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1.
Mater Sci Eng C Mater Biol Appl ; 106: 110268, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753373

RESUMO

Microfiber yarns (MY) have been widely employed to construct tendon tissue grafts. However, suboptimal ultrastructure and inappropriate environments for cell interactions limit their clinical application. Herein, we designed a modified electrospinning device to coat poly(lactic-co-glycolic acid) PLGA nanofibers onto polylactic acid (PLA) MY to generate PLGA/PLA hybrid yarns (HY), which had a well-aligned nanofibrous structure, resembling the ultrastructure of native tendon tissues and showed enhanced failure load compared to PLA MY. PLGA/PLA HY significantly improved the growth, proliferation, and tendon-specific gene expressions of human adipose derived mesenchymal stem cells (HADMSC) compared to PLA MY. Moreover, thymosin beta-4 (Tß4) loaded PLGA/PLA HY presented a sustained drug release manner for 28 days and showed an additive effect on promoting HADMSC migration, proliferation, and tenogenic differentiation. Collectively, the combination of Tß4 with the nano-topography of PLGA/PLA HY might be an efficient strategy to promote tenogenesis of adult stem cells for tendon tissue engineering.


Assuntos
Nanofibras/química , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Timosina/química , Engenharia Tecidual , Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tendões/citologia , Tendões/metabolismo , Timosina/metabolismo , Timosina/farmacologia , Tecidos Suporte/química
2.
Ann N Y Acad Sci ; 1457(1): 128-141, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31407357

RESUMO

Metallacarboranes are anionic boron clusters with high affinity to serum albumin, ability to cross biological membranes, and no apparent toxicity in vitro and in vivo. Thus, conjugation with cobalt bis(1,2-dicarbollide), [COSAN]- , ([3,3'-Co(1,2-C2 B9 H11 )2 ]- ) may improve the properties of therapeutic peptides or proteins at both molecular and systemic levels. Here, we conjugated [COSAN]- with the therapeutic peptide thymosin ß4 (Tß4), which has a pleiotropic activity that results in enhanced healing and regeneration of injured tissues. Using fluorescence quenching of human serum albumin and surface plasmon resonance techniques, we showed that the conjugates have a high affinity to human serum albumin. Using an in vitro wound closure assay, we showed that conjugation with [COSAN]- enhances the activity of Tß4 toward fibroblasts (MSU1.1 cell line). These results indicate an application of metallacarboranes in the development of analogs of various therapeutic peptides/proteins with superior pharmacological properties.


Assuntos
Albuminas/análise , Boranos/química , Membrana Celular/metabolismo , Cobalto/química , Metais/química , Peptídeos/química , Ânions/química , Linhagem Celular , Dicroísmo Circular , Complexos de Coordenação/química , Fibroblastos/metabolismo , Humanos , Cinética , Estrutura Terciária de Proteína , Albumina Sérica/química , Albumina Sérica Humana/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Ressonância de Plasmônio de Superfície , Timosina/química
3.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140252, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31325636

RESUMO

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 µM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R1, R1ρ, R2 and ηxy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , DNA/química , Complexos Multiproteicos/química , Proteínas de Neoplasias/química , Precursores de Proteínas/química , Timosina/análogos & derivados , Humanos , Domínios Proteicos , Espalhamento a Baixo Ângulo , Timosina/química , Difração de Raios X
4.
Fish Shellfish Immunol ; 86: 516-524, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30468890

RESUMO

The ß-thymosin (Tß) proteins participate in numerous biological processes, such as cell proliferation and differentiation, anti-inflammatory and antimicrobial mechanism. To date, Tß proteins have been well studied in vertebrates, especially mammals. While limited Tß or Tß-like proteins have been reported in invertebrates. Moreover, rare information of Tß or Tß-like proteins is available in scallop species yet. In the present study, two Tß homologues, AiTß and CfTß, were identified and characterized from two scallop species bay scallop Argopecten irradians and Zhikong scallop Chlamys farreri. They were both 41 amino acid peptide and contained one THY domain, a highly conserved actin-binding motif and two conserved helix forming regions. Tissue distribution and expression profiles of their mRNA transcripts were roughly similar yet different in detail, while their recombinant proteins exhibited different immunomodulation activity on the downstream immune parameters. These results collectively indicated that the function of Tß family in scallop were functionally differentiated.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Pectinidae/genética , Pectinidae/imunologia , Timosina/genética , Timosina/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Timosina/química
5.
Fish Shellfish Immunol ; 84: 244-251, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30292805

RESUMO

Thymosins ß are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus, and named as Mjthymosin2, Mjthymosin3, Mjthymosin4, and Mjthymosin5 according the number of their thymosin beta actin-binding motifs. Mjthymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mjthymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mjthymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mjthymosins played important roles in the antibacterial immune response of kuruma shrimp.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Timosina/genética , Timosina/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Timosina/química , Vibrio/fisiologia
6.
Biochemistry ; 57(48): 6645-6648, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30430826

RESUMO

It was recently reported that human linker histone H1.0 and its chaperone prothymosin-α (ProTα) form an extremely disordered 1:1 complex with an ultrahigh affinity (equilibrium dissociation constant KD of ∼2 × 10-12 M) measured using a single-molecule Förster resonance energy transfer method. It was hypothesized that the ultrahigh affinity and extreme disorder may be required for the chaperone function of ProTα, in which it displaces the linker histone from condensed chromatin. Here, we measure the binding affinity for the ProTα-H1.0 complex using isothermal titration calorimetry and report a KD value of (4.6 ± 0.5) × 10-7 M. In addition, we show that ProTα facilitates the formation of the H1.0-nucleosome complex in vitro. The results of our study contrast with those of the previous report and provide new insights into the chaperone function of ProTα. Possible causes for the observed discrepancy in binding affinity are discussed.


Assuntos
Histonas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Sequência de Aminoácidos , Calorimetria , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Histonas/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Timosina/química , Timosina/genética , Timosina/metabolismo
7.
Bioconjug Chem ; 29(11): 3509-3515, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30365887

RESUMO

Anionic boron clusters are man-made, inorganic compounds with potential applications in therapeutic peptides modification to improve their biological activity and pharmacokinetics, e.g., by enabling complexation with serum albumin. However, the conjugation of anionic boron clusters and peptides remains poorly understood. Here, we report a solid-state, thermal reaction to selectively conjugate carboxylic groups in the peptide thymosin ß4 (Tß4) with cyclic oxonium derivatives of anionic boron clusters (dodecaborate anion [B12H12]2- and cobalt bis(1,2-dicarbollide), [COSAN]- [3,3'-Co(1,2-C2B9H11)2]-). Modification of the carboxylic groups retains the negative charge at the modification site and leads to the formation of ester bonds. The ester bonds in the conjugates undergo hydrolysis at different rates depending on the site of the modification. We obtained conjugates with dramatically different stabilities (τ1/2 from 3-836 h (Tß4-[B12H12]2- conjugates) and 9-1329 h (Tß4-[COSAN]- conjugates)) while retaining or improving the prosurvival activity of Tß4 toward cardiomyocytes (H9C2 cell line).


Assuntos
Boro/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Timosina/química , Ânions/química , Linhagem Celular , Complexos de Coordenação/farmacocinética , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Miócitos Cardíacos/efeitos dos fármacos , Albumina Sérica/química
8.
J Biol Inorg Chem ; 23(8): 1255-1263, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30187212

RESUMO

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Zinco/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Proteínas Intrinsicamente Desordenadas/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ácido Poliglutâmico/síntese química , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Timosina/química , Timosina/metabolismo
9.
Biochimie ; 154: 99-106, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30096371

RESUMO

Thymosin α1 (Tα1), a hormone containing 28 amino acids, has been approved in several cancer therapies, but the lack of tumor-targeting hinders its full use in tumor treatment. We designed a new peptide by connecting Tα1 and RGDR, generating a product, Tα1-RGDR, where RGDR is located in the C-end with both tumor-homing and cell internalizing properties (C-end rule peptides, a consensus R/KXXR/K motif). This work aimed to study the antitumor and immunological activities of Tα1-RGDR, and its differences compared with the wild-type Tα1. The antitumor and immunological activities of Tα1-RGDR were measured using the B16F10 tumor and immunologic suppression models. Tα1-RGDR treatment led to significant inhibition of tumor growth at a dose at which Tα1 showed a slight effect in the B16F10 tumor growth model. In the immunologic suppression model, Tα1-RGDR shared almost equivalent immunomodulatory effect with Tα1. These results demonstrated the better therapeutic effects after treatment with Tα1-RGDR compared with Tα1. Moreover, both Tα1-RGDR and Tα1 shared a helical conformation in the presence of trifluoroethanol based on CD spectroscopy. Our dock information of Tα1-RGDR when combined with integrin αvß3 or neuropilin-1 further confirmed previous experimental results. All these findings suggest that Tα1-RGDR might be a useful therapy for tumors by overcoming its wild type limitation of tumor homing.


Assuntos
Antineoplásicos/química , Melanoma/metabolismo , Timosina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Timalfasina , Timosina/química , Timosina/farmacologia
10.
Expert Opin Biol Ther ; 18(sup1): 177-184, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063851

RESUMO

OBJECTIVES: Hepatic stellate cells (HSC) trans-differentiation is central to the development of liver fibrosis, marked by the expression of pro-fibrogenic genes and the proliferation and migration of activated HSC. Therefore, preventing and/or reverting the activation, proliferation, and migration of HSC may lead to new therapies for treating fibrosis/cirrhosis. Thymosin ß4 (Tß4) inhibits PDGF-BB-induced fibrogenesis, proliferation and migration of HSC by blocking Akt phosphorylation. Here, we utilized Tß4-derived peptides: amino-terminal-Ac-SDKPDMAEIEKFDKS (1-15aa) and actin-binding-LKKTETQ (17-23aa) to investigate the molecular mechanisms in the anti-fibrogenic actions of Tß4. METHODS: We used RT-PCR, Western blot, and proliferation and migration assays in early passages of human HSC cultures treated with PDGF-BB and/or Tß4 peptides. RESULTS: We showed that 17-23aa but not 1-15aa inhibited PDGF-BB-dependent up-regulation of PDGFß receptor, α-SMA, and collagen 1. It also blunted the phosphorylation of Akt at T 308 and S473, resulting in the inhibition of phosphorylation of PRAS40, and HSC proliferation and migration. Interestingly, 1-15aa blocked Akt phosphorylation at S473, but not T308 by inhibiting mTOR phosphorylation, thus, it did not have any effect on HSC proliferation and migration. CONCLUSION: These findings suggest that while 1-15aa has a minor effect on Akt phosphorylation, the anti-fibrogenic actions of Tß4 are exerted via 17-23aa.


Assuntos
Actinas/metabolismo , Becaplermina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Timosina/farmacologia , Animais , Células Cultivadas , Células Estreladas do Fígado/fisiologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/prevenção & controle , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/fisiologia , Timosina/química , Timosina/metabolismo
11.
Expert Opin Biol Ther ; 18(sup1): 199-203, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063862

RESUMO

OBJECTIVES: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5. RESULTS: We detected more than 100 monoisotopic masses corresponding to thymosin ß4 and truncated forms of ubiquitin, prothymosin α, thymosin ß4, and thymosin ß9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3. CONCLUSION: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.


Assuntos
Espectrometria de Massas/métodos , Timosina/análogos & derivados , Animais , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Humanos , Precursores de Proteínas/análise , Precursores de Proteínas/isolamento & purificação , Proteômica/métodos , Timosina/análise , Timosina/química , Timosina/isolamento & purificação , Fatores de Tempo , Ubiquitina/análise , Ubiquitina/isolamento & purificação
12.
Int J Pharm ; 547(1-2): 611-620, 2018 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-29933059

RESUMO

Tumor-targeted therapy is an attractive strategy for cancer treatment. Peptide hormone thymosin α1 (Tα1) has been used against several diseases, including cancer, but its activity is pleiotropic. Herein, we designed a fusion protein Tα1-iRGD by introducing the tumor homing peptide iRGD to Tα1. Results show that Tα1-iRGD can promote T-cell activation and CD86 expression, thereby exerting better effect and stronger inhibitory against melanoma and lung cancer, respectively, than Tα1 in vivo. These effects are indicated by the reduced densities of tumor vessels and Tα1-iRGD accumulation in tumors. Moreover, compared with Tα1, Tα1-iRGD can attach more B16F10 and H460 cells and exhibits significantly better immunomodulatory activity in immunosuppression models induced by hydrocortisone. Circular dichroism spectroscopy and structural analysis results revealed that Tα1 and Tα1-iRGD both adopted a helical confirmation in the presence of trifluoroethanol, indicating the structural basis of their functions. These findings highlight the vital function of Tα1-iRGD in tumor-targeted therapy and suggest that Tα1-iRGD is a better antitumor drug than Tα1.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Oligopeptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Timosina/análogos & derivados , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antígeno B7-2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Feminino , Humanos , Tolerância Imunológica/efeitos dos fármacos , Neoplasias Pulmonares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Nus , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timalfasina , Timosina/química , Timosina/farmacologia , Timosina/uso terapêutico , Trifluoretanol/química , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nature ; 555(7694): 61-66, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29466338

RESUMO

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.


Assuntos
Histonas/química , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Sítios de Ligação , Humanos , Ligação Proteica , Eletricidade Estática , Timosina/química , Timosina/metabolismo
14.
Chemphyschem ; 19(7): 848-856, 2018 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-29274195

RESUMO

Wide-line 1 H NMR measurements were extended and all results were reinterpreted in a new thermodynamics-based approach to study aqueous solutions of thymosin-ß4 (Tß4 ), stabilin C-terminal domain (CTD) and their 1:1 complex. The energy distributions of the potential barriers, which control motion of protein-bound water molecules, were determined. Heterogeneous and homogeneous regions were found at the protein-water interface. The measure of heterogeneity gives a quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to 20 % of the whole proteins. About 40 % of the binding sites of free Tß4 become involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tß4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.


Assuntos
Moléculas de Adesão Celular Neuronais/química , Fragmentos de Peptídeos/química , Timosina/química , Água/química , Humanos , Movimento (Física) , Estrutura Quaternária de Proteína , Espectroscopia de Prótons por Ressonância Magnética , Termodinâmica
15.
Toxicol In Vitro ; 47: 269-273, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29262310

RESUMO

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that it binds to rat IEC-6 and human Caco-2 intestinal epithelial cells with high affinity (Kd 3.6 and 3.7nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α1 and 131-135 in IFN-α2, but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (Ki>10µM). Thus, TM-α1, IFN-α2, and the peptide: LKEKK bind with high affinity and specificity to the cholera toxin receptor on IEC-6 and Caco-2 cells. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the nitric oxide production and the soluble guanylate cyclase activity in IEC-6 and Caco-2 cells.


Assuntos
Toxina da Cólera/metabolismo , Gangliosídeo G(M1)/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Sítios de Ligação , Ligação Competitiva , Células CACO-2 , Linhagem Celular , Toxina da Cólera/farmacologia , Gangliosídeo G(M1)/agonistas , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Humanos , Interferon-alfa/química , Interferon-alfa/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Radioisótopos do Iodo , Cinética , Ligantes , Óxido Nítrico/agonistas , Óxido Nítrico/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ratos , Receptores de Superfície Celular/agonistas , Timosina/química , Timosina/metabolismo
16.
Molecules ; 22(11)2017 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-29077041

RESUMO

Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression.


Assuntos
Sequência de Aminoácidos , Ácido Hialurônico/química , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , Timosina/análogos & derivados , Espectroscopia de Ressonância Magnética , Ligação Proteica , Timalfasina , Timosina/química
17.
Fish Shellfish Immunol ; 69: 90-98, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28803959

RESUMO

Thymosin beta belongs to the thymosin family, which consists of a series of highly conserved peptides involved in various biological processes. In teleosts, understanding of the immunological functions of thymosin beta is limited, particularly in vivo, which is essentially unknown. In the current study, we cloned and identified thymosin beta 4 from the teleost fish Golden pompano (Trachinotus ovatus), which we have named TroTß4. We investigated the expression patterns and functions of TroTß4 in both in vivo and in vitro assays. TroTß4 is composed of 44 amino acids and shares high sequence identities with known thymosin ß4 species in other teleosts, which contains a highly conserved actin-binding motif (LKKTET). The expression of TroTß4 was most abundant in immune organs, and was significantly up-regulated in response to infection bacterial with one of a number of bacteria (including Edwardsiella tarda, Vibrio harveyi, and Streptococcus agalactiae). Purified recombinant TroTß4 (rTroTß4) inhibited the growth of bacteria, as measured using an automatic growth curve analyzer, indicating that TroTß4 has antimicrobial functions. When administered in vivo, overexpression of TroTß4 in T. ovatus, bacterial colonization of tissues was significantly reduced. In contrast, when a DNA vector-based siRNA technology was used to knock down TroTß4 expression, bacterial dissemination and colonization of tissues increased significantly. Taken together, these results provide the first in vivo evidence to indicate that teleost thymosin beta 4 plays a significant role in innate antibacterial immune responses in addition to in vitro bacteriostatic activity. This provides valuable information regarding the biological functions of teleost thymosin beta.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata , Perciformes , Timosina/genética , Timosina/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/fisiologia , Timosina/química , Vibrio/fisiologia , Vibrioses/imunologia
18.
Curr Med Chem ; 24(17): 1747-1760, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28521686

RESUMO

BACKGROUND/OBJECTIVE: Prothymosin alpha (proTα) is a ubiquitous polypeptide first isolated by Haritos in 1984, whose role still remains partly elusive. We know that proTα acts both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similarly to molecules termed as "alarmins". Our research team pioneered the elucidation of the mechanisms underlying the observed activities of proTα. RESULTS: We were the first to demonstrate that proTα levels increase during normal and abnormal cell proliferation. We showed that proTα acts pleiotropically, inducing immunomodulatory effects on immune cell populations. We revealed that the immunoreactive region of proTα is the carboxyterminal decapeptide proTα(100-109) and both molecules stimulate innate immune responses, signaling through Toll-like receptors (TLRs), specifically TLR-4. We reported that proTα and proTα(100-109) bind on the surface of human neutrophils on sites involving TLR-4, and cell activation is complemented by cytoplasmic calcium ion influx. Further, we showed that proTα and proTα(100-109) act as adjuvants upstream of lymphocyte stimulation and, in the presence of antigen, promote the expansion of antigen-reactive effectors. Most recently, we reported that proTα(100-109) may accumulate in experimentally inflamed sites and can serve as a surrogate biomarker in severe bacterial infections, proposing that extracellular release of proTα or proTα(100- 109) alerts the immune system during conditions of danger. CONCLUSION: We, therefore, suggest that proTα, and likely proTα(100-109), act as alarmins, being important immune mediators as well as biomarkers, and could eventually become targets for new therapeutic/diagnostic approaches in immune-related diseases like cancer, inflammation, and sepsis.


Assuntos
Alarminas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Alarminas/química , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Humanos , Imunidade Inata/efeitos dos fármacos , Células Matadoras Naturais/citologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Precursores de Proteínas/química , Precursores de Proteínas/uso terapêutico , Sepse/metabolismo , Sepse/patologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Timosina/química , Timosina/metabolismo , Timosina/uso terapêutico , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo
19.
Proteins ; 85(2): 296-311, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27936491

RESUMO

Proteins that lack tertiary stability under normal conditions, known as intrinsically disordered, exhibit a wide range of biological activities. Molecular descriptions for the biology of intrinsically disordered proteins (IDPs) consequently rely on disordered structural models, which in turn require experiments that assess the origins to structural features observed. For example, while hydrodynamic size is mostly insensitive to sequence composition in chemically denatured proteins, IDPs show strong sequence-specific effects in the hydrodynamic radius (Rh ) when measured under normal conditions. To investigate sequence-modulation of IDP Rh , disordered ensembles generated by a hard sphere collision model modified with a structure-based parameterization of the solution energetics were used to parse the contributions of net charge, main chain dihedral angle bias, and excluded volume on hydrodynamic size. Ensembles for polypeptides 10-35 residues in length were then used to establish power-law scaling relationships for comparison to experimental Rh from 26 IDPs. Results showed the expected outcomes of increased hydrodynamic size from increases in excluded volume and net charge, and compaction from chain-solvent interactions. Chain bias representing intrinsic preferences for α helix and polyproline II (PPII ), however, modulated Rh with intricate dependence on the simulated propensities. PPII propensities at levels expected in IDPs correlated with heightened Rh sensitivity to even weak α helix propensities, indicating bias for common (φ, ψ) are important determinants of hydrodynamic size. Moreover, data show that IDP Rh can be predicted from sequence with good accuracy from a small set of physicochemical properties, namely intrinsic conformational propensities and net charge. Proteins 2017; 85:296-311. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Precursores de Proteínas/química , Proteínas Proto-Oncogênicas c-mdm2/química , Timosina/análogos & derivados , Humanos , Hidrodinâmica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Eletricidade Estática , Termodinâmica , Timosina/química
20.
Biochemistry (Mosc) ; 81(8): 871-5, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27677554

RESUMO

The synthetic peptide LKEKK corresponding to sequence 16-20 of human thymosin-α1 and 131-135 of human interferon-α2 was labeled with tritium to specific activity 28 Ci/mol. The [3H]LKEKK bound with high affinity (Kd = 3.7 ± 0.3 nM) to donor blood T-lymphocytes. Treatment of cells with trypsin or proteinase K did not abolish [3H]LKEKK binding, suggesting the non-protein nature of the peptide receptor. The binding was inhibited by thymosin-α1, interferon-α2, and cholera toxin B subunit (Ki = 2.0 ± 0.3, 2.2 ± 0.2, and 3.6 ± 0.3 nM, respectively). Using [3H]LKEKK, we demonstrated the existence of a non-protein receptor common for thymosin-α1, interferon-α2, and cholera toxin B-subunit on donor blood T-lymphocytes.


Assuntos
Interferon-alfa , Peptídeos , Linfócitos T/metabolismo , Timosina/análogos & derivados , Humanos , Interferon-alfa/química , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Linfócitos T/citologia , Timalfasina , Timosina/química , Timosina/metabolismo , Timosina/farmacologia
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