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1.
Yakugaku Zasshi ; 139(11): 1403-1415, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31685737

RESUMO

For my Ph.D. research topic, I isolated endogenous morphine-like analgesic dipeptide, kyotorphin, which mediates Met-enkephalin release, and discovered kyotorphin synthetase, a putative receptor and antagonist. Furthermore, I succeeded in purifying µ-opioid receptor and functional reconstitution with purified G proteins. After receiving my full professor position at Nagasaki University in 1996, I worked on two topics of research, molecular mechanisms of chronic pain through lysophosphatidic acid (LPA) and identification and characterization of neuroprotective protein, prothymosin α. In a series of studies, we have shown that LPA signaling defines the molecular mechanisms of neuropathic pain and fibromyalgia in terms of development and maintenance. Above all, the discovery of feed-forward system in LPA production and pain memory may contribute to better understanding of chronic pain and future analgesic drug discovery. Regarding prothymosin α, we first discovered it as neuronal necrosis-inhibitory molecule through two independent mechanisms, such as toll-like receptor and F0/F1 ATPase, both which protect neurons through indirect mechanisms. Prothymosin α is released by non-classical and non-vesicular mechanisms on various stresses, such as ischemia, starvation, and heat-shock. Thus it may be called a new type of neuroprotective damage-associated molecular patterns (DAMPs)/Alarmins. Heterozygotic mice showed a defect in memory-learning and neurogenesis as well as anxiogenic behaviors. Small peptide, P6Q derived from prothymosin α retains neuroprotective actions, which include blockade of cerebral hemorrhage caused by late treatment with tissue plasminogen activator in the stroke model in mice.


Assuntos
Dor Crônica/etiologia , Dor Crônica/genética , Fármacos Neuroprotetores , Precursores de Proteínas , Receptores de Ácidos Lisofosfatídicos/fisiologia , Transdução de Sinais/fisiologia , Timosina/análogos & derivados , Animais , Endorfinas , Humanos , Camundongos , Precursores de Proteínas/metabolismo , ATPases Translocadoras de Prótons , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores Opioides , Acidente Vascular Cerebral , Timosina/metabolismo , Receptores Toll-Like
2.
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
3.
Cancer Sci ; 110(4): 1208-1219, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30719818

RESUMO

Prothymosin-α (PTMA) is a small, acidic protein that is usually transported into the nucleus and involves many cellular and immunological functions. Previous studies demonstrated that aberrant location of PTMA expression exists in human bladder cancer, but the role of PTMA protein expression remains elusive. In this study, we created ectopic nuclear or cytoplasmic PTMA expression in human bladder cancer cells by infecting lentiviruses carrying wild type or deleted nuclear localization signal of the PTMA gene. The in vivo tumorigenesis assay showed PTMA protein with deleted nuclear localization signal promotes J82 xenograft tumor growth in mice and shortens their survival more so than the wild type. Chromatin immunoprecipitation showed that wild-type PTMA protein binds to the PTEN promoter and enhances phosphatase and tensin homolog (PTEN) expression. Through immunoblot proteomics and in vivo ubiquitination studies, PTMA protein can bind with tripartite motif-containing protein 21 (TRIM21) and block its ubiquitination. Also, TRIM21 can downregulate both forms of PTMA protein. In human bladder tumors, loss of nuclear PTMA expression was an unfavorable prognostic indicator for shorter disease-free survival (hazard ratio, 1.54; P = 0.009). Our data support that nuclear PTMA protein serves as a tumor suppressor in bladder cancer through upregulating PTEN and orchestrating TRIM21 for the regulation of Nrf2 signaling.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Precursores de Proteínas/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Timosina/análogos & derivados , Neoplasias da Bexiga Urinária/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , PTEN Fosfo-Hidrolase/genética , Prognóstico , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Timosina/genética , Timosina/metabolismo , Ubiquitinação , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia
4.
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
5.
J Phys Chem B ; 122(49): 11262-11270, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30230839

RESUMO

The malleability of intrinsically disordered proteins (IDPs) has generated great interest in understanding how their conformations respond to crowded cellular environments. Experiments can report gross properties such as fluorescence resonance energy transfer (FRET) efficiency but cannot resolve the conformational ensembles of IDPs and their interactions with macromolecular crowders. Computation can in principle provide the latter information but in practice has been hampered by the enormous expense for realistic modeling of IDPs and crowders and for sufficient conformational sampling. Here, taking advantage of a powerful method called FMAP (fast Fourier transform-based modeling of atomistic protein-crowder interactions), we computed how the conformational ensembles of three IDPs are modified in concentrated polyethylene glycol (PEG) 6000 solutions. We represented the IDPs at the all-atom level and the PEG molecules at a coarse-grained level and calculated the experimental observable, i.e., FRET efficiency. Whereas accounting for only steric repulsion of PEG led to overestimation of crowding effects, quantitative agreement with experimental data was obtained upon including mild IDP-PEG attraction. The present work demonstrates that realistic modeling of IDPs under crowded conditions for direct comparison with experiments is now achievable.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Transferência Ressonante de Energia de Fluorescência , Integrase de HIV/química , HIV-1/química , Simulação de Dinâmica Molecular , Coativador 3 de Receptor Nuclear/química , Polietilenoglicóis/química , Conformação Proteica , Domínios Proteicos , Precursores de Proteínas/química , Timosina/análogos & derivados
6.
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
7.
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
8.
Expert Opin Biol Ther ; 18(sup1): 89-94, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063859

RESUMO

OBJECTIVES: Prothymosin α (ProTα) was reported to inhibit the neuronal necrosis by facilitating the plasma membrane localization of endocytosed glucose transporter 1/4 through an activation of putative Gi-coupled receptor. The present study aims to identify a novel ProTα target, which may lead to an activation of Gi-coupled receptor. METHODS: We used Gi-rich lipid rafts fraction of retinal cell line N18-RE-105 cells for affinity cross-linking. The biological confirmation that F0/F1 ATPase is a target protein complex was performed by cell-free experiments using ELISA-based binding assay, surface plasmon resonance assay and quartz crystal microbalance assay, and cell-based experiments to measure extracellular ATP level in the HUVECs culture. RESULTS: From the cross-linking study and above-mentioned protein-protein interaction assays, ATP5A1 and ATP5B, F1 ATPase subunits were found to ProTα binding target proteins. In the culture of HUVEC cells, furthermore, ProTα increased the extracellular ATP levels in a reversible manner by anti-ATP5A1- and ATP5B-antibodies. CONCLUSION: The present study suggests that ProTα may activate ecto-F0/F1 ATPase and produced ATP. This study leads to next subjects whether produced ATP and its metabolites, ADP or adenosine may activate corresponding Gi-coupled receptors.


Assuntos
Adenosina Trifosfatases/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Receptores Imunológicos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Reagentes para Ligações Cruzadas/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Necrose , Ligação Proteica , Precursores de Proteínas/metabolismo , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/fisiologia , Timosina/análogos & derivados , Timosina/metabolismo
9.
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
10.
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
11.
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
12.
Nihon Yakurigaku Zasshi ; 151(1): 15-19, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29321391

RESUMO

Prothymosin alpha (ProTα) has been identified as an anti-necrotic factor from the conditioned medium of primary cultured of rat cortical neurons under the serum-free starving condition. ProTα is released in a non-vesicular manner from neurons or astrocytes by the help of cargo protein S100A13. Thus released ProTα is found to have robustness roles in the brain under the condition of neuronal necrosis or apoptosis. ProTα inhibits necrosis by plasma membrane-translocation of glucose transporters endocytosed by ischemia/starving stress, through an activation of unidentified G protein-coupled receptor and protein kinase Cß. In the cerebral or retinal ischemia model, systemic injection of ProTα protects brain or retina from ischemic damages by converting necrosis to apoptosis, which is in turn blocked by neurotrophic factors. In the retinal ischemia model, ProTα prevents the damages by another mechanism through toll-like receptor 4 (TLR4) and downstream TRIF signaling. The direct interaction between ProTα and TLR4/MD2 is also evidenced by the study of molecular dynamics and protein-protein interaction. All these findings indicate that ProTα could be called a cytoprotective member of damage-associated molecular patterns (DAMPs) or alarmins. ProTα and its modified peptide fragment, NEVDQE (P6Q) show the vasculoprotective actions by itself in a model of cerebral ischemia as well as neuroprotective actions. The concomitant administration of these peptides abolishes the cerebral hemorrhage induced tissue plasminogen activator (tPA), which is treated late after cerebral ischemia models. Thus, ProTα and P6Q seem to have promising therapeutic potencies to directly protect neurons and inhibit the hemorrhage by late treatment with tPA against stroke.


Assuntos
Alarminas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Animais , Apoptose , Desenho de Fármacos , Humanos , Necrose , Transdução de Sinais , Timosina/metabolismo
13.
J Viral Hepat ; 25(1): 4-9, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29052304

RESUMO

Hepatitis B virus (HBV) causes both acute and chronic hepatitis and infects large numbers of individuals worldwide. Unfortunately, prediction of typical clinical outcome is problematic and there is considerable variability in the frequency, duration and severity of disease progression. The mainstay of HBV treatment is directed towards the suppression of HBV replication by nucleos(t)ide analogs (NUCs). The use of immunomodulators such as α-Interferon and thymosin α1 can, in select patients, results in elimination of both HBsAg and HBeAg. Given the observation that viral clearance is most effective in the presence of a strong immune response, this review summarizes data suggesting that the use of a combination of an immune modulator such as Tα1 with a highly effective NUC may result in a more successful therapeutic approach in patients with chronic hepatitis B (CHB). Results from small studies using combination Tα1 and NUCs are encouraging, and ongoing clinical trials combining entecavir with Tα1 are anticipated to provide important data assessing the use of a combination of Tα1 with a NUC to achieve resolution of CHB.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antivirais/administração & dosagem , Hepatite B Crônica/terapia , Imunoterapia/métodos , Timosina/análogos & derivados , Quimioterapia Combinada/métodos , Humanos , Timalfasina , Timosina/administração & dosagem , Resultado do Tratamento
14.
Zygote ; 25(6): 760-770, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29173229

RESUMO

Prothymosin alpha (PTMA) is a highly acidic, intrinsically disordered protein that was first extracted from rat thymus and characterized as an immunogenic factor but soon detected in a variety of mammalian tissues. The presence of a nuclear localization signal and the adoption of a peculiar random-coil conformation are among the reasons behind its interaction with several molecular partners, hence at this time PTMA is known to be a very conserved and widely expressed molecule, involved in numerous and diverse biological processes. Only few studies have tried to weigh its possible involvement in reproduction, specifically in male gametogenesis: first reports have suggested that PTMA might be associated with the proliferative and early-meiotic phases of mammal spermatogenesis. Some years later, a comparative project on vertebrate spermatogenesis reported the isolation, for the first time, of prothymosin in a non-mammalian species, the amphibian Pelophylax esculentus. PTMA transcript and protein are localized in the germinal compartment, from spermatocytes to spermatozoa. A congruent pattern has been highlighted in studies on the fish Torpedo marmorata and Danio rerio, and in the mammal Rattus norvegicus, in which the expression of PTMA has been found in meiotic and post-meiotic germ cells inside testicular cysts and tubules. Moreover, its presence has been confirmed in rat and human spermatozoa (associated with the acrosome); its retention in the apical region of the head after the acrosome reaction revealed a striking conservation of the pattern during phylogenesis and suggested a possible role for the protein in gametogenesis and in fertilization.


Assuntos
Precursores de Proteínas/metabolismo , Espermatogênese/fisiologia , Testículo/fisiologia , Timosina/análogos & derivados , Animais , Masculino , Testículo/citologia , Timosina/metabolismo , Vertebrados
15.
Arch Biochem Biophys ; 635: 74-86, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29106904

RESUMO

Prothymosin α (ProTα) is an acidic protein with a nuclear role related to the chromatin activity through its interaction with histones in mammalian cells. ProTα acts as an anti-apoptotic factor involved in the control of the apoptosome activity in the cytoplasm, however the mechanisms underlying this function are still known. ProTα shares similar biological functions with acidic nuclear-cytoplasmic shuttling proteins included in SET and ANP32 family members. Using affinity chromatography, co-immunoprecipitation and chemical cross-linking, we demonstrate that ProTα interacts with SET, ANP32A and ANP32B proteins. The study by mass spectrometry of the complexes stabilized by chemical cross-linking showed that associations of ProTα consist of six highly acidic ProTα-complexes, which corresponds to differentiated interactions of ProTα either with SET or ANP32 proteins. The presence in the ProTα-complexes of cytoplasmic proteins involved in membrane remodeling and proteins implicated in the mitochondrial permeability, seems to indicate that they could be related to a cytoplasmic-mitochondrial activity. According to the cellular function of the characterized targets of ProTα, and the evolution in the composition of the diverse ProTα-complexes when proliferation activity was reduced or apoptosis induced, leads to hypothesized that ProTα interactions might be related to the proliferation activity and control of the cell survival.


Assuntos
Sobrevivência Celular/fisiologia , Citoplasma/metabolismo , Chaperonas de Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Fatores de Transcrição/metabolismo , Proliferação de Células/fisiologia , Humanos , Células Jurkat , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA , Transdução de Sinais/fisiologia , Timosina/metabolismo
16.
Biochemistry (Mosc) ; 82(9): 1036-1041, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28988532

RESUMO

In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (Kd = 3.3 nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (Ki 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (Ki > 1 µM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.


Assuntos
Toxina da Cólera/farmacologia , Interferon-alfa , Guanilil Ciclase Solúvel/genética , Linfócitos T/efeitos dos fármacos , Timosina/análogos & derivados , Toxina da Cólera/toxicidade , Humanos , Linfócitos T/metabolismo , Timalfasina , Regulação para Cima
17.
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
18.
Neurosci Bull ; 33(6): 675-684, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28780644

RESUMO

In early life, the immune system plays an essential role in brain development. In our study, the immunopotentiator thymosin alpha-1 (Ta1) was peripherally administered to neonatal mice to explore whether the peripheral immunopotentiator affects neurodevelopment and cognition, and to further investigate the relevant mechanism. Compared with the control group, the Ta1 mice displayed better cognitive abilities in early life. The numbers of 5-bromodeoxyuridine (BrdU)+, nestin+, T-box transcription factor 2 (Tbr2)+, BrdU+/doublecortin (DCX)+, BrdU+/ionized calcium-binding adaptor molecule 1 (Iba1)+, and BrdU+/neuronal nuclei (NeuN)+ cells in the hippocampus were increased in the Ta1 group, accompanied by increased interleukin-4 (IL-4), interferon-gamma, brain-derived neurotrophic factor, nerve growth factor, and insulin-like growth factor-1 as well as decreased IL-6 and tumor necrosis factor-α. Furthermore, the Ta1-group showed a Th1-polarized immune response, and the neurotrophic factors were positively associated with the Th1/Th2 ratio. More importantly, administration of Ta1 blocked lipopolysaccharide-induced impairment of hippocampal neurogenesis in early life. These findings suggest that peripheral Ta1 contributes to neurogenesis and cognition probably through a systemic Th1 bias, as well as neuroprotection against LPS infection by Ta1.


Assuntos
Adjuvantes Imunológicos/farmacologia , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Citocinas/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurogênese/efeitos dos fármacos , Timosina/análogos & derivados , Adjuvantes Imunológicos/administração & dosagem , Animais , Animais Recém-Nascidos , Citocinas/sangue , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/sangue , Timalfasina , Timosina/administração & dosagem , Timosina/farmacologia
19.
Zhonghua Yi Xue Za Zhi ; 97(25): 1942-1946, 2017 Jul 04.
Artigo em Chinês | MEDLINE | ID: mdl-28693071

RESUMO

Objective: To investigate the effect of transcatheter arterial chemoembolization(TACE)combined with thymosin alpha1(Tα1)on the autophagy of immune cells from advanced hepatocellular carcinoma. Methods: A total of 30 patients with advanced liver cancer enrolled in Lishui Central Hospital from September 2015 to June 2016 were collected in this study. The average age of patients was 16-75(56±12) years. All patients were treated with TACE after enrolled in hospital in a week. Patients were divided into TACE group and TACE+ Tα1 treatment group(15 cases in each group). Patients in TACE group received a conventional treatment, without any immunotherapy, while the TACE+ Tα1 treatment group accepted TACE following a subcutaneously injection of 1.6 mg Tα1 twice a week for 4 weeks. Flow cytometry was used to detect the T cell subsets in two groups both before and after TACE treatment for 1, 4 weeks and at 3 months follow-up. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. The expression of Beclin-1, LC3 protein and mRNA were detected by Western blot (WB) and PCR respectively. Results: There was no statistical difference of the percentage of CD3(+) , CD4(+) , CD8(+) T cell subsets and Beclin-1, LC3 protein and mRNA expression between the two groups before TACE treatment (P>0.05). The percentage of CD3(+) , CD4(+) , CD8(+) T cell subsets in TACE+ Tα1 group at 1 week post-TACE treatment (58.45%±16.34%, 38.33%±15.16%, 27.31%±12.54%), at 4 weeks post-TACE treatment (62.38%±18.62%, 43.19%±13.86%, 29.54%±10.33%) and 3 months follow-up (64.15%±13.76%, 41.28%±14.65%, 29.38%±15.65%) were statistically higher than those in TACE group at 1 week post-TACE treatment (53.71%±11.17%, 32.12%±10.53%, 24.45%±13.72%) at 4 weeks post-TACE treatment (52.12%±14.26%, 31.16%±15.43%, 23.39%±15.33%) and 3 months follow-up (54.28%±13.15%, 32.17%±14.98%, 24.34%±14.12%) (P<0.05). The Beclin-1, LC3 protein and mRNA expression in TACE+ Tα1 group at 1 week post-TACE treatment (protein: 0.57±0.08, 2.26±0.36, mRNA: 0.62±0.11, 2.69±0.27), at 4 weeks post-TACE treatment (protein: 0.66±0.09, 3.11±0.45, mRNA: 0.78±0.13, 3.43±0.61) were higher than those in TACE group at 1 week post-TACE treatment (protein: 0.45±0.16, 1.43±0.30, mRNA: 0.52±0.15, 1.15±0.37), at 4 weeks post-TACE treatment (protein: 0.51±0.13, 1.81±0.35, mRNA: 0.56±0.10, 1.98±0.41) ( P<0.05). But there was no statistically significant difference in the expression of Beclin-1 and LC3 in two groups at 3 months follow-up (P>0.05). Conclusions: TACE combined with Tα1 significantly increase the level of autophagy in the immune cells of patients with advanced primary hepatocellular carcinoma.


Assuntos
Autofagia , Carcinoma Hepatocelular/terapia , Quimioembolização Terapêutica , Neoplasias Hepáticas/terapia , Timosina/análogos & derivados , Adolescente , Adulto , Idoso , Terapia Combinada , Feminino , Humanos , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , Timalfasina , Timosina/uso terapêutico , Adulto Jovem
20.
Int Immunopharmacol ; 50: 279-282, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28719851

RESUMO

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that its binding to T and B lymphocytes from the blood of healthy donors was high-affinity (Kd 2.8 and 3.0nM, 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 CT-B receptor on donor blood T and B lymphocytes. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the soluble guanylate cyclase activity in T and B lymphocytes.


Assuntos
Linfócitos B/metabolismo , Células Sanguíneas/metabolismo , Toxina da Cólera/metabolismo , Guanilato Ciclase/metabolismo , Linfócitos T/metabolismo , Linfócitos B/imunologia , Células Sanguíneas/imunologia , Células Cultivadas , Toxina da Cólera/imunologia , Ativação Enzimática , Humanos , Interferon-alfa/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Linfócitos T/imunologia , Timalfasina , Timosina/análogos & derivados , Timosina/metabolismo
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