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BACKGROUND: Aspergillus oryzae protease can release the opioid peptide ß-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. RESULTS: A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with µ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. CONCLUSION: Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.
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Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.
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Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , ARN/genética , Proteínas de Unión al ADN/genética , Endorribonucleasas/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , ARN/químicaRESUMEN
A Q-body capable of detecting target molecules in solutions could serve as a simple molecular detection tool. The position of the fluorescent dye in a Q-body affects sensitivity and therefore must be optimized. This report describes the development of Nef Q-bodies that recognize Nef protein, one of the human immunodeficiency virus (HIV)'s gene products, in which fluorescent dye molecules were placed at various positions using an in vivo unnatural amino acid incorporation system. A maximum change in fluorescence intensity of 2-fold was observed after optimization of the dye position. During the process, some tryptophan residues of the antibody were found to quench the fluorescence. Moreover, analysis of the epitope indicated that some amino acid residues of the antigen located near the epitope affected the fluorescence intensity.
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Aminoácidos/análisis , Aminoácidos/química , Productos del Gen nef/química , Antígenos VIH/química , Secuencia de Aminoácidos , Animales , Epítopos/análisis , Epítopos/química , Fluorescencia , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Productos del Gen nef/análisis , Antígenos VIH/análisis , Humanos , Ratones , ConejosRESUMEN
Among imaging techniques, fluorescence microscopy is a unique method to noninvasively image individual molecules in whole cells. If the three-dimensional spatial precision is improved to the angstrom level, various molecular arrangements in the cell can be visualized on an individual basis. We have developed a cryogenic reflecting microscope with a numerical aperture of 0.99 and an imaging stability of 0.05 nm in standard deviation at a temperature of 1.8 K. The key optics to realize the cryogenic performances is the reflecting objective developed by our laboratory. With this cryogenic microscope, an individual fluorescent molecule (ATTO647N) at 1.8 K was localized with standard errors of 0.53 nm (x), 0.31 nm (y), and 0.90 nm (z) when 106 fluorescence photons from the molecule were accumulated in 5 min.
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OBJECTIVES: We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole. RESULTS: We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM. CONCLUSIONS: We found that the addition of the common sequence, which was employed for ribosome display, makes the affinity of a selected peptide as strong as the previously reported peptide.
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Aptámeros de Péptidos/metabolismo , Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Aptámeros de Péptidos/química , Bovinos , Fluorescencia , Espectroscopía de Resonancia Magnética , Unión Proteica , Solubilidad , Resonancia por Plasmón de SuperficieRESUMEN
Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.
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Proteínas Bacterianas/química , Escherichia coli/citología , Escherichia coli/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Thermus thermophilus/genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos/genética , Thermus thermophilus/químicaRESUMEN
Female athletes who endure intense training are at risk of developing the 'female athlete triad,' making energy intake management crucial. However, the fluctuations in estradiol and progesterone levels throughout the menstrual cycle present a challenge in maintaining consistent energy intake. This study aimed to uncover the underlying factors associated with appetite regulation linked to menstrual phases and exercise using proteomic approach. Five female athletes engaged in 60 min of bicycle exercise, followed by 90 min of rest, during both the follicular and luteal phases. Serum samples were collected before, during, and after exercise, and the serum proteome was analyzed using 2D-gel electrophoresis. A total of 511 spots were detected in the subjects' serum profiles, with significant decreases observed in haptoglobin during the luteal phase and complement component 3 during bicycle training. Unsupervised learning with a generalized estimating equation analysis showed that serum peptide YY (PYY), an appetite suppressor, significantly influenced the fluctuations of serum proteins induced by exercise (p < 0.05). Regression analysis demonstrated a positive correlation between PYY and serum IgM (R = 0.87), implying that the intestinal environment and the immune response in female athletes may contribute to appetite regulation.
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Apetito , Proteómica , Humanos , Femenino , Apetito/fisiología , Proyectos Piloto , Progesterona , Ciclo Menstrual/fisiología , Atletas , Péptido YYRESUMEN
Atg9 is a transmembrane protein that is essential for autophagy. In the budding yeast Saccharomyces cerevisiae, it has recently been revealed that Atg9 exists on cytoplasmic small vesicles termed Atg9 vesicles. To identify the components of Atg9 vesicles, we purified the Atg9 vesicles and subjected them to mass spectrometry. We found that their protein composition was distinct from other organellar membranes and that Atg9 and Atg27 in particular are major components of Atg9 vesicles. In addition to these two components, Trs85, a specific subunit of the transport protein particle III (TRAPPIII) complex, and the Rab GTPase Ypt1 were also identified. Trs85 directly interacts with Atg9, and the Trs85-containing TRAPPIII complex facilitates the association of Ypt1 onto Atg9 vesicles. We also showed that Trs85 and Ypt1 are localized to the preautophagosomal structure in an Atg9-dependent manner. Our data suggest that Atg9 vesicles recruit the TRAPPIII complex and Ypt1 to the preautophagosomal structure. The vesicle-tethering machinery consequently acts in the process of autophagosome formation.
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Autofagia , Vesículas Citoplasmáticas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Relacionadas con la Autofagia , Vesículas Citoplasmáticas/genética , Proteínas de la Membrana/genética , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca(2+) concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D (1)H-(15)N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg(2+)-bound state, and then gradually converted to the Ca(2+)-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca(2+) into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.
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Calcio/metabolismo , Citosol/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Proteína G de Unión al Calcio S100/metabolismo , Estrés Fisiológico , Calbindinas , Células HeLa , Humanos , Magnesio/metabolismo , Modelos Moleculares , Proteína G de Unión al Calcio S100/análisisRESUMEN
Lymphocyte enhancer factor 1 (LEF1), a member of the LEF/T-cell-specific factor (TCF) family of the high mobility group domain transcription factors, acts downstream in canonical Wnt signaling. Aberrant transactivation of LEF1 contributes to the tumorigenesis of colonic neoplasms, sebaceous skin tumors, and lymphoblastic leukemia. LEF1-associated proteins are crucial for regulating its transcriptional activity. In this study, glutathione-S-transferase pull-down assay and mass spectrometry enabled identification of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel interaction partner for LEF1. The interaction between LEF1 and DNA-PKcs was confirmed using in vivo co-immunoprecipitation. Furthermore, double immunofluorescence observations showed that LEF1 and DNA-PKcs colocalized in the nuclei of colon adenocarcinoma cell lines. Identification of the interaction between LEF1 and DNA-PKcs may provide clues for a novel therapy for cancer treatment as well as for understanding LEF1-mediated transcriptional regulation.
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Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Vía de Señalización Wnt/genética , Dominio Catalítico/genética , Línea Celular Tumoral , Proteína Quinasa Activada por ADN/genética , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa , Humanos , Inmunoprecipitación , Espectrometría de MasasRESUMEN
INTRODUCTION: Cardiopulmonary resuscitation (CPR) can sometimes induce organ injury, however, such an occurrence is rare. We herein report a case of liver injury due to CPR with life-threatening pulmonary embolization (PE) that required the patient to undergo surgical hemostasis and antithrombotic therapy. PRESENTATION OF CASE: A woman in her 70s fell off her bicycle. She suffered cardiopulmonary arrest and underwent CPR. She was diagnosed with PE and underwent catheter treatment and anticoagulant therapy; however, her blood pressure did not increase. Contrast-enhanced computed tomography revealed injury to the liver and inferior phrenic artery. Hemostasis could not be completely achieved by transcatheter arterial embolization alone. She was therefore transferred to our hospital and underwent damage control surgery (DCS). Definitive surgery (DS) performed 33 h after DCS showed right hepatic subcapsular hematoma and left hepatic subcapsular hematoma. We cut away the capsules and removed the hematomas. There were lacerations and oozing under the capsule in the left lobe. We sutured the laceration. At 72 h after undergoing DS, antithrombotic therapy was started. On day 19, the patient was discharged home by herself without any neurological damage. DISCUSSION: For a case of liver injury due to CPR with life-threatening PE, treatment with both hemostasis and antithrombotic therapy should be performed. Antithrombotic therapy was started appropriately in this case by accurately identifying the liver laceration and suturing it. CONCLUSION: Hemostasis following both DCS and DS with appropriate anticoagulant therapy was effective for the management of liver injury due to CPR with life-threatening PE.
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Even though hematopoietic stem cell transplantation (HSCT) allows successful treatment for many malignant and non-malignant disorders, its curative potential remains limited by severe side effects, including infections and other transplant-related complications such as graft-versus-host disease (GvHD). This study examined changes in serum proteome via high-performance two-dimensional gel electrophoresis (2-DE) during HSCT to search for diagnostic biomarkers for post-HSCT complications. Longitudinal proteomic analysis revealed proteins related to metabolic complications and hemolytic anemia. Retinol-binding protein 4 (RBP4), a reliable marker of insulin resistance, was identified, and is possibly associated with the onset mechanism of acute graft-versus-host disease (aGvHD) and/or skin GvHD. Although the cause of insulin resistance is not fully understood, it is thought to be associated with adipocytes inflammation induced by RBP4, iron overload and hemolytic anemia after HSCT, as observed in this study. The present study has demonstrated that insulin resistance and metabolic complications could be immediate complications after transplantation and are associated with aGvHD. The biomarkers revealed in this study are promising tools to be used for improving the early diagnosis of HSCT-associated complications, especially aGvHD, possibly even before clinical manifestations.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Resistencia a la Insulina , Biomarcadores , Enfermedad Injerto contra Huésped/patología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Proteómica , Proteínas Plasmáticas de Unión al Retinol , Trasplante Homólogo/efectos adversosRESUMEN
Neurodegeneration is a process involving both cell autonomous and non-cell autonomous neuron loss, followed by a collapse of neural networks, but its pathogenesis is poorly understood. We have previously demonstrated that Eomes-positive helper T (Eomes + Th) cells recognizing LINE-1(L1)-derived prototypic antigen ORF1 mediate neurotoxicity associated with the neurodegenerative pathology of experimental autoimmune encephalomyelitis (EAE). Here, we show that Eomes + Th cells accumulate in the CNS of mouse models of authentic neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), and secrete the neurotoxic granzyme B after encounter with ORF1 antigen. Multimodal derepression of neuronal L1 transcription is observed in EAE and ALS/AD models during neurodegeneration in active and cell cycle-mediated manner, respectively. These data suggest that the adventitious concurrence of immune-mediated neurodegenerative traits by Eomes + Th cells and ectopic expression of L1-derived antigen(s) in the inflamed CNS may materialize a communal and previously unappreciated pathogenesis of neurodegeneration.
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Rib fractures can cause injury to some organs. We herein report a case of hemorrhagic shock due to intercostal artery injury that occurred during initial trauma care (ITC) treated by resuscitative thoracotomy (RT) and transcatheter arterial embolization (TAE) with multiple displaced rib fractures (RFs) and traumatic head injury (THI). A man in his 50s who was injured in a traffic accident was transferred to our institution by helicopter for emergency medical treatment. He underwent left thoracic drainage on site. On admission, he was diagnosed with multiple RF, THI, pelvic fracture and right humerus fracture. His D-dimer and fibrin degradation products (FDP) level were extremely elevated. However, contrast enhance CT (CECT) revealed no extravasation. At 2 h after arrival, massive hemorrhaging from his thoracic tube suddenly occurred and his blood pressure decreased to approximately 40s mmHg. CECT performed after volume resuscitation and massive transfusion revealed extravasation from the intercostal artery. Because his blood pressure could not be maintained by massive transfusion, we performed RT and TAE followed by RT. He then received intensive care and several surgical procedures were performed, including craniotomy for removal of hematoma, rib fixation and humerus fixation. He was transferred to another hospital for rehabilitation on day 63, with a GCS of 15. Hemorrhagic shock due to intercostal artery injury may occur at any time from arrival in cases with displaced RF, especially when complicated by THI.
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Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminal region of TH affects this stability. TH molecules phosphorylated at their Ser31 and Ser40 were localized predominantly in the cytoplasm of PC12D cells. However, those molecules phosphorylated at Ser19 were found mainly in the nucleus, whereas they seemed to be negligible in the cytoplasm. The inhibition of proteasomes increased the quantity of TH molecules phosphorylated at their Ser19 and Ser40, although it did not increase that of TH molecules or that of TH phosphorylated at its Ser31. The inhibition of autophagy did not affect the amount of the TH molecule or that of its three phosphorylated forms. Deletion mutants of human TH type-1 lacking the N-terminal region containing the three phosphorylation sites possessed high stability of the enzyme in PC12D cells. These results suggest that the phosphorylation of the N-terminal portion of TH regulates the degradation of this enzyme by the ubiquitin-proteasome pathway.
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Complejo de la Endopetidasa Proteasomal/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina/metabolismo , Animales , Autofagia , Humanos , Lisosomas/enzimología , Células PC12 , Fosforilación , Estructura Terciaria de Proteína/genética , Ratas , Eliminación de Secuencia , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG(1) Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG(1) revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.
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Anticuerpos Monoclonales/química , Carcinoma/inmunología , Neoplasias/inmunología , Animales , Antígenos de Neoplasias/química , Antineoplásicos/farmacología , Carcinoma/diagnóstico , Línea Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Inmunoterapia/instrumentación , Inmunoterapia/métodos , Ratones , Ratones Desnudos , Modelos Biológicos , Neoplasias/diagnóstico , Biblioteca de PéptidosRESUMEN
Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP-CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein-protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP-CaM interaction, including a 3D model of the complex between full-length proteins.
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Calmodulina/química , Proteína Básica de Mielina/química , Animales , Encéfalo , Bovinos , Células Cultivadas , Humanos , Ligandos , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Porcinos , TermodinámicaRESUMEN
The cytoplasmic tail of mammalian CD8alpha binds the kinase LCK in a zinc-dependent manner. In analogy with a previous study for humans (Kim et al., 2003) peptides were synthesized from rainbow trout CD8alpha and LCK. Surface plasmon resonance (SPR) analysis indicated that also in fish these molecules bind to each other in a zinc-dependent manner.
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Antígenos CD8/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Oncorhynchus mykiss/metabolismo , Zinc/fisiología , Animales , Sitios de Unión , Pollos , Humanos , Ratones , Oncorhynchus mykiss/inmunología , Alineación de Secuencia , Resonancia por Plasmón de Superficie , Zinc/metabolismoRESUMEN
N-myristoylation, one of the co- or post-translational modifications of proteins, has so far been regarded as necessary for anchoring of proteins to membranes. Recently, we have revealed that N(alpha)-myristoylation of several brain proteins unambiguously regulates certain protein-protein interactions that may affect signaling pathways in brain. Comparison of the amino acid sequences of myristoylated proteins including those in other organs suggests that this regulation is involved in signaling pathways not only in brain but also in other organs. Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions. An algorithm we have developed to identify myristoylated proteins in cells predicts the presence of hundreds of myristoylated proteins. Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins. If the proteins functionally regulated by myristoylation, a posttranslational protein modification, were understood as cross-talk points within the intracellular signal transduction system, known signaling pathways could thus be linked to each other, and a novel map of this intracellular network could be constructed. On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.
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Células/citología , Células/metabolismo , Espacio Intracelular/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Calmodulina/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas/químicaRESUMEN
BACKGROUND: Flotillin-2 (Flot2) is a lipid raft scaffold protein that is thought to be related to neural differentiation. Flot2 is phosphorylated by Fyn, a Src kinase, and causes raft-dependent endocytosis; however, the exact role of Flot2 in neural differentiation remains unclear. To reveal the roles of lipid raft-associated proteins during neural differentiation, we tried to analyze the expression and localization. RESULTS: In this study, we found that the expression levels of the Flot2 and Fyn proteins increased in whole-cell lysates of P19C6 cells after neural differentiation. In addition, sucrose density fractionation and immunofluorescence experiments revealed an increase in the localization of Flot2 and Fyn to lipid rafts after neural differentiation. We also found that Fyn partially colocalized with Flot2 lipid rafts in neural cells. CONCLUSION: The observed distribution of Fyn and level of inactivated Fyn and/or c-Src in detergent-resistant membrane (DRM) fractions suggests that the amount of activated Fyn might increase in DRM fractions after neural differentiation. Overall these findings suggest that Flot2 lipid rafts are associated with Fyn, and that Fyn phosphorylates Flot2 during neural differentiation of P19C6 cells.