layers and concomitant increased epidermal EGFR expression in patients harbouring CTSZ variants. Accordingly, human keratinocytes transfected with constructs expressing PPK-causing variants in CTSZ displayed reduced cathepsin Z enzymatic activity, as well as increased EGFR expression. In line with the role played by EGFR in the regulation of keratinocyte proliferation, human keratinocytes transfected with the PPK-causing variants showed significantly increased proliferation that was abolished upon exposure to erlotinib, an EGFR inhibitor. Similarly, downregulation of CTSZ resulted in increased EGFR expression and increased proliferation in human keratinocytes, suggestive of a loss-of-function effect of the pathogenic variants. Finally, three-dimensional organotypic skin equivalents grown from CTSZ-downregulated cells showed increased epidermal thickness and EGFR expression as seen in patient skin; here, too, erlotinib was found to rescue the abnormal phenotype. CONCLUSIONS: Taken collectively, these observations attribute to cathepsin Z a hitherto unrecognized function in epidermal differentiation.

Asunto(s)

Catepsina Z , Queratodermia Palmoplantar , Humanos , Clorhidrato de Erlotinib , Catepsina Z/genética , Catepsina Z/metabolismo , Queratodermia Palmoplantar/genética , Queratodermia Palmoplantar/patología , Receptores ErbB/genética , Piel/patología

RESUMEN

TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.

Asunto(s)

Antioxidantes , Canales de Potencial de Receptor Transitorio , Adenosina Trifosfatasas/metabolismo , Antioxidantes/metabolismo , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Quinasa de la Caseína II/metabolismo , Catepsina D/metabolismo , Catepsina Z/genética , Catepsina Z/metabolismo , Cloruros/metabolismo , Cromatina/metabolismo , Disulfuros , Guanosina Trifosfato/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hexosaminidasas/genética , Hexosaminidasas/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Nucleosomas/metabolismo , Nucleótidos/metabolismo , PPAR gamma/genética , Piridinas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Sirolimus , Tiorredoxina Reductasa 1/genética , Tiorredoxina Reductasa 1/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo

RESUMEN

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.

Asunto(s)

Neoplasias Encefálicas/metabolismo , Catepsina Z/metabolismo , Glioblastoma/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Microambiente Tumoral , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Catepsina Z/antagonistas & inhibidores , Catepsina Z/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Regulación hacia Arriba

RESUMEN

Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1ß production. Cathepsin Z has been associated with the development of inflammation and IL-1ß production; however, the mechanism of how cathepsin Z leads to IL-1ß production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1ß release was determined using enzyme-linked immunosorbent assay or IL-1ß bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1ß expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1ß through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.

Asunto(s)

Catepsina Z , Inflamasomas , Animales , Catepsina Z/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Integrina alfa5/metabolismo , Interleucina-1beta/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Dióxido de Silicio/farmacología , Silicosis/metabolismo

RESUMEN

Cathepsin Z (CTSZ) is a cysteine protease responsible for the adhesion and migration of both immune and tumor cells. Due to its dual role, we hypothesized that the site of CTSZ expression could be determinant of the pro- or anti-tumorigenic effects of this enzyme. To test this hypothesis, we analyzed CTSZ expression data in healthy and tumor tissues by bioinformatics and evaluated the expression levels of CTSZ mRNA in the blood cells of prostate cancer (PCa) patients by qRT-PCR compared with healthy subjects, evaluating its diagnostic and prognostic implications for this type of cancer. Immune cells present in the blood of healthy patients overexpress CTSZ. In PCa, we found decreased CTSZ mRNA levels in blood cells, 75% lower than in healthy subjects, that diminished even more during biochemical relapse. CTSZ mRNA in the blood cells had an area under the curve for PCa diagnosis of 0.832, with a 93.3% specificity, and a positive likelihood ratio of 9.4. The site of CTSZ mRNA expression is fundamental to determine its final role as a protective determinant in PCa, such as CTSZ mRNA in the blood cells, or a malignant determinant, such as found for CTSZ expressed in high levels by different types of primary and metastatic tumors. Low CTSZ mRNA expression in the total blood is a possible PCa marker complementary to prostate-specific antigen (PSA) for biopsy decisions, with the potential to eliminate unnecessary biopsies.

Asunto(s)

Catepsina Z , Neoplasias de la Próstata , Células Sanguíneas , Humanos , Masculino , Pronóstico , Antígeno Prostático Específico , Neoplasias de la Próstata/diagnóstico , ARN Mensajero

RESUMEN

Human cathepsin X belongs to the cathepsin family of 11 lysosomal cysteine proteases. We expressed recombinant procathepsin X in Pichia pastoris in vitro and cleaved it into its active mature form using aspartic cathepsin E. We found, using size exclusion chromatography, X-ray crystallography, and small-angle X-ray scattering, that cathepsin X is a biologically active homodimer with a molecular weight of ~53 kDa. The novel finding that cathepsin X is a dimeric protein opens new horizons in the understanding of its function and the underlying pathophysiological mechanisms of various diseases including neurodegenerative disorders in humans.

Asunto(s)

Catepsina K/genética , Catepsina Z/genética , Proteínas Recombinantes/química , Secuencia de Aminoácidos/genética , Catepsina K/ultraestructura , Catepsina Z/ultraestructura , Cristalografía por Rayos X , Humanos , Pichia/química , Pichia/genética , Proteínas Recombinantes/genética , Saccharomycetales/química , Saccharomycetales/genética

RESUMEN

Cathepsin Z (CTSZ) is a cysteine protease responsible for the adhesion and migration of both immune and tumor cells. Due to its dual role, we hypothesized that the site of CTSZ expression could be determinant of the pro- or anti-tumorigenic effects of this enzyme. To test this hypothesis, we analyzed CTSZ expression data in healthy and tumor tissues by bioinformatics and evaluated the expression levels of CTSZ mRNA in the blood cells of prostate cancer (PCa) patients by qRT-PCR compared with healthy subjects, evaluating its diagnostic and prognostic implications for this type of cancer. Immune cells present in the blood of healthy patients overexpress CTSZ. In PCa, we found decreased CTSZ mRNA levels in blood cells, 75% lower than in healthy subjects, that diminished even more during biochemical relapse. CTSZ mRNA in the blood cells had an area under the curve for PCa diagnosis of 0.832, with a 93.3% specificity, and a positive likelihood ratio of 9.4. The site of CTSZ mRNA expression is fundamental to determine its final role as a protective determinant in PCa, such as CTSZ mRNA in the blood cells, or a malignant determinant, such as found for CTSZ expressed in high levels by different types of primary and metastatic tumors. Low CTSZ mRNA expression in the total blood is a possible PCa marker complementary to prostate-specific antigen (PSA) for biopsy decisions, with the potential to eliminate unnecessary biopsies.

Asunto(s)

Humanos , Masculino , Neoplasias de la Próstata/diagnóstico , Catepsina Z , Pronóstico , Células Sanguíneas , ARN Mensajero , Antígeno Prostático Específico

Asunto(s)

Enfermedad de la Arteria Coronaria , Inflamasomas/metabolismo , Proteómica , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Catepsina Z/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Selectina E/sangre , Femenino , Humanos , Inflamación/sangre , Inflamación/tratamiento farmacológico , Interleucina-6/sangre , Masculino , Metaloproteinasa 9 de la Matriz/sangre

RESUMEN

Brown adipose tissue (BAT) generates heat to maintain body temperature and suppress obesity. Agonists for nuclear receptors PPARα and PPARγ both affect brown adipocyte function, yet the interplay between these factors in BAT is uncertain. Here, we report that PPARα shares most genomic binding sites with PPARγ, and these common binding sites are more related to BAT function than PPARγ-selective sites without PPARα. Integrating PPARα and PPARγ genomic occupancy with cold-responsive BAT transcriptomes identifies a subset of 16 genes with potential relevance to BAT function. Among these, we focused on the lysosomal protease cathepsin Z (CTSZ) and showed it is necessary for mitochondrial respiration in both mouse and human brown adipocytes. Thus, CTSZ is a shared PPARα/γ target gene in BAT and a regulator of brown adipocyte thermogenic function.

Asunto(s)

Adipocitos Marrones/metabolismo , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Termogénesis/genética , Tejido Adiposo Pardo/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Catepsina Z/genética , Catepsina Z/metabolismo , Frío , Genoma , Humanos , Masculino , Ratones Endogámicos C57BL , PPAR alfa/agonistas , PPAR gamma/agonistas , Unión Proteica

RESUMEN

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major, and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N-glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida: E. piscicida and Streptococcus iniae: S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ, and the leukocytes of red sea bream also induced apoptosis and viability reduction.

Asunto(s)

Catepsina Z/genética , Catepsina Z/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Dorada/genética , Dorada/inmunología , Secuencia de Aminoácidos , Animales , Catepsina Z/química , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Edwardsiella/fisiología , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/veterinaria , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Iridoviridae/fisiología , Filogenia , Alineación de Secuencia/veterinaria , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus iniae/fisiología

RESUMEN

Osteoporosis, one of the most prevalent chronic ageing-related bone diseases, often goes undetected until the first fragility fracture occurs, causing patient suffering and cost to health/social care services. Osteoporosis arises from imbalanced activity of osteoclasts and osteoblasts. Since these cell lineages produce the protease, cathepsin Z, the aim of this study was to investigate whether altered cathepsin Z mRNA levels are associated with osteoporosis in clinical samples. Cathepsin Z mRNA in human peripheral blood mononuclear cells was significantly differentially-expressed among non-osteoporotic controls, osteopenia and osteoporosis patients (p < 0.0001) and in female osteoporosis patients over the age of 50 years (P = 0.0016). Cathepsin Z mRNA level strongly correlated with low bone mineral density (BMD) (g/cm2), lumbar spine L2-L4 and femoral neck (T-scores) (P = 0.0149, 0.0002 and 0.0139, respectively). Importantly, cathepsin Z mRNA was significantly associated with fragility fracture in osteoporosis patients (P = 0.0018). The levels of cathepsin Z mRNA were not significantly higher in patients with chronic inflammatory disorders in these two groups compared to those without (P = 0.774 and 0.666, respectively). ROC analysis showed that cathepsin Z mRNA has strong diagnostic value for osteoporosis and osteoporotic fracture. The results show for the first time that cathepsin Z could be a future diagnostic biomarker for osteoporosis including female osteoporosis patients over the age of 50 years.

Asunto(s)

Catepsina Z/genética , Osteoporosis/genética , Adulto , Anciano , Biomarcadores , Densidad Ósea/genética , Enfermedades Óseas Metabólicas/diagnóstico , Enfermedades Óseas Metabólicas/genética , Catepsina Z/metabolismo , Femenino , Fracturas Óseas/diagnóstico , Fracturas Óseas/etiología , Fracturas Óseas/metabolismo , Expresión Génica , Humanos , Inflamación/genética , Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Osteoporosis/complicaciones , Osteoporosis/diagnóstico , Osteoporosis/metabolismo , Pronóstico , ARN Mensajero/genética , Curva ROC

RESUMEN

Accumulating evidence supports the notion that epigenetic modifiers are abnormal in carcinogenesis and have a fundamental role in cancer progression. Among these aberrant epigenetic modifiers, the function of histone methyltransferase KMT2A in somatic tumors is not well known. By analyzing KMT2A expression in patient tissues, we demonstrated that KMT2A was overexpressed in colorectal cancer tissues in comparison with adjacent normal tissues and its expression was positively correlated with cancer stages. In KMT2A-knockdown HCT116 and DLD1 cells, cell invasion and migration were consequently suppressed. In addition, KMT2A depletion effectively suppressed cancer metastasis in vivo. Mechanistically, cathepsin Z (CTSZ) was demonstrated to be an important downstream gene of KMT2A. Further studies showed that p65 could recruit KMT2A on the promoter region of the downstream gene CTSZ and knockdown of p65 could reduce the KMT2A on the promoter of CTSZ. Finally, our present study revealed that KMT2A epigenetically promotes cancer progression by targeting CTSZ, which has specific functions in cancer invasion and metastasis.

Asunto(s)

Catepsina Z/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Activación Transcripcional , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epigenómica , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Unión Proteica , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Myocardial infarction (MI) is a disease characterized by high morbidity and mortality rates. MI biomarkers are frequently used in clinical diagnosis; however, their specificity and sensitivity remain unsatisfactory. Urinary proteome is an easy, efficient and noninvasive source to examine biomarkers associated with various diseases. The present study, to the best of the authors' knowledge, is the first to examine the urinary proteome using the isobaric tags for relative and absolute quantitation (iTRAQ) technology to identify potential diagnostic biomarkers of MI. The urinary proteome was analyzed within 12 h following the first symptoms of early­onset MI and at day 7 following percutaneous coronary intervention via iTRAQ labeling and two­dimensional liquid chromatography­tandem mass spectrometry. Candidate biomarkers were validated by multiple reaction monitoring (MRM) analysis. A total of 233 urinary proteins were differentially expressed. Gene enrichment analysis identified that the urinary proteome in patients with MI was associated with atherosclerosis, abnormal coagulation and abnormal cell metabolism. In total, 12 differentially expressed urinary proteins were validated by MRM analysis, five of which were associated with MI for the first time in the present study. Binary logistic regression analysis suggested that the combination of five urinary proteins (antithrombin­III, complement C3, α­1­acid glycoprotein 1, serotransferrin and cathepsin Z) may be used to diagnose MI with 94% sensitivity and 93% specificity. In addition, the protein expression levels of three proteins were significantly restored to normal levels following surgical treatment. The verified candidate biomarkers may be used for the diagnosis of MI, and for monitoring the disease status and the effects of treatments for MI. The present results may facilitate future clinical applications of the urinary proteome to diagnose MI.

Asunto(s)

Proteoma/análisis , Proteómica/métodos , Adulto , Antitrombina III/orina , Biomarcadores/orina , Estudios de Casos y Controles , Catepsina Z/orina , Cromatografía Líquida de Alta Presión , Complemento C3/orina , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/metabolismo , Espectrometría de Masas en Tándem , Transferrina/orina

RESUMEN

Cathepsin Z (CTSZ) is a lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. In this study, we reported the identification of SmCTSZ, a CTSZ homolog from turbot (Scophthalmus maximus L.). SmCTSZ was 317 residues in length and contains a Pept-C1 domain. In multiple species comparison, SmCTSZ shared 65-93% overall sequence identities with the CTSZ counterparts from human, rat, and several fish species. In the phylogenetic analysis, SmCTSZ showed the closest relationship to Cynoglossus semilaevis. The syntenic analysis revealed the similar neighboring genes of CTSZ across all the selected species, which suggested the synteny encompassing CTSZ region during vertebrate evolution. Subsequently, SmCTSZ was constitutively expressed in various tissues, with the lowest and highest levels in brain and intestine respectively. In addition, SmCTSZ was significantly up-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSZ showed strong binding ability to all the examined microbial ligands, and the agglutination effect to different bacteria. Taken together, these results indicated SmCTSZ could play important roles in mucosal immune response in the event of bacterial infection in teleost. However, the knowledge of CTSZ are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Asunto(s)

Catepsina Z/genética , Catepsina Z/inmunología , Enfermedades de los Peces/inmunología , Peces Planos/genética , Peces Planos/inmunología , Regulación de la Expresión Génica/inmunología , Streptococcus iniae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catepsina Z/química , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Inmunidad Innata/genética , Inmunidad Mucosa , Alineación de Secuencia/veterinaria , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Vibrio/fisiología , Vibriosis/inmunología , Vibriosis/veterinaria

RESUMEN

Our recent genome-wide association study found that the NELFCD/CTSZ locus was significantly associated with progression of primary biliary cholangitis (PBC) to jaundice stage in the Japanese population. In this study, we investigated the role of cathepsin Z in the etiology and pathology of PBC. Serum cathepsin Z levels were measured using enzyme-linked immunosorbent assay. The expression and localization of cathepsin Z in liver specimens were analyzed by western blotting and immunohistochemistry. In PBC patients, serum cathepsin Z levels were significantly increased with disease progression. In addition, its levels were positively correlated with alanine transaminase, aspartate transaminase and total bilirubin, and were negatively correlated with platelet count and albumin. Cathepsin Z expression was markedly increased in hepatocytes at later stages of PBC, and its localization was altered from the peri-bile canaliculus to the cytoplasm, where a fraction was no longer colocalized with endosomal/lysosomal vesicles. Similar altered expression of cathepsin Z was observed in end-stage of other cholestatic liver diseases including sepsis, obstructive jaundice, and Alagille syndrome. Our results indicate that altered expression and localization of cathepsin Z in hepatocytes are characteristic features of PBC and other cholestatic liver diseases, and are implicated in the progression of PBC.

Asunto(s)

Células Sanguíneas/enzimología , Catepsina Z/biosíntesis , Regulación Enzimológica de la Expresión Génica , Hepatocitos/enzimología , Ictericia/enzimología , Cirrosis Hepática Biliar/enzimología , Adulto , Anciano , Células Sanguíneas/patología , Endosomas/enzimología , Endosomas/patología , Femenino , Estudio de Asociación del Genoma Completo , Hepatocitos/patología , Humanos , Inmunohistoquímica , Ictericia/patología , Cirrosis Hepática Biliar/patología , Lisosomas/enzimología , Lisosomas/patología , Masculino , Persona de Mediana Edad

RESUMEN

Lung cancer is the leading cause of cancer-related death among both men and women every year, mainly due to metastasis. Although natural compound deguelin has been reported to inhibited cell migration and invasion in various cancer cells, the details of this regulation progress remain to be fully elucidated. In this study, we investigated the underlying mechanism of deguelin-suppressed metastasis of non-small cell lung cancer (NSCLC) cells. Our results demonstrate that deguelin inhibits NSCLC cell migration, invasion, and metastasis both in vitro and in vivo. These inhibitory effects of deguelin were mediated by suppressing of Cathepsin Z (CtsZ) expression and interrupting the interaction of CtsZ with integrin ß3. Moreover, deguelin inhibits the activation of CtsZ downstream FAK/Src/Paxillin signaling. Knockdown of CtsZ mimicked the effect of deguelin on NSCLC cells migration and invasion. Our study reveals that deguelin exerts its anti-metastatic effect both in vitro and in vivo is partly dependent on the suppression of CtsZ signaling. Deguelin would be a potential anti-metastasis agent against NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Catepsina Z/genética , Quinasa 1 de Adhesión Focal/genética , Neoplasias Pulmonares/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Rotenona/análogos & derivados , Transducción de Señal/efectos de los fármacos , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia/genética , Rotenona/farmacología , Transducción de Señal/genética

RESUMEN

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.

Asunto(s)

Catepsinas/metabolismo , Glioblastoma/patología , Nicho de Células Madre , Adulto , Anciano , Anciano de 80 o más Años , Arteriolas , Catepsina B/análisis , Catepsina B/metabolismo , Catepsina K , Catepsina Z/análisis , Catepsina Z/metabolismo , Catepsinas/análisis , Glioblastoma/enzimología , Glioblastoma/metabolismo , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Proteolisis

RESUMEN

Approximately 10-20% of patients with primary biliary cholangitis (PBC) progress to jaundice stage regardless of treatment with ursodeoxycholic acid and bezafibrate. In this study, we performed a GWAS and a replication study to identify genetic variants associated with jaundice-stage progression in PBC using a total of 1,375 patients (1,202 early-stage and 173 jaundice-stage) in a Japanese population. SNP rs13720, which is located in the 3'UTR of cathepsin Z (CTSZ), showed the strongest association (odds ratio [OR] = 2.15, P = 7.62 × 10-7) with progression to jaundice stage in GWAS. High-density association mapping at the CTSZ and negative elongation factor complex member C/D (NELFCD) loci, which are located within a strong linkage disequilibrium (LD) block, revealed that an intronic SNP of CTSZ, rs163800, was significantly associated with jaundice-stage progression (OR = 2.16, P = 8.57 × 10-8). In addition, eQTL analysis and in silico functional analysis indicated that genotypes of rs163800 or variants in strong LD with rs163800 influence expression levels of both NELFCD and CTSZ mRNA. The present novel findings will contribute to dissect the mechanism of PBC progression and also to facilitate the development of therapies for PBC patients who are resistant to current therapies.

Asunto(s)

Catepsina Z/genética , Ictericia/patología , Cirrosis Hepática Biliar/complicaciones , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Factores de Transcripción/genética , Progresión de la Enfermedad , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Japón/epidemiología , Ictericia/epidemiología , Ictericia/genética , Desequilibrio de Ligamiento , Cirrosis Hepática Biliar/epidemiología , Cirrosis Hepática Biliar/genética , Masculino , Persona de Mediana Edad , Pronóstico