RESUMO
Sperm motility-initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species-specific reproductive environment. In the present study, we identified the gene of C. ensicauda SMIS (CeSMIS) and examined the mechanism of SMIS action with reference to that of the closely related Cynops species. The CeSMIS was identified by a 176-amino acid sequence including seven amino acids critical for the initiation of sperm motility. The amino acid sequence showed 91% homology to the whole sequence of C. pyrrhogaster SMIS (CpSMIS). By immunostaining with an anti-CpSMIS antibody, CeSMIS was shown to be localized in the outer layer of the egg jelly. A peptide presenting the active site of SMIS was observed to bind to the axial rod of the midpiece in C. ensicauda sperm. The localization and binding patterns of CeSMIS were fundamentally similar to those of CpSMIS. However, the SMIS peptide did not induce forward motility of C. ensicauda sperm, although it induced a fast wave of the undulating membrane. Forward sperm motility was induced in the egg jelly extract containing CeSMIS. These results suggest that the mechanism of initiation of sperm motility is differentiated between C. ensicauda and C. pyrrhogaster.
Assuntos
Proteínas do Ovo/fisiologia , Salamandridae/fisiologia , Motilidade dos Espermatozoides/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Animais , Feminino , Masculino , Oviductos/fisiologiaRESUMO
Dr. Sumiko Kimura, a former professor of biology at Chiba University, was known as a distinguished biochemist who contributed considerably to our knowledge about the cytoskeleton of muscle cells, especially through her work on connectin (also called titin) and actin regulatory proteins. Sadly, she suddenly passed away in Tokyo on November 1, 2018 at the age of 71. She succumbed to multiple organ failure caused by a bacterial infection following a third operation on her heart. Dr. Kimura had been continuing her research into connectin right up until several months before her decease.
Assuntos
Pesquisa Biomédica/educação , Músculos/fisiologia , HumanosRESUMO
Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca2+-dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca2+-independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles.
Assuntos
Cordados não Vertebrados , Músculo Liso/metabolismo , Tropomiosina , Animais , Cordados não Vertebrados/genética , Cordados não Vertebrados/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismoRESUMO
Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn13, Asn16 and Asn23) for N-linked glycosylation in its extracellular amino-terminus which may modulate its reactivity. Site-directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N-linked glycosylation sites in MCHR1 were glycosylated, and that N-linked glycosylation of Asn23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N-linked glycosylation of the MCHR1.
Assuntos
Receptores do Hormônio Hipofisário/química , Receptores do Hormônio Hipofisário/metabolismo , Animais , Asparagina/química , Sítios de Ligação , Sinalização do Cálcio , Linhagem Celular , Glicosilação , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Ratos , Receptores do Hormônio Hipofisário/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Herp is an endoplasmic reticulum (ER)-stress-inducible membrane protein, which has a ubiquitin-like domain (ULD). However, its biological function is as yet unknown. Previously, we reported that a high expression level of Herp in cells increases the generation of amyloid beta-protein (Abeta) and that Herp interacts with presenilin (PS). Here, we addressed the role of the ULD of Herp in Abeta generation and intracellular Herp stability. We found that the ULD is not essential for the enhancement of Abeta generation by Herp expression and the interaction of Herp with PS, but is involved in the rapid degradation of Herp, most likely via the ubiquitin/proteasome pathway. Thus, the ULD of Herp most likely plays a role in the regulation of the intracellular level of Herp under ER stress.
Assuntos
Acetilcisteína/análogos & derivados , Peptídeos beta-Amiloides/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ubiquitina/química , Acetilcisteína/farmacologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/biossíntese , Animais , Linhagem Celular , Células Cultivadas , Retículo Endoplasmático/metabolismo , Fibroblastos , Humanos , Proteínas de Membrana/genética , Camundongos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Deleção de Sequência/genética , Fatores de Tempo , Transfecção , Ubiquitina/metabolismoRESUMO
Multiple sclerosis is a chronic demyelinating disease of the central nervous system leading to progressive cognitive and motor dysfunction, which is characterized by neuroinflammation, demyelination, astrogliosis, loss of oligodendrocytes, and axonal pathologies. Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. In this study, we investigated the effects of cPA on cuprizone-induced demyelination, which is a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, astrocyte and microglial activation, and motor dysfunction. Simultaneous administration of cPA effectively attenuated cuprizone-induced demyelination, glial activation, and motor dysfunction. These data indicate that cPA may be a useful treatment to reduce the extent of demyelination and the severity of motor dysfunction in multiple sclerosis. cPA is a potential lead compound in the development of drugs for the treatment of this devastating disease.
Assuntos
Cuprizona/toxicidade , Doenças Desmielinizantes/prevenção & controle , Modelos Animais de Doenças , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Transtornos das Habilidades Motoras/prevenção & controle , Ácidos Fosfatídicos/uso terapêutico , Animais , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Transtornos das Habilidades Motoras/induzido quimicamente , Transtornos das Habilidades Motoras/patologia , Ácidos Fosfatídicos/farmacologia , Resultado do TratamentoRESUMO
Vertebrate striated muscle contains the giant elastic protein connectin that maintains the position of the A-band at the center of the sarcomere during repeated muscular contraction and relaxation. Connectin-like molecules may perform conserved functions in vertebrate and invertebrate striated and oblique muscles, although less is known about the structure of invertebrate connectins at present. The protein that maintains such a structure is present not only in vertebrate striated muscle, but also in invertebrate striated and oblique muscle. In the present study, we analyzed the partial primary structure of a 1200K-protein, which is a connectin-like protein that is expressed in Neanthes sp. body wall muscle that is in turn composed of oblique muscle. Antibody screening of a cDNA library of Neanthes sp. body wall muscle identified two different clones. Both clones coded for a sequence predominantly comprised of the four amino acids proline (P), glutamate (E), valine (V) and lysine (K). One clone included a PEVK-like repeat sequence flanked by an Ig domain, while the other clone comprised a distinct 14 amino acid repeat rich in PEVK residues, flanked by a non-repetitive unique sequence. The PEVK region is found in vertebrate connectin and is thought to generate elasticity and be responsible for passive tension of the muscle. The antibodies produced against a portion of each clone both reacted with bands corresponding to 1200 kDa present in Neanthes sp. body wall muscle. Therefore, our results demonstrate that this 1200K-protein is a connectin-like elastic protein and includes specific PEVK-like fragment. We suggest that this 1200K-protein plays a major role in maintaining the structure of oblique muscle in invertebrates.
Assuntos
Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Poliquetos/genética , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Western Blotting , Clonagem Molecular , Conectina , Sequência Consenso/genética , DNA Complementar/química , DNA Complementar/genética , Variação Genética , Invertebrados/genética , Dados de Sequência Molecular , Peso Molecular , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Poliquetos/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos/genética , Análise de Sequência de DNA , Vertebrados/genéticaRESUMO
Presenilin (PS) is essential for gamma-cleavage, which is required for the generation of amyloid beta-protein (Abeta) from the beta-amyloid precursor protein. However, it remains to be clarified how gamma-cleavage is regulated. To elucidate the regulation of PS-mediated gamma-cleavage, we developed a new functional screening method for identifying cDNA that enhances gamma-cleavage. This screening system utilizes our own developed cell line, where the expression of cDNA that enhances gamma-cleavage confers puromycin resistance. The cDNA library is retrovirally delivered to the above-mentioned cell line, allowing the identification of our target cDNAs by a combination of puromycin resistance selection and Abeta assay screening. With this screening method, we isolated several cDNAs enhancing gamma-cleavage, including the previously reported Herp. Here we also demonstrate that Rab1A, identified with this screening, can be a regulator of Abeta generation. Thus, our established screening method is a powerful tool for identifying multiple regulators involved in gamma-cleavage in the Abeta generation pathway, including modulators of gamma-secretase activity or the intracellular trafficking of factors necessary for gamma-cleavage.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Técnicas Genéticas , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Endopeptidases/metabolismo , Biblioteca Gênica , Hipocampo/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , Modelos Genéticos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Retroviridae/genética , Fatores de Transcrição HES-1 , Proteínas rab1 de Ligação ao GTP/metabolismoRESUMO
Presenilin (PS) is essential for the gamma-cleavage required for the generation of the C terminus of amyloid beta-protein (Abeta). However, the mechanism underlying PS-mediated gamma-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of gamma-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of Abeta generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Abeta generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in Abeta accumulation, including the formation of senile plaques and vascular Abeta deposits.