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1.
J Sep Sci ; 43(1): 313-336, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31631532

RESUMO

More than 300 different protein post-translational modifications are currently known, but only a few have been extensively investigated because modified proteoforms are commonly present in sub-stoichiometry amount. For this reason, improvement of specific enrichment techniques is particularly useful for the proteomic characterization of post-translationally modified proteins. Enrichment proteomic strategies could help the researcher in the challenging issue to decipher the complex molecular cross-talk existing between the different factors influencing the cellular pathways. In this review the state of art of the platforms applied for the enrichment of specific and most common post-translational modifications, such as glycosylation and glycation, phosphorylation, sulfation, redox modifications (i.e. sulfydration and nitrosylation), methylation, acetylation, and ubiquitinylation, are described. Enrichments strategies applied to characterize less studied post-translational modifications are also briefly discussed.

2.
Arch Oral Biol ; 98: 148-155, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30496935

RESUMO

OBJECTIVE: In the present study the salivary proteome of burning mouth syndrome patients and healthy subjects was characterized by a top-down proteomic approach and compared to highlight possible qualitative and quantitative differences that may give suggestions about the causes of this pathology which are still unknown. MATERIALS AND METHODS: Resting and stimulated whole saliva, stimulated parotid and submandibular/sublingual saliva samples were collected from burning mouth syndrome patients (n = 16) and age- and gender-matched healthy subjects (n = 14). An equal volume of 0.2% trifluoroacetic acid was added to each sample immediately after collection and the supernatants were analysed by liquid chromatography coupled to electrospray-ionisation mass spectrometry. Proteins and peptides were quantified using a label-free approach measuring the extracted ion current peak areas of the main salivary proteins and peptides. RESULTS: The quantitation of the main salivary proteins and peptides revealed a higher concentration of cystatin SN in resting saliva of burning mouth syndrome patients with respect to healthy controls and no other conspicuous changes. CONCLUSIONS: The reported data showed that the salivary protein profile was not affected, in composition and relative abundance, by the burning mouth syndrome, except for the cystatin SN, a protein up-regulated in several pathological conditions, that might be considered potentially indicative of the disease.


Assuntos
Síndrome da Ardência Bucal/complicações , Síndrome da Ardência Bucal/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Saliva/química , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/análise , Idoso , Cromatografia Líquida , Feminino , Humanos , Pessoa de Meia-Idade , Glândula Parótida/metabolismo , Salivação , Espectrometria de Massas por Ionização por Electrospray , Xerostomia/complicações
3.
J Proteome Res ; 17(9): 3292-3307, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30064219

RESUMO

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.


Assuntos
Processamento de Proteína Pós-Traducional , Saliva/química , Proteínas Salivares Ricas em Prolina/metabolismo , Adulto , Sequência de Aminoácidos , Cromatografia Líquida , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Glândula Parótida/química , Glândula Parótida/metabolismo , Peptídeos/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteólise , Proteômica/métodos , Proteínas Salivares Ricas em Prolina/química , Proteínas Salivares Ricas em Prolina/genética , Proteínas Salivares Ricas em Prolina/isolamento & purificação , Espectrometria de Massas em Tandem
4.
Biopolymers ; 106(5): 714-25, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27272460

RESUMO

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.


Assuntos
Fármacos Anti-HIV/química , Peptídeos Catiônicos Antimicrobianos/química , Simulação de Dinâmica Molecular , Proteínas Salivares Ricas em Prolina/química , Domínios de Homologia de src , Humanos , Ressonância de Plasmônio de Superfície
5.
J Proteomics ; 146: 48-57, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27321913

RESUMO

The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC­ESI­MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100 A6, S100 A7, S100 A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and α-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100 A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain. SIGNIFICANCE: Differential protein expression analysis of the acid insoluble fraction of saliva from preterm, at-term newborns and adults has been performed in this study by coupling 2-DE analysis and high-resolution tandem mass spectrometry in order to complete the information previously obtained by top-down LC­MS only on the acid-soluble proteome. Several proteins identified in the acid insoluble fraction of both preterm newborn and adult saliva are not of glandular origin, being only prolactin-inducible protein, salivary cystatins, α-amylase and polymeric immunoglobulin receptor exclusive of salivary glands. Three proteins resulted increased in at-term newborns with respect to preterm newborns and adults: BPI fold-containing family A member 1, two proteoforms of annexin A1 and keratin type 1 cytoskeletal 13, while several proteins were significantly increased in adults.


Assuntos
Recém-Nascido Prematuro/metabolismo , Proteoma/metabolismo , Saliva/metabolismo , Adolescente , Adulto , Anexina A1/análise , Eletroforese em Gel Bidimensional , Glicoproteínas/análise , Humanos , Lactente , Recém-Nascido , Queratina-1/análise , Pessoa de Meia-Idade , Fosfoproteínas/análise , Proteoma/análise , Saliva/química , Espectrometria de Massas em Tandem , Adulto Jovem
6.
PLoS One ; 11(1): e0147925, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26814504

RESUMO

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.


Assuntos
Cálcio/metabolismo , Peptídeos/metabolismo , Progesterona/farmacologia , Glândulas Salivares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Citosol/metabolismo , Humanos , Íons/química , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Peptídeos/síntese química , Peptídeos/química , Progestinas/farmacologia , Domínios Proteicos Ricos em Prolina , Ligação Proteica , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Espectrometria de Massas por Ionização por Electrospray
7.
J Proteomics ; 134: 47-56, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26375204

RESUMO

UNLABELLED: The most heterogeneous family of human salivary proteins is represented by proline-rich proteins (PRPs) divided in acidic, basic, and basic glycosylated (aPRPs, bPRPs, gPRPs). They are encoded by six genes, clustered on chromosome 12p13.2: PRH1-2 encode aPRPs, PRB1-4 encode bPRPs and gPRPs. Each gene exists in different allelic forms: two for PRH2, three for PRH1, PRB2, and PRB4, four for PRB1, and PRB3. During granule maturation, PRP proproteins undergo proteolysis by the action of convertases and carboxypeptidases. Differently from bPRPs, proteolysis of aPRPs is not complete, and, besides fragments, entire protein species are also secreted. Maturation process generates ten aPRPs (PRP-1, PRP-2, PIF-s, Db-s, Pa, PRP-3, PRP-4, PIF-f, Db-f, P-C), and at least 18 bPRPs (II-2, P-E, IB-6, Ps-1, Ps-2, IB-1, P-J, IB-8a, P-F, P-H, P-D, II-1, protein glycosylated A, CD-IIg, and Gl1-4). In addition, single nucleotide and length polymorphisms, and differentially spliced transcripts originate several natural variants. Phosphorylation, N-pyroglutaminylation, dimerization, and N-/O-glycosylation also occur during maturation, enlarging the number of protein species, further increased by proteolytic events governed by carboxy- and endo-peptidases during and after secretion, and giving rise to a huge number of small peptides. The PRP functional role is still poorly understood. SIGNIFICANCE: The high polymorphism of PRPs gives an important contribution to the high heterogeneity and inter-individual variability of the human salivary proteome. The products of six genes clustered on chromosome 12p13.2 comprise a mixture of entire, truncated, phosphorylated, glycosylated and dimerized protein/peptide species, sharing large part of their sequences, and possibly involved in different biological activities. Whatever the role of PRP species is, it should be crucial, given that PRPs are the most conserved oral salivary proteins among mammals.


Assuntos
Peptídeos , Processamento de Proteína Pós-Traducional/fisiologia , Proteólise , Proteínas Salivares Ricas em Prolina , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Humanos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Salivares Ricas em Prolina/genética , Proteínas Salivares Ricas em Prolina/metabolismo
8.
Mol Biosyst ; 11(6): 1668-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25909245

RESUMO

A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to ß- and α-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes.


Assuntos
Astrocitoma/química , Neoplasias Encefálicas/química , Meduloblastoma/química , Proteoma/análise , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Humanos , Meduloblastoma/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteômica
9.
J Proteome Res ; 14(4): 1666-77, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25761918

RESUMO

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Modelos Biológicos , Proteoma/metabolismo , Proteômica/métodos , Saliva/química , Fenômenos Cronobiológicos/genética , Humanos , Recém-Nascido Prematuro , Proteoma/genética , Saliva/metabolismo , Fatores de Tempo
10.
Bioanalysis ; 6(4): 563-81, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24568357

RESUMO

Comprehensive analysis and characterization of the human salivary proteome is an important step towards the possible use of saliva for diagnostic and prognostic purposes. The contribution of the different sources to whole saliva, and the evaluation of individual variability and physiological modifications have been investigated by top-down proteomic approaches, disclosing the faceted and complex profile of the human salivary proteome. All this information is essential to develop saliva protein biomarkers. In this Review the major results obtained in the field by top-down platforms, and the improvements required to allow a more complete picture, will be discussed.


Assuntos
Proteoma/análise , Proteômica , Saliva/metabolismo , Biomarcadores/análise , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Processamento de Proteína Pós-Traducional
11.
J Comp Physiol B ; 183(7): 905-19, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23632627

RESUMO

The hemoglobin system of the serpent eel Ophisurus serpens was structurally and functionally characterized with the aim of comparing it to the hemoglobin system of other fish species, as oxygen loading under the severe habitat conditions experienced by O. serpens could have necessitated specific adaptation mechanisms during evolution. The hemoglobin system of O. serpens includes one cathodic and four anodic components. The molecular mass of the α and ß chains of the cathodic component as well as the 2 α and 4 ß of the anodic components were determined. Analysis of the intact α and ß chains from cathodic hemoglobin and their proteolytic digestion products by high-resolution MS and MS/MS experiments resulted in 92 and 95 % sequence coverage of the α and ß globins, respectively. The oxygen binding properties of both hemoglobin components were analyzed with respect to their interactions with their physiological effectors. Stripped cathodic hemoglobin displayed the highest oxygen affinity among Anguilliformes with no significant effect of pH on O2-affinity. In the presence of both chloride and organic phosphates, O2-affinity was strongly reduced, and cooperativity was enhanced; moreover, cathodic hemoglobin contains two indistinguishable GTP-binding sites. Stripped anodic hemoglobins exhibited both low O2-affinity and low cooperativity and a larger Bohr effect than cathodic hemoglobin. The cathodic hemoglobin of O. serpens and the corresponding component of Conger conger share the greatest structural and functional similarity among hemoglobin systems of Anguilliformes studied to date, consistent with their phylogenetic relationship.


Assuntos
Enguias/sangue , Hemoglobinas/química , Trifosfato de Adenosina/sangue , Sequência de Aminoácidos , Animais , Eritrócitos/metabolismo , Guanosina Trifosfato/sangue , Hemoglobinas/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/química , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
12.
J Proteome Res ; 12(2): 917-26, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23278499

RESUMO

Analysis by a HPLC-ESI-MS top-down proteomic platform of specimens of human preterm newborn whole saliva evidenced high relative amounts of cystatin B and its S-glutathionylated,S-cysteinylated, and S-S 2-mer (on Cys(3)) derivatives, decreasing as a function of postconceptional age (PCA). The percentage of S-unmodified cystatin B was higher than the S-modified isoforms in the early PCA period, differently from adults where cystatin B was detectable only as S-modified derivatives. The percentage of S-modified derivatives increased as a function of PCA, reaching at the normal term of delivery values similar to those determined in at-term newborns, babies, and adults. Moreover, in the early PCA period, high relative amounts of the 1-53 and 54-98 cystatin B fragments were detected, decreasing as a function of PCA and disappearing at the normal term of delivery. In agreement with intact cystatin B, fragment 1-53 was detectable as S-unmodified and S-modified derivatives, and their percentages changed accordingly with the percentages of intact proteins, suggesting that the fragmentation process could be subsequent to and independent from the S-modification of the protein. This study highlights specific enzymatic activity in the oral cavity of preterm newborns not present in at-term newborns and adults, which can be a clue to specialized pathways occurring during fetal oral development.


Assuntos
Cistatina B/análise , Cisteína/metabolismo , Glutationa/metabolismo , Fragmentos de Peptídeos/análise , Saliva/química , Adulto , Cromatografia Líquida de Alta Pressão , Cistatina B/metabolismo , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Pessoa de Meia-Idade , Boca/química , Espectrometria de Massas por Ionização por Electrospray
13.
Biochim Biophys Acta ; 1810(12): 1272-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21763402

RESUMO

BACKGROUND: HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population. METHODS: Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined. RESULTS: Sequencing of the γ globin genes revealed the TGT→CGT transition at codon 93 in one of the two (G)γ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position ß93 are known to affect the arrangement of a salt bridge at the α1ß2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity. GENERAL SIGNIFICANCE: Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T→R transition of the HbF tetramer. Substitutions at the F9 position of the (G)γ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport.


Assuntos
Hemoglobina Fetal/fisiologia , Arginina/química , Cromatografia Líquida de Alta Pressão , Cisteína/química , Hemoglobina Fetal/química , Hemoglobina Fetal/genética , Humanos , Espectrometria de Massas por Ionização por Electrospray
14.
Trends Biotechnol ; 29(8): 409-18, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21620493

RESUMO

Although very attractive for noninvasive specimen collection, saliva has not yet been considered a relevant bodily fluid for the diagnosis and prognosis of diseases. The functional roles of specific salivary peptides and proteins have also not yet been studied in detail. Recent proteomic analysis of human whole saliva has shown that salivary biomarkers could contribute to the detection of local and systemic diseases, provided the standardization of proper sampling procedures exists. Recently, interesting and novel functions for different families of specific secretory peptides and proteins have been demonstrated, which could be a basis for the design of peptidomimetics with relevant biotechnological applications. In this review, we focus on the most recent advances in analysing salivary proteins and their potential application in biotechnology.


Assuntos
Biotecnologia/métodos , Proteoma/química , Proteoma/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/metabolismo , Humanos , Proteômica/métodos
15.
Mol Cell Proteomics ; 10(1): M110.003467, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20943598

RESUMO

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins ß(4) and ß(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.


Assuntos
Recém-Nascido Prematuro/metabolismo , Proteoma/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Recém-Nascido , Masculino , Peso Molecular , Proteoma/química , Proteínas e Peptídeos Salivares/química , Espectrometria de Massas por Ionização por Electrospray
17.
Biochem Biophys Res Commun ; 398(3): 477-81, 2010 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20599699

RESUMO

RP-HPLC-ESI-MS profile of saliva samples from human preterm newborn showed a protein peak in the elution range 26.6-27.6min. Deconvolution of ESI-MS spectra revealed the presence of two proteins with average molecular mass (Mav) values of 17,239+/-3Da and 18,065+/-3Da in 9 samples, with Mav value of 17,239+/-3Da in 4 samples and Mav value of 18,065+/-3Da in 2 samples. MALDI-TOF-MS analysis of tryptic digest allowed identifying the proteins as two isoforms of small proline-rich protein 3 and cDNA amplification of RNA extracts from oral mucosa, parotid and submandibular gland samples, obtained at fetal autopsy, provided two nucleotide sequences in agreement with those reported in the literature. The two proteins differ for an octapeptide repeat (GCTKVPEP) and the substitution Leu-->Val, at position 148 and 140 of the mature form of the 18,065 and 17,239Da protein, respectively. During maturation the two proteins undergo two post-translational modifications, corresponding to N-terminal acetylation and removal of the initiator methionine. cDNA amplification did not allow to clarify if the proteins found in saliva originated from cellular shedding of the epithelium and/or secretion.


Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/química , Recém-Nascido Prematuro/metabolismo , Mucosa Bucal/metabolismo , Glândula Parótida/metabolismo , Saliva/metabolismo , Glândula Submandibular/metabolismo , Cromatografia Líquida de Alta Pressão , Proteínas Ricas em Prolina do Estrato Córneo/genética , Feto/metabolismo , Humanos , Recém-Nascido , Mucosa Bucal/embriologia , Glândula Parótida/embriologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Espectrometria de Massas por Ionização por Electrospray , Glândula Submandibular/embriologia
18.
J Pept Sci ; 16(6): 269-75, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20474038

RESUMO

This study describes the identification and structural characterization of Sus scrofa statherin. HPLC-electrospray ionization mass spectrometry analysis on pig parotid secretory granule extracts evidenced a peptide with a molecular mass value of 5381.1 +/- 0.6 Da and its truncated form, devoid of the C-terminal Ala residue, with a molecular mass value of 5310.1 +/- 0.6 Da. The complete sequence of pig statherin gene was determined by sequencing the full-length cDNA obtained by rapid amplification of cDNA ends. The gene is 549 base pairs long and contains an open reading frame of 185 nucleotides, encoding a 42-amino acid secretory polypeptide with a signal peptide of 19 residues. This sequence presents some typical features of the four statherins characterized till now, showing the highest degree of amino acid identity with bovine (57%) and human statherin (39%). Pig statherin is mono-phoshorylated on Ser-3, while primate statherins already characterized are di-phosphorylated on Ser-2 and Ser-3. This difference, probably connected to the Asp-4 --> Glu substitution, suggests the involvement of the Golgi-casein kinase, which strictly recognizes the SX(E/pS) consensus sequence.


Assuntos
Grânulos Citoplasmáticos/química , Glândula Parótida/química , Proteínas e Peptídeos Salivares/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Dados de Sequência Molecular , Peso Molecular , Glândula Parótida/citologia , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Sus scrofa
19.
Protein Expr Purif ; 69(2): 219-25, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19651217

RESUMO

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria-Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47mg per liter of cell culture, while 112mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP-HPLC corresponded to 0.6mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.


Assuntos
Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas e Peptídeos Salivares/isolamento & purificação , Proteínas e Peptídeos Salivares/metabolismo , Animais , Caseína Quinases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Complexo de Golgi/enzimologia , Humanos , Glândula Parótida/citologia , Fosforilação , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Suínos
20.
J Comp Physiol B ; 179(8): 971-83, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19554331

RESUMO

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC-electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major beta (beta1 16,022 Da, beta2 16,036 Da, beta3 16,036 Da, labeled according to their progressive elution times) and two major alpha globins (alpha1 15,345 Da, alpha2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that beta2 globin differs from beta1 for the substitution Val126 --> Leu, while beta3 globin differs from beta2 for the isobaric substitution Lys65 --> Gln. The alpha2 globin differs from the alpha1 for the substitution Ser15 --> Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC-ESI-MS analysis revealed that the fraction with higher pI (HbI) contained beta1, beta2 and both the alpha globins, and the fraction with lower pI (HbII) contained beta3 and both the alpha globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA(0), with HbII showing the more pronounced differences. With respect to HbA(0), either the substitution Probeta5 --> Gly or the Probeta5 --> Ala is present in all the cetacean beta globins sequenced so far, and it has been hypothesized that position 5 of beta globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lysbeta65 --> Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA(0).


Assuntos
Golfinhos Comuns/sangue , Hemoglobinas/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Golfinhos Comuns/genética , DNA Complementar/química , Hemoglobinas/genética , Hemoglobinas/isolamento & purificação , Hemoglobinas/metabolismo , Ponto Isoelétrico , Mar Mediterrâneo , Dados de Sequência Molecular , Oxigênio/metabolismo , Fragmentos de Peptídeos/química , Ligação Proteica , Isoformas de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Temperatura
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