RESUMO
In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.
RESUMO
As an approach to investigate the suspected involvement of cellular factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns from hemocytes collected from susceptible and resistant snails. This proteomic approach revealed that twelve hemocytic proteins exhibited significant differences in their apparent abundance. The genes corresponding to five of them were characterized by a combination of mass spectrometry and molecular cloning. They encode an aldolase, an intermediate filament protein, a cytidine deaminase, the ribosomal protein P1 and the histone H4. Furthermore, we investigated their expression in parasite-exposed or -unexposed snails. These last experiments revealed changes in transcript levels corresponding to intermediate filament and histone H4 proteins post-infection.
Assuntos
Biomphalaria/parasitologia , Echinostoma/fisiologia , Hemócitos/metabolismo , Proteínas/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Modelos Biológicos , Dados de Sequência Molecular , Proteínas/químicaRESUMO
In the deoxyribonucleoprotein complex of Methanosarcina sp. CHTI 55, DNA is associated with two proteins, named MC1 (methanogen chromosomal protein 1) (Mr 10,760) and MC2 (Mr 17,000). Protein MC1, the most abundant of these proteins, is closely related to the Methanosarcina barkeri MS protein MC1. The effect of Methanosarcina sp. CHTI 55 protein MC1 on the thermal stability of DNA has been studied in native deoxyribonucleoprotein complex, as well as in reconstituted complexes, and it has been compared to the effect of E. coli DNA-binding protein II. Both proteins are able to protect DNA against thermal denaturation, but the differences observed in the melting profiles suggest that they interact by different mechanisms. Moreover, our studies indicate that one molecule of protein MC1 protects eight base pairs of DNA.
Assuntos
Archaea/análise , Proteínas Arqueais , Bactérias/análise , Proteínas de Bactérias/farmacologia , DNA Bacteriano/efeitos dos fármacos , Ribonucleoproteínas , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Desoxirribonucleoproteínas/isolamento & purificação , Desoxirribonucleoproteínas/metabolismo , Estabilidade de Medicamentos , Escherichia coli/análise , Temperatura Alta , Peso Molecular , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Renaturação de Ácido NucleicoRESUMO
Two protamines, St1 and St2, were isolated from stallion sperm nuclei, where they represent about 75 and 25%, respectively, of the total basic protein complement. The primary structure of protamine St1 (49 residues; Mr approximately equal to 6600) has been determined. The structure of this protamine is compared to the amino-acid sequence of other mammalian protamines already known.
Assuntos
Cavalos/metabolismo , Protaminas/isolamento & purificação , Espermatozoides/análise , Sequência de Aminoácidos , Animais , MasculinoRESUMO
Basic, low-molecular-weight DNA-binding proteins were isolated from the unicellular cyanobacterium Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum) and from the chloroplasts of spinach (Spinacia oleacera). In Synechococcus, two major proteins which bind to double-strand DNA (10 and 16 kDa, respectively) were purified. The 10 kDa protein, named HAq, resembles strongly, in amino-acid composition, eubacterial HU-type proteins. The 16 kDa protein is slightly basic. Its characteristics are compared to those of E. coli protein H1 and 17K. In spinach chloroplasts, a major protein HC (10 kDa), which also binds to ds-DNA, was purified. As observed for known archaebacterial and mitochondrial DNA-binding proteins, its amino-acid composition differs significantly from those of eubacterial HU. The comparison of the amino-terminal sequence (27 residues) with other chloroplast peptidic sequences is discussed.
Assuntos
Cloroplastos/análise , Cianobactérias/análise , Proteínas de Ligação a DNA/análise , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Cianobactérias/genética , DNA/análise , Eletroforese em Gel de Poliacrilamida , Escherichia coli/análise , Dados de Sequência Molecular , Peso Molecular , Filogenia , Células Vegetais , Plantas/análiseRESUMO
Dog-fish sperm nuclei contain four low molecular weight basic proteins called scylliorhinines. Protein Z3 is a typical arginine-rich protamine, whilst the three other components, Z1, Z2 and S4, are characterized by high arginine and cysteine contents. In contrast to protamine Z3, which can be directly solubilized by 0.25 M HCl, the three other protamines must be reduced and alkylated before acid extraction. They were further purified by ion-exchange chromatography on carboxymethyl-cellulose. The amino acid compositions and the N-terminal sequences reveal significant differences between scylliorhinines, particularly in their molecular size and amino acid diversity. Moreover, they show no common feature with other sperm-specific protamines previously described.
Assuntos
Protaminas/isolamento & purificação , Espermatozoides/análise , Aminoácidos/análise , Animais , Núcleo Celular/análise , Cromatografia por Troca Iônica , Cação (Peixe) , Masculino , Peso MolecularRESUMO
Two electrophoretic forms of rat alpha-fetoprotein were purified using immunosorbent chromatography and preparative electrophoresis on polyacrylamide gel slabs. Some of their respective chemical properties and their affinity for the Ricinus communis lectin (RCAI) were compared. Electrophoresis on polyacrylamide gradient gel in the presence of sodium dodecyl sulfate indicated a slight difference in molecular 74 000 for the slow alpha-fetoprotein (AFPA) and 72000 for the fat alpha-fetoprotein (AFPB). no significant difference in amino acid composition between AFPA and AFPB was found. A residue of valine was identified at the C-germinal end of both alpha-fetoproteins. The analysis of the CNRr-cleavage products reveals slight differences between AFPS and AFPB. The slow moving alpha-fetoprotein could be further fractionated on RCAI-sepharose column in two components, AFPA1 and AFPA2 differing by their sialic acid content.
Assuntos
Variação Genética , Lectinas , alfa-Fetoproteínas , Sequência de Aminoácidos , Aminoácidos/análise , Líquido Amniótico , Animais , Carboidratos/análise , Cromatografia de Afinidade , Feminino , Imunoeletroforese , Lectinas de Plantas , Plantas Tóxicas , Gravidez , Ratos , Ricinus , alfa-Fetoproteínas/isolamento & purificaçãoRESUMO
Proteolytic digestion of calf thymus histone H1 with Staphylococcus aureus V8-proteinase under structuring conditions generates one major limit peptide P1 which consists of approx. 170 residues. Edman degradation establishes the N-terminal sequence as: Leu-Ile-Thr-Lys-Ala-Val-Ala-Ala-Ser-Lys. Chymotryptic fingerprinting shows that the C-terminal part of the H1 molecule is fully preserved. The peptide therefore comprises the residues H1 (42-210). The Glu-41 cleavage is extremely unusual as it occurs in the structured G-domain which is known to be resistant to proteinases (Hartman, P. G., Chapman, G. E., Moss, T. and Bradbury, E. M. (1977) Eur. J. Biochem. 77, 45-71; Böhm, L., Sautière, P., Cary, P. D. and Crane-Robinson, C. (1982) Biochem. J. 203, 577-582). The V8-proteinase cleavage product H1 (42-210) shows only 20% folding as compared to 95-99% folding shown by the peptides H1 (34-121), H1 (31-210) and H1 (33-210). Folding of the G-domain thus critically depends upon the presence of the eight residues 33-41 amongst which the Gly-Pro-Pro sequence at position 36-38 and a beta-turn predicted at position 35 are considered to be particularly important. The location of the cleavage site in the G-domain renders Staphylococcus aureus V8-proteinase suitable as a structural probe.
Assuntos
Histonas , Serina Endopeptidases , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Quimotripsina , Fragmentos de Peptídeos , Conformação Proteica , Staphylococcus aureus/enzimologia , TimoRESUMO
Cyanogen bromide (CNBr) cleavage of total rat liver histone H1 generates a C-terminal peptide which originates from a methionine-containing subfraction. This subfraction comprises approx. 20% of the whole rat liver H1 population, resembles calf thymus CTL-1 in size but contains methionine and histidine, higher proportions of serine and less alanine and proline. Edman degradation established the N-terminal sequence of the CNBr peptide as Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val-Glu. By alignment with calf thymus CTL-1, methionine was identified as residue 30 replacing alanine in a non-conservative replacement. Residue 40 is deleted but sequence homology near the double proline sequence in the G-domain is retained. The CNBr peptide is estimated at 177-181 residues and comprises the complete G- and C-domain and two arginines from the basic cluster in the N-domain. Removal from H1 of all but two residues of the N-domain does not abolish secondary and tertiary folding. This GC-peptide opens new approaches to the study of the function of H1 in chromatin.
Assuntos
Histonas/isolamento & purificação , Fígado/análise , Metionina , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Fracionamento Celular , Núcleo Celular/análise , Quimotripsina , Brometo de Cianogênio , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Fragmentos de Peptídeos/análise , RatosRESUMO
Human sperm is characterized by a high heterogeneity of its basic nuclear protein complement of pro-protamines, protamines and histones. This heterogeneity is increased by the persistence of phosphorylated protamines in mature spermatozoa. Alkaline phosphatase treatment of whole protein indicated that protamines HP1 and HP2 were phosphorylated to various degrees. Presence of non-phosphorylated and phosphorylated protamines HP1 and HP2 was further demonstrated by electrospray mass spectrometry. Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were identified by automatic Edman degradation of the protein after phosphoserine derivatization to S-ethylcysteine. In both phosphorylated forms, Ser-10 was found phosphorylated; in the di-phosphorylated form, Ser-8 was identified as the second site of phosphorylation. In protamine HP2, the unique site of phosphorylation (Ser-14) was located after limited acid hydrolysis of enzymic peptides and thin-layer electrophoresis.
Assuntos
Protaminas/metabolismo , Espermatozoides/metabolismo , Fosfatase Alcalina , Sequência de Aminoácidos , Sítios de Ligação , Quimotripsina , Humanos , Masculino , Espectrometria de Massas/métodos , Metaloendopeptidases , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Protaminas/química , Protaminas/isolamento & purificaçãoRESUMO
The DNA of the thermophilic archaebacterium Methanosarcina sp. CHTI 55 has been shown to be associated with two proteins called MC1 and MC2, of molecular mass 11 kDa and 17 kDa (Chartier et al. (1988) Biochim. Biophys. Acta 951, 149-156). The most abundant of these proteins, protein MC1, can protect DNA against thermal denaturation. In the present paper we report the covalent structure of protein MC1 and its effect on transcription of DNA in vitro. The covalent structure was determined from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid and arginine residues. The amino-acid sequence of protein MC1 from Methanosarcina sp. CHTI 55 is closely related to that of the protein MC1 (previously called HMb) isolated from Methanosarcina barkeri strain MS: among the nine substitutions observed between the two proteins seven are conservative. Transcription of DNA in vitro is stimulated by protein MC1 at low protein-to-DNA ratio but is inhibited at a ratio higher than 0.1 (w/w), which is the one determined in the bacterial deoxyribonucleoprotein complex.
Assuntos
Archaea/metabolismo , Proteínas Arqueais , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Nucleoproteínas/metabolismo , Ribonucleoproteínas , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , DNA Bacteriano/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Conformação Proteica , Espectrofotometria Ultravioleta , Transcrição GênicaRESUMO
With the view of investigating the metabolism of inter-alpha-inhibitor, a plasma serine-proteinase inhibitor, in an animal model of inflammatory syndrome, we isolated inter-alpha-inhibitor from pig plasma. A high yield was obtained (140 mg/liter) with a two-step procedure: anion-exchange chromatography followed by affinity chromatography on heparin-Sepharose. In contrast to bovine inter-alpha-inhibitor was highly similar to human inter-alpha-inhibitor: its heavy chains are homologous to the human H1 and H2 heavy chains, as shown by chromatographic and electrophoretic properties, cross-immunoreactivity and N-terminal sequencing. Pig may therefore represent a good animal model to study inter-alpha-inhibitor metabolism and elucidate its physiological role.
Assuntos
alfa-Globulinas/química , Inibidores de Serina Proteinase/química , alfa-Globulinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Humanos , Hidroxilamina , Hidroxilaminas , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , SuínosRESUMO
Highly purified histone H2B from rat chloroleukaemia has been isolated by preparative electrophoresis at pH 2.7 in polyacrylamide slab gel, using the fraction F2b of Johns (Johns E. W. (1964) Biochem, J. 92, 55-59) as starting material. This histone was characterized by amino acid analysis and end groups determination. Comparative studies with homologous calf thymus histone show similarity of the amino acid compositions and of the amino terminal groups. the carboxyl terminal sequence presents two conservative substitutions.
Assuntos
Histonas/isolamento & purificação , Sarcoma Experimental/análise , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Eletroforese em Gel de Poliacrilamida , RatosRESUMO
Chicken erythrocyte histone H5 has been cleaved by acetic acid hydrolysis at the two aspartic acid residues 65 and 99 and the 4 peptides (1-65), (66-185) (1-99) (100-185) recovered in a pure form. 270 MHz magnetic resonance and circular dichroic studies show that the two C-terminal peptides are unable to form secondary or tertiary structure. The N-terminal peptides however, form both secondary and tertiary structure. In particular, the peptide (1-99) at high ionic strength possesses a similar number of helical residues to intact histone H5 and also had a closely related nuclear magnetic resonance spectrum. It is concluded that the peptide (1-99) contains most, but not quite all of the residues that are included in the globular segment of histone H5.
Assuntos
Eritrócitos/análise , Histonas , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Galinhas , Dicroísmo Circular , Histonas/sangue , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/análise , Conformação ProteicaRESUMO
Methanogen chromosomal protein MC1 is a polypeptide of 93 amino acid residues (Mr 10,757) which represents the major protein associated with the DNA of the archaebacterium Methanosarcina barkeri and can protect DNA against thermal denaturation. The conformation of protein MC1 has been investigated by means of predictive methods, infrared spectroscopy, circular dichroism and tryptophan fluorescence studies. Protein MC1 has a low amount of alpha-helix but contains antiparallel beta-sheet strands. The larger hydrophobic cluster which contains tryptophan at position 61 appears buried in the protein. Addition of salts induces the unfolding of the protein and makes the tryptophan indole ring more rigid. With respect to its primary structure and its conformation, protein MC1 appears radically different from the chromosomal DNA-binding protein II (also called HU-type protein) in eubacteria.
Assuntos
Archaea , Proteínas Arqueais , Bactérias , Proteínas de Bactérias , Nucleoproteínas , Ribonucleoproteínas , Sequência de Aminoácidos , Archaea/análise , Bactérias/análise , Proteínas de Bactérias/isolamento & purificação , Dicroísmo Circular , Proteínas de Ligação a DNA , Dados de Sequência Molecular , Nucleoproteínas/isolamento & purificação , Conformação Proteica , Espectrometria de Fluorescência , Espectrofotometria InfravermelhoRESUMO
Digestion of sea urchin sperm nuclei with micrococcal nuclease yields nucleosomal monomer fragments of 151 and 164 base pairs. Prior trypsin treatment of the sperm chromatin does not alter the size of these monomer DNA fragments despite the fact that the H1 histone is reduced to a limit globular peptide of about 83 residues. Heterologous reconstitution experiments show that this peptide is capable of protecting an extra 22 base pairs beyond the core particle in a chromatosome. Nuclease digestion of reconstitutes from DNA and sperm core histones yields a core monomer of about 141 base pairs. It is concluded that this sperm chromatin contains a chromatosome of 164 bp essentially similar to that observed in the more usual chromatins. Edman degradation of the H1 limit peptide shows its sequence to be closely analogous to the corresponding peptide of calf H1 and chicken H5. Circular dichroism studies of histone H1 from the sperm of three sea urchin species demonstrate the presence of trypsin-sensitive helical regions outside the globular domain that are absent in calf H1 and chicken H5.
Assuntos
Cromatina/ultraestrutura , Histonas/fisiologia , Nucleossomos/ultraestrutura , Ouriços-do-Mar/ultraestrutura , Espermatozoides/ultraestrutura , Sequência de Aminoácidos , Animais , DNA/análise , Masculino , Nuclease do Micrococo , Conformação Proteica , Relação Estrutura-Atividade , Tripsina/metabolismoRESUMO
Highly purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins are in fact the same protein with different degrees of phosphorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.
Assuntos
Núcleo Celular/análise , Proteínas/isolamento & purificação , Espermatozoides/análise , Sequência de Aminoácidos , Aminoácidos/análise , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Microscopia Eletrônica , Fosfoproteínas/isolamento & purificação , Protaminas/isolamento & purificaçãoRESUMO
Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.
Assuntos
Epitopos/análise , Histonas/análise , Aminoácidos/análise , Animais , Anticorpos Monoclonais , Bovinos , Galinhas , Ensaio de Imunoadsorção Enzimática , Eritrócitos/análise , Timo/análiseRESUMO
We have redetermined the primary structure for dog-fish protamine using automated amino-acid sequencing associated to mass spectrometry techniques and report, on the basis of these findings, that the previously published amino-acid sequence is incorrect. The correct protamine sequence is 37 amino acids long and differs from the original published sequence by the C-terminal hexapeptide Arg32-Gly-Arg-Arg-Ser-Arg37.
Assuntos
Cação (Peixe)/metabolismo , Protaminas/química , Protaminas/isolamento & purificação , Sequência de Aminoácidos , Animais , Quimotripsina , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Protaminas/análise , TripsinaRESUMO
NMR structures of a new toxin from the scorpion Leiurus quinquestriatus hebraeus (Lqh III) have been investigated in conjunction with its pharmacological properties. This toxin is proposed to belong to a new group of scorpion toxins, the alpha-like toxins that target voltage-gated sodium channels with specific properties compared with the classical alpha-scorpion toxins. Electrophysiological analysis showed that Lqh III inhibits a sodium current inactivation in the cockroach axon, but induces in addition a resting depolarization due to a slowly decaying tail current atypical to other alpha-toxin action. Binding studies indicated that radiolabeled Lqh III binds with a high degree of affinity (Ki=2.2 nM) on cockroach sodium channels and that the alpha-toxin from L quinquestriatus hebraeus highly active on insects (LqhalphaIT) and alpha-like toxins compete at low concentration for its receptor binding site, suggesting that the alpha-like toxin receptor site is partially overlapping with the receptor site 3. Conversely, in rat brain, Lqh III competes for binding of the most potent anti-mammal alpha-toxin from Androctonus australis Hector venom (AaH II) only at very high concentration. The NMR structures were used for the scrutiny of the similarities and differences with representative scorpion alpha-toxins targeting the voltage-gated sodium channels of either mammals or insects. Three turn regions involved in the functional binding site of the anti-insect LqhalphaIT toxin reveal significant differences in the Lqh III structure. The electrostatic charge distribution in the Lqh III toxin is also surprisingly different when compared with the anti-mammal alpha-toxin AaH II. Similarities in the electrostatic charge distribution are, however, recognized between alpha-toxins highly active on insects and the alpha-like toxin Lqh III. This affords additional important elements to the definition of the new alpha-like group of scorpion toxins and the mammal versus insect scorpion toxin selectivities.