RESUMEN
Earlier it was shown that a group of extracellular low-specific metallopeptidases is present in the mammalian brain Kropotova and Mosevitsky (2016) [1]. These enzymes are weakly connected to the axonal ends of neurons. They were named Neuron bound Extracellular MetalloPeptidases (NEMP). The enzyme named NEMP3 turned out to be a unique exopeptidase that exhibits two activities: it removes the dipeptide from the N-end of the peptide, and it can also remove the tripeptide from the C-end of the peptide. Therefore, NEMP3 possesses the activities of dipeptidylaminopeptidase and of tripeptidylcarboxypeptidase. Mass spectrometry has revealed a homology of NEMP3 with DPP3 (DPP III, EC3.4.14.4), known as cytosolic dipeptidylaminopeptidase. We isolated DPP3 from rat and bovine liver and brain by the procedures used for this purpose by other authors. The effect of DPP3 on test peptides is the same as that of NEMP3. In particular, all DPP3 samples delete the tripeptide (AKF) from the C-end of the test peptide blocked at the N-end. The data obtained show that NEMP3 and DPP3 are the same protein (enzyme). Thus, DPP3 has two exopeptidase activities: the previously known activity of dipeptidylaminopeptidase and the activity of tripeptidylcarboxypeptidase discovered in this study. Another discovery is the extracellular activity of DPP 3 in the mammalian brain near synapses, which controls neuropeptides. DPP3 is involved in various processes, but in many cases its role remains to be clarified. The results obtained in this study will be useful for solving these questions.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Neuropéptidos , Animales , Bovinos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Metaloproteasas/metabolismo , Neuronas/enzimología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Péptidos/metabolismo , RatasRESUMEN
The main obstacle to the use of many therapeutic peptides in practice is their rapid destruction by extracellular peptidases. Earlier we have found that active in the extracellular medium of mammalian brain exopeptidases are unable to break the bonds formed by ß-alanine. We have designed several modified forms of opioid peptide enkephalin (Tyr-Gly-Gly-Phe-Met; Enk) with end ßAla: ModEnk1 (ßAla-Tyr-Gly-Gly-Phe-Met-ßAla), ModEnk2 (ßAla-Tyr-Gly-Gly-Phe-NH2), ModEnk3 (ßAla-Tyr-Gly-Phe-NH2). These modifications are much more stable than Enk in the suspension of isolated axonal endings (synaptosomes) that mimics the brain extracellular medium. ModEnk1-3 have been tested in standard "pain" experiment "tail flick" on rats using intranasal peptide administration. ModEnk1 and ModEnk2 (but not ModEnk3) have fully preserved pain-relieving properties of Enk, but their efficiency was maintained for much longer. Compared to ModEnk1, ModEnk2 is more stable and provides longer analgesia because it is less accessible for endopeptidases. They are potent non-toxic analgesics.
Asunto(s)
Analgésicos/farmacología , Encéfalo/efectos de los fármacos , Diseño de Fármacos , Encefalinas/farmacología , Péptido Hidrolasas/metabolismo , Analgesia , Analgésicos/síntesis química , Analgésicos/química , Animales , Encéfalo/metabolismo , Bovinos , Relación Dosis-Respuesta a Droga , Encefalinas/síntesis química , Encefalinas/química , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Estructura Molecular , Manejo del Dolor , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
We have found that isolated from mammalian brain (rat, bovine) axonal endings (synaptosomes) degrade peptides of different composition. With the use of low concentration of non ionic detergent Triton X-100 (0.05-0.1 %) four low specific metallopeptidases were detached from synaptosomes. These peptidases were named Neuronal EctoMetalloPeptidases (NEMPs). Using specially designed test-peptides they were characterized as: carboxypeptidase (NEMP1), aminopeptidase (NEMP2) and endopeptidases NEMP3 and NEMP4. NEMPs are true peptidases (oligopeptidases), because they are able efficiently degrade peptides containing less than 40 amino acid residues. Specific properties of some NEMPs were revealed. NEMP1 is a small protein (molecular mass of about 10 kDa), which tends to dynamic oligomerization. NEMP3 needs activation. Some amino acids activate this enzyme. As far as we know, these properties were not ascribed to the known similarly localized peptidases. A possible physiological function of low specific NEMPs is participation in control of wide range of neuropeptides secreted in the synaptic cleft. However, NEMPs also due to their low specificity can destroy introduced in brain therapeutic peptides. The data obtained in this study open new opportunities for the protection of synthetic therapeutic peptides in brain and, possibly, in other tissues.
Asunto(s)
Encéfalo/enzimología , Metaloproteasas/metabolismo , Neuronas/enzimología , Animales , Bovinos , Espacio Extracelular/enzimología , Neuropéptidos/metabolismo , Péptido Hidrolasas/metabolismo , Ratas , Sinaptosomas/metabolismoRESUMEN
All-trans-retinoic acid (atRA), the oxidized form of vitamin A (retinol), regulates a wide variety of biological processes, such as cell proliferation and differentiation. Multiple alcohol, retinol and retinaldehyde dehydrogenases (ADHs, RDHs, RALDHs) as well as aldo-keto reductases (AKRs) catalyze atRA production. The reduced atRA biosynthesis has been observed in several human tumors, including colorectal cancer. However, subsets of atRA-synthesizing enzymes have not been determined in colorectal tumors. We investigated the expression patterns of genes involved in atRA biosynthesis in normal human colorectal tissues, primary carcinomas and cancer cell lines by RT-PCR. These genes were identified using transcriptomic data analysis (expressed sequence tags, RNA-sequencing, microarrays). Our results indicate that each step of the atRA biosynthesis pathway is dysregulated in colorectal cancer. Frequent and significant decreases in the mRNA levels of the ADH1B, ADH1C, RDHL, RDH5 and AKR1B10 genes were observed in a majority of colorectal carcinomas. The expression levels of the RALDH1 gene were reduced, and the expression levels of the cytochrome CYP26A1 gene increased. The human colon cancer cell lines showed a similar pattern of changes in the mRNA levels of these genes. A dramatic reduction in the expression of genes encoding the predominant retinol-oxidizing enzymes could impair atRA production. The most abundant of these genes, ADH1B and ADH1C, display decreased expression during progression from adenoma to early and more advanced stage of colorectal carcinomas. The diminished atRA biosynthesis may lead to alteration of cell growth and differentiation in the colon and rectum, thus contributing to the progression of colorectal cancer.
Asunto(s)
Adenoma/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Bases de Datos Factuales , Perfilación de la Expresión Génica , Tretinoina/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/genética , Adenoma/patología , Alcohol Deshidrogenasa/genética , Oxidorreductasas de Alcohol/genética , Aldehído Reductasa/genética , Aldo-Ceto Reductasas , Estudios de Casos y Controles , Colon/metabolismo , Neoplasias Colorrectales/patología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Recto/metabolismoRESUMEN
Protein BASP1 was discovered in brains of mammals and birds. In presynaptic area of synapses, BASP1 is attached to plasma membrane owing to N-terminal myristoylation as well as to the positively charged "effecter domain". BASP1 interactions with other proteins as well as with lipids contribute to membrane traffic, axon outgrowth and synaptic plasticity. BASP1 is present also in other tissues, where it was found not only in cytoplasm, but also in nucleus. Nuclear BASP1 suppresses activity of transcription factor WT1 and acts as tumor suppressor. BASP1 deficiency in a cell leads to its transformation. Previously it was shown that in BASP1 samples prepared from different animals and different tissues, six BASP1 N-end myristoylated fragments (BNEMFs) are present. Together, they amount to 30 % of the whole molecules. BNEMFs presence in different species and tissues demonstrates their physiological significance. However BNEMFs remain unexplored. In this paper, the time of appearance and dynamics of both BASP1 and BNEMFs during rat development from embryo to adult animals were determined. In rat brain, the amounts of all BASP1 forms per cell systematically increase during development and remain at the highest levels in adult animals. BNEMFs appear during embryogenesis non-simultaneously and accumulate with different dynamics. These results say for formation of six BNEMFs in the course of different processes and, possibly, using different mechanisms.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Encéfalo/embriología , Proteínas de Unión a Calmodulina/genética , Proteínas del Citoesqueleto/genética , Femenino , Proteínas del Tejido Nervioso/genética , Embarazo , Ratas , Ratas WistarRESUMEN
The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.