RESUMEN

Chronic kidney disease can develop from kidney injury incident to chemotherapy with cisplatin, which complicates the prognosis of cancer patients. MicroRNAs regulate gene expression by pairing with specific sets of messenger RNAs. Therefore, elucidating direct physical interactions between microRNAs and their target messenger RNAs can help decipher crucial biological processes associated with cisplatin-induced kidney injury. Through intermolecular ligation and transcriptome-wide sequencing, we here identify direct pairs of microRNAs and their target messenger RNAs in the kidney of male mice injured by cisplatin. We find that a group of cisplatin-induced microRNAs can target select messenger RNAs that affect the mitochondrial metabolic pathways in the injured kidney. Specifically, a cisplatin-induced microRNA, miR-429-3p, suppresses the pathway that catabolizes branched-chain amino acids in the proximal tubule, leading to cell death dependent on lipid peroxidation, called ferroptosis. Identification of miRNA-429-3p-mediated ferroptosis stimulation suggests therapeutic potential for modulating the branched-chain amino acid pathway in ameliorating cisplatin-induced kidney injury.

Asunto(s)

Ferroptosis , MicroARNs , Insuficiencia Renal Crónica , Humanos , Masculino , Ratones , Animales , MicroARNs/genética , MicroARNs/metabolismo , Cisplatino/farmacología , Cisplatino/metabolismo , Ferroptosis/genética , Riñón/metabolismo , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo

RESUMEN

The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids (RNAs). Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts. Existing methods for probing exosome cargo molecules rely on a prior exosome isolation procedure. Here we report an in situ labelling approach for exosome cargo identification, which bypasses the exosome isolation steps. In this methodology, a variant of the engineered ascorbate peroxidase APEX, fused to an exosome cargo protein such as CD63, is expressed specifically in exosome-generating vesicles in live cells or in secreted exosomes in the conditioned medium, to induce biotinylation of the proteins in the vicinity of the APEX variant for a short period of time. Mass spectrometry analysis of the proteins biotinylated by this approach in exosomes secreted by kidney proximal tubule-derived cells reveals that oxidative stress can cause ribosomal proteins to accumulate in an exosome subpopulation that contains the CD63-fused APEX variant.

Asunto(s)

Exosomas , Ascorbato Peroxidasas/análisis , Transporte Biológico , Comunicación Celular , Exosomas/química , Proteínas/análisis

RESUMEN

While urine-based liquid biopsy has expanded to the analyses of extracellular nucleic acids, the potential of transfer RNA (tRNA) encapsulated within extracellular vesicles has not been explored as a new class of urine biomarkers for kidney injury. Using rat kidney and mouse tubular cell injury models, we tested if extracellular vesicle-loaded tRNA and their m1A (N1-methyladenosine) modification reflect oxidative stress of kidney injury and determined the mechanism of tRNA packaging into extracellular vesicles. We determined a set of extracellular vesicle-loaded, isoaccepting tRNAs differentially released after ischemia-reperfusion injury and oxidative stress. Next, we found that m1A modification of extracellular vesicle tRNAs, despite an increase of the methylated tRNAs in intracellular vesicles, showed little or no change under oxidative stress. Mechanistically, oxidative stress decreases tRNA loading into intracellular vesicles while the tRNA-loaded vesicles are accumulated due to decreased release of the vesicles from the cell surface. Furthermore, Maf1-mediated transcriptional repression of the tRNAs decreases the cargo availability for extracellular vesicle release in response to oxidative stress. Taken together, our data support that release of extracellular vesicle tRNAs reflects oxidative stress of kidney tubules which might be useful to detect ischemic kidney injury and could lead to rebalance protein translation under oxidative stress.

Asunto(s)

Lesión Renal Aguda/metabolismo , Vesículas Extracelulares/metabolismo , Isquemia/metabolismo , Riñón/irrigación sanguínea , Riñón/metabolismo , Estrés Oxidativo , ARN de Transferencia/metabolismo , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Vesículas Extracelulares/genética , Vesículas Extracelulares/patología , Isquemia/genética , Isquemia/patología , Riñón/patología , Metilación , Ratones , ARN de Transferencia/genética , Ratas Sprague-Dawley , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transcripción Genética

RESUMEN

Exosomes, vehicles for intercellular communication, are formed intracellularly within multivesicular bodies (MVBs) and are released upon fusion with the plasma membrane. For their biogenesis, proper cargo loading to exosomes and vesicle traffic for extracellular release are required. Previously we showed that the L-type lectin, LMAN2, limits trans-Golgi Network (TGN)-to-endosomes traffic of GPRC5B, an exosome cargo protein, for exosome release. Here, we identified that the protein deacetylase sirtuin 2 (SIRT2) as a novel interactor of LMAN2. Loss of SIRT2 expression resulted in exosomal release of LMAN2, a Golgi resident protein, along with increased exosomal release of GPRC5B. Furthermore, knockout of SIRT2 increased total number of extracellular vesicles (EVs), indicating increased MVB-to-EV flux. While knockout of SIRT1 increased EV release with enlarged late endolysosome, knockout of SIRT2 did not exhibit endolysosome enlargement for increased EV release. Taken together, our study suggests that SIRT2 regulates cargo loading to MVBs and MVB-to-EV flux through a mechanism distinct from that of SIRT1.

Asunto(s)

Vesículas Extracelulares/metabolismo , Sirtuina 1/fisiología , Sirtuina 2/fisiología , Acetilación , Células HEK293 , Humanos , Lectinas de Unión a Manosa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Unión Proteica , Transporte de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Sirtuina 1/genética , Sirtuina 2/genética

RESUMEN

Because exosomes have gained attention as a source of biomarkers, we investigated if miRNAs in exosomes (exo-miRs) can report the disease progression of organ injury. Using rat renal ischemia-reperfusion injury (IRI) as a model of acute kidney injury (AKI), we determined temporally-released exo-miRs in urine during IRI and found that these exo-miRs could reliably mirror the progression of AKI. From the longitudinal measurements of miRNA expression in kidney and urine, we found that release of exo- miRs was a regulated sorting process. In the injury state, miR-16, miR-24, and miR-200c were increased in the urine. Interestingly, expression of target mRNAs of these exo-miRs was significantly altered in renal medulla. Next, in the early recovery state, exo-miRs (miR-9a, miR-141, miR-200a, miR-200c, miR-429), which share Zeb1/2 as a common target mRNA, were upregulated together, indicating that they reflect TGF-ß-associated renal fibrosis. Finally, release of exo-miRs (miR-125a, miR-351) was regulated by TGF-ß1 and was able to differentiate the sham and IRI even after the injured kidneys were recovered. Altogether, these data indicate that exo-miRs released in renal IRI are associated with TGF-ß signaling. Temporal release of exo-miRs which share targets might be a regulatory mechanism to control the progression of AKI.

Asunto(s)

Lesión Renal Aguda/diagnóstico , Exosomas/genética , Médula Renal/fisiología , MicroARNs/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Progresión de la Enfermedad , Fibrosis , Perfilación de la Expresión Génica , Humanos , Médula Renal/patología , Masculino , MicroARNs/análisis , Técnicas de Diagnóstico Molecular , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Orina/química

RESUMEN

Matrix metalloproteinases and their natural inhibitors (TIMPs) are important elements in a wide range of oncology settings. Elevated levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) have often been associated with increased tumorigenesis. This has been demonstrated in a number of clinical and experimental models which include breast, gastric, colorectal and non-small cell lung carcinoma (NSCLC). Our earlier studies have identified increased angiogenic activity and aggressive tumor kinetics in TIMP-1 overexpressing H2009 lung adenocarcinoma cells. TIMP-1 overexpression has also been implicated in antiapoptotic responses, inducing a significant upregulation of Bcl-2. These TIMP-1 functions have been shown to be MMP-independent and provide insight into its pleiotropic activities. The current study examines microRNA (miRNA) interactions with this molecule. We have sought to define the relationship between TIMP-1 and miRNA by knocking down TIMP-1 in high TIMP-1 expressing lung adenocarcinoma cell lines. TIMP-1 knockdown resulted in increased expression of miR-125a-5p with a concomitant increase in apoptosis and attenuation of the tumorigenic features of these cells. We have identified TIMP-1 as a bona fide target of miR-125a-5p, and their interaction resulted in an increase in p53 expression. We further corroborated our in vitro data with patient samples, which exhibited an inverse correlation between TIMP-1 and miR-125a-5p expression. Our study lends support to the notion that elevated TIMP-1 levels, which are frequently associated with poor prognosis, cause aberrant modulation of miRNAs.

RESUMEN

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.

Asunto(s)

Adenocarcinoma/patología , Apoptosis/genética , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/fisiología , Adenocarcinoma del Pulmón , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Permeabilidad , Estaurosporina/farmacología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

α-Synuclein, a protein central to Parkinson's disease, is frequently expressed in melanoma tissues, but not in non-melanocytic cutaneous carcinoma and normal skin. Thus, α-synuclein is not only related to Parkinson's disease, but also to melanoma. Recently, epidemiologists reported co-occurrence of melanoma and Parkinson's disease in patients, suggesting that these diseases could share common pathogenetic components and that α-synuclein might be one of these. In Parkinson's disease, phosphorylation of α-synuclein at Ser129 plays an important role in the pathobiology. However, its role in melanoma is not known. Here, we show the biological relevance of Ser129 phosphorylation in human melanoma cells. First, we have identified an antibody that reacts with Ser129-unphosphorylated α-synuclein but not with Ser129-phosphorylated α-synuclein. Using this and other antibodies to α-synuclein, we investigated the role of Ser129 phosphorylation in human melanoma SK-MEL28 and SK-MEL5 cells. Our immunofluorescence microscopy showed that the Ser129-phosphorylated form, but not the Ser129-unphosphorylated form, of α-synuclein localizes to dot-like structures at the cell surface and the extracellular space. Furthermore, immuno-electron microscopy showed that the melanoma cells release microvesicles in which Ser129-phosphorylated α-synuclein localizes to the vesicular membrane. Taken together, our studies suggest that the phosphorylation of Ser129 leads to the cell surface translocation of α-synuclein along the microtubule network and its subsequent vesicular release in melanoma cells.

Asunto(s)

Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , alfa-Sinucleína/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Mapeo Epitopo , Humanos , Melanoma/patología , Melanoma/ultraestructura , Datos de Secuencia Molecular , Fosforilación , Serina/metabolismo , Neoplasias Cutáneas/patología , alfa-Sinucleína/inmunología

RESUMEN

The medulla of the adrenal gland is a neuroendocrine tissue in which catecholamine-storing chromaffin cells exist. The chromaffin cells are derived from neural crest cells and distinctly differentiated into two types of cells, epinephrine (E) (adrenaline)-storing and norepinephrine (NE) (noradrenaline)-storing cells. Using histochemical or immunostaining methods, the two types of chromaffin cells have been differentially distinguished. However, difficulties and/or drawbacks of the procedures have somewhat restricted the progress of research in differential functions of E-storing and NE-storing cells. Here, we show a new method for the differential demonstration of these two cell types. We found that mouse and rat adrenomedullary cells are heterogeneously stained with Harris hematoxylin after treatment with citrate buffer at pH 6. The cell clusters stained with hematoxylin were positive for tyrosine hydroxylase, which is an enzyme involved in catecholamine biosynthesis. Furthermore, the cell clusters were negative for phenylethanolamine-N-methyl transferase, which is an enzyme responsible for the conversion from NE to E and expresses in E-storing chromaffin cells. Moreover, we found that the cell clusters stained with hematoxylin can also be stained with nitroblue tetrazolium at pH 11, using Hopsu and Mäkinen's method by which NE-storing chromaffin cells are stained. These observations indicate that the cytoplasm of NE-storing chromaffin cells is specifically stained with hematoxylin after treatment with citrate buffer at pH 6. This method will allow us to facilitate cell-type specific research of chromaffin cells. Indeed, this method revealed that α-synuclein selectively expresses in E-storing chromaffin cells, but not in NE-storing chromaffin cells.

Asunto(s)

Médula Suprarrenal/metabolismo , Células Cromafines/metabolismo , Norepinefrina/metabolismo , Médula Suprarrenal/citología , Animales , Células Cromafines/química , Femenino , Histocitoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Coloración y Etiquetado , alfa-Sinucleína/metabolismo

RESUMEN

α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.

Asunto(s)

Immunoblotting/métodos , alfa-Sinucleína/análisis , Animales , Línea Celular , Formaldehído , Humanos , Immunoblotting/normas , Ratones , Fosforilación , Polímeros , Distribución Tisular

RESUMEN

PURPOSE: To investigate the short-term effects of panretinal photocoagulation (PRP) combined with an intravitreal injection of Avastin(bevacizumab) as an adjuvant to high-risk proliferative diabetic retinopathy (PDR). METHODS: The data was collected retrospectively from the eyes of high-risk PDR patients, which were divided into two groups. One eye was treated with only PRP (PRP only group) and the fellow eye of same patient was treated with both PRP and intravitreal bevacizumab injection (Adjuvant group). Best corrected visual acuity (BCVA), IOP (intraocular pressure), and new vessel (NV) size in fluorescein angiography were recorded immediately and at the six-week follow-up visit. Adverse events associated with intravitreal injection were investigated. RESULTS: Of 12 patients with high-risk PDR, five were male and seven were female. There were no statistically significant BCVA or IOP changes after treatment in either group (p=0.916, 0.888). The reduction of NV size was found in both groups, but NV size in the adjuvant group showed a greater decrease than that of the PRP only group (p=0.038). Three patients had adverse events after intravitreal injection. Two patients had mild anterior uveitis and one patient had a serious complication of branched retinal artery obstruction (BRAO). CONCLUSIONS: Intravitreal bevacizumab injection with PRP resulted in marked regression of neovascularization compared with PRP alone. One serious side effect, BRAO, was noted in this study. Further studies are needed to determine the effect of repeated intravitreal bevacizumab injections and the proper number of bevacizumab injections as an adjuvant.

Asunto(s)

Inhibidores de la Angiogénesis/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Retinopatía Diabética/terapia , Coagulación con Láser/métodos , Vitreorretinopatía Proliferativa/terapia , Anticuerpos Monoclonales Humanizados , Bevacizumab , Retinopatía Diabética/diagnóstico , Femenino , Angiografía con Fluoresceína , Estudios de Seguimiento , Fondo de Ojo , Humanos , Inyecciones , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Vitreorretinopatía Proliferativa/diagnóstico , Cuerpo Vítreo

RESUMEN

The pepstatin-insensitive carboxyl proteinase grifolisin was purified from fruiting bodies of the fungus Grifola frondosa, a maitake mushroom. The enzyme had an optimum pH of 3.0 for the digestion of hemoglobin and 2.8 for milk casein digestion. Its molecular mass was determined to be 43kDa by SDS-PAGE and 40kDa by gel chromatography on Superose 12, and its isoelectric point was found to be 4.6 by isoelectric focusing. The enzyme hydrolyzed four major bonds in the oxidized insulin B-chain: Phe1-Val2, Ala14-Leu15, Gly20-Glu21 and Phe24-Phe25 at pH 3.0. The first 15 amino acid residues in the N-terminal region were AVPSSCASTITPACL, and the coding region of the grifolisin gene (gfrF) has a 1960-base pair cDNA. The predicted mature grifolisin protein consisted of 365 residues and was 26% identical to that of sedolisin from Pseudomonas sp. 101 and 34% identical to that of aorsin from Aspergillus oryzae. Grifolisin is a member of the sedolisin S53 family and is not inhibited by pepstatin.

Asunto(s)

Carboxipeptidasas/química , Carboxipeptidasas/metabolismo , Grifola/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Carboxipeptidasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

The NF-kB family transcription factor c-Rel is a critical molecule for inducing expression of cytokine genes by T cells. Here, we report that a deletion of the C-terminal end, similar to the deletion in the highly oncogenic chicken v-Rel gene, renders c-Rel hyperactive toward cytokine gene promoters. At the same time, this mutation dramatically reduced c-Rel activity in induction of IkB-alpha mRNA expression. Moreover, ectopic expression of IkB-alpha, along with the C-terminal truncated c-Rel, abrogates hyperactivity of this mutant. IkB-alpha co-expression did not affect the function of wild-type c-Rel. The data demonstrate that the C-terminal end of c-Rel has specific activity for IkB-alpha mRNA expression and is dispensable for IL-2 gene expression.

Asunto(s)

Regulación de la Expresión Génica , Proteínas I-kappa B/genética , Interleucina-2/genética , Proteínas Proto-Oncogénicas c-rel/química , Proteínas Proto-Oncogénicas c-rel/fisiología , Humanos , Proteínas I-kappa B/fisiología , Interleucina-2/metabolismo , Células Jurkat , Mutación/genética , Inhibidor NF-kappaB alfa , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-rel/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Eliminación de Secuencia/genética , Linfocitos T/química , Linfocitos T/metabolismo , Transfección

RESUMEN

Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn(2+)- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.

Asunto(s)

Calor , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Mutagénesis Sitio-Dirigida , Zinc/metabolismo , Secuencia de Aminoácidos , Aspergillus/enzimología , Aspergillus/genética , Secuencia de Bases , Catálisis , Dicroismo Circular , Disulfuros , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Punto Isoeléctrico , Cinética , Metaloendopeptidasas/química , Metaloendopeptidasas/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Penicillium/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrofotometría Atómica , Espectrofotometría Ultravioleta , Especificidad por Sustrato

RESUMEN

An intracellular aspartic proteinase obtained from the hepatopancreas (liver) of Japanese common squid (Todarodes pacificus) was purified to homogeneity. The molecular mass of the enzyme was 36,500 Da on SDS-PAGE, and the isoelectric point was 8.29 by isoelectric focusing. The enzyme activity was optimal at pH 3.5, pH 2.2 and pH 3.0 for the substrates acid-denatured hemoglobin, acid-denatured casein, and MOCAc-GKPILFFRLK(Dnp)-D-R-NH2, respectively. Enzyme activity decreased rapidly at 50 degrees C. The Km and kcat values of the enzyme were estimated to be 3.2 mM and 46 s(-1) with MOCAc-GKPILFFRLK(Dnp)-D-R-NH2, and 1.7 mM and 1.1 s(-1) with MOCAc-SEVNLDAEFRK(Dnp)RR-NH2. The enzyme activity was strongly inhibited by pepstatin A, but only partially inhibited by DAN and EPNP. The Ki values for pepstatin A, DAN and EPNP were 0.5 nM, 0.5 mM and 0.2 mM, respectively. A cDNA encoding the enzyme was cloned by RT-PCR and subjected to nucleotide sequencing. The entire open reading frame was 1179 bp and coded for a protein of 392 amino acid residues. The mature enzyme consisted of 334 amino acids. The deduced amino acid sequence of the enzyme showed a high degree of identity to the sequences of cathepsins D found in various species.

Asunto(s)

Catepsina D/genética , Decapodiformes/enzimología , Hepatopáncreas/enzimología , Animales , Secuencia de Bases , Catepsina D/aislamiento & purificación , ADN Complementario , Inhibidores Enzimáticos , Cinética , Datos de Secuencia Molecular , Péptidos/metabolismo , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

The substrate specificities of deuterolysin, a 19-kDa zinc-protease (EC 3.4.24.39) from Aspergillus oryzae, were investigated at pH 9.0 with various fluorogenic acyl-peptide-4-methylcoumaryl-7-amides (peptide-MCAs). N-Butoxycarbonyl-Arg-Val-Arg-Arg-MCA was the best substrate for deuterolysin. We therefore measured its kinetic parameters. Deuterolysin had high activity toward the peptide bonds next to pairs of basic residues in calf thymus histone H4. The specificity of cobalt-substituted deuterolysin (Co-deuterolysin) for peptide-MCAs was similar to that of native deuterolysin. The CD spectrum of Co-deuterolysin was similar to that of the native deuterolysin. The metal coordination sphere of Co-deuterolysin was analyzed by Q-band (33.9570 GHz) electron paramagnetic resonance (EPR) spectroscopy. Using computer simulation of EPR, we found the g principal values to be g(xx) = 5.20, g(yy) = 4.75, and g(zz) = 2.24; the metal center was a divalent cobalt ion in a high spin state.

Asunto(s)

Aspergillus oryzae/enzimología , Cobalto/química , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Zinc/metabolismo , Amidas/química , Amidas/metabolismo , Secuencia de Aminoácidos , Aminoácidos Básicos/metabolismo , Animales , Sitios de Unión , Bovinos , Dicroismo Circular , Cobalto/metabolismo , Simulación por Computador , Espectroscopía de Resonancia por Spin del Electrón , Estabilidad de Enzimas , Histonas/metabolismo , Calor , Cinética , Datos de Secuencia Molecular , Especificidad por Sustrato , Timo/química

RESUMEN

The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-alpha-d-mannosidase were studied by site-directed mutagenesis and kinetic analyses. We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme. Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb. D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity. This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense. Km values of E273D, E414D, and E474D mutants were greatly increased to 17-31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate. Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A. saitoi 1,2-alpha-d-mannosidase. EDTA inhibits the Ca2+-free 1,2-alpha-d-mannosidase as a competitive inhibitor, not as a chelator. We deduce that the Glu-124 residue of A. saitoi 1,2-alpha-d-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved. Ca2+ is not essential for the activity. The catalytic mechanism of 1,2-alpha-d-mannosidase may deviate from that typical glycosyl hydrolase.

Asunto(s)

Aspergillus/enzimología , Calcio/metabolismo , Manosidasas/química , Manosidasas/genética , Western Blotting , Dominio Catalítico , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Temperatura , Factores de Tiempo

RESUMEN

A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae. Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH 4.0. The specificity of aorsin appeared to require a basic residue at the P(1) position and to prefer paired basic residues. Aorsin activated plasminogen and converted trypsinogen to trypsin. The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases. To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established. The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases. Several putative catalytic residues were mutated. The k (cat)/ K(m) values of the mutant enzymes Glu(86)-->Gln, Asp(211)-->Asn and Ser(354)-->Thr were 3-4 orders of magnitude lower and Asp(90)-->Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis. Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin.

Asunto(s)

Aspergillus oryzae/enzimología , Serina Endopeptidasas/metabolismo , Tripsina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato