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1.
Biosci Trends ; 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32173687

RESUMO

4-anilinoquinazoline-containing inhibitors of the epidermal growth factor receptor (EGFR) are widely used in non-small cell lung cancer patients with mutated EGFR, but they are less effective in multiple myeloma (MM), a fatal malignancy derived from plasma cells. The present study designed a series of novel compounds by conjugating a peroxide bridge to the 4-anilinoquinazoline pharmacophore. Further studies showed that these agents such as 4061 and 4065B displayed potent activity to induce MM cell apoptosis by upregulating pro-apoptotic p53 and Bax while downregulating pro-survival Bcl-2. The mechanistic analysis revealed that both 4061 and 4065B inhibited IGF1-R, AKT and mTOR activation in a concentration dependent manner but had no effects on the expression of their total proteins, suggesting the conjugates of endoperoxide and 4-anilinoquinazoline may exert its anti-myeloma activity by targeting the IGF1-R/AKT/mTOR pathway.

2.
J Comput Chem ; 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31970817

RESUMO

Metal azides have attracted increasing attention as precursors for synthesizing polymeric nitrogen. In this article, we report the amorphous polymerization of nitrogen by compressing cupric azide. The ab initio molecular dynamics simulations show that crystalline cupric azide transforms into a disordered network composed of singly bonded nitrogen at a hydrostatic pressure of 40 GPa and room temperature. The transformation manifests the formation of a π delocalization along the disordered Cu-N network, thus resulting in a semiconductor-metal transition. The estimated heat of formation of the amorphous polymeric nitrogen system is comparable to conventional high-energy-density materials. The amorphization provides an alternative route to the polymerization of nitrogen under moderate conditions.

3.
Acta Pharmacol Sin ; 41(3): 394-403, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31645658

RESUMO

RNF6, a RING-type ubiquitin ligase, has been identified as an oncogene in various cancers but its role in multiple myeloma (MM) remains elusive. In the present study we first showed that the expression levels of RNF6 in MM were significantly elevated compared with the bone marrow cells of healthy donors. Overexpression of RNF6 in LP1 and PRMI-8266 MM cell lines promoted cell proliferation, whereas knockdown of RNF6 led to apoptosis of MM cells. Furthermore, we revealed that RNF6, as a ubiquitin ligase, interacted with glucocorticoid receptor (GR) and induced its K63-linked polyubiquitination. Different from current knowledge, RNF6 increased GR stability at both endogenous and exogenous contexts. Such an action greatly promoted GR transcriptional activity, which was confirmed by luciferase assays and by the increased expression levels of prosurvival genes including Bcl-xL and Mcl-1, two typical downstream genes of the GR pathway. Consistent with these findings, ectopic expression of RNF6 in MM cells conferred resistance to dexamethasone, a typical anti-myeloma agent. In conclusion, we demonstrate that RNF6 promotes MM cell proliferation and survival by inducing atypical polyubiquitination to GR, and RNF6 could be a promising therapeutic target for the treatment of MM.

4.
J Biol Chem ; 295(7): 2084-2096, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31822558

RESUMO

The Maf proteins, including c-Maf, MafA, and MafB, are critical transcription factors in myelomagenesis. Previous studies demonstrated that Maf proteins are processed by the ubiquitin-proteasome pathway, but the mechanisms remain elusive. This study applied MS to identify MafB ubiquitination-associated proteins and found that the ubiquitin-specific protease USP7 was present in the MafB interactome. Moreover, USP7 also interacted with c-Maf and MafA and blocked their polyubiquitination and degradation. Consistently, knockdown of USP7 resulted in Maf protein degradation along with increased polyubiquitination levels. The action of USP7 thus promoted Maf transcriptional activity as evidenced by luciferase assays and by the up-regulation of the expression of Maf-modulated genes. Furthermore, USP7 was up-regulated in myeloma cells, and it was negatively associated with the survival of myeloma patients. USP7 promoted myeloma cell survival, and when it was inhibited by its specific inhibitor P5091, myeloma cell lines underwent apoptosis. These results therefore demonstrated that USP7 is a deubiquitinase of Maf proteins and promotes MM cell survival in association with Maf stability. Given the significance of USP7 and Maf proteins in myeloma genesis, targeting the USP7/Maf axle is a potential strategy to the precision therapy of MM.

5.
Phys Chem Chem Phys ; 21(35): 19414-19422, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31460524

RESUMO

Self-aggregation of water vapour molecules under external electric fields is systemically investigated by using molecular dynamics simulations. It is found that small water clusters aggregate into one-dimensional water nanowires along the electric field direction. The electric field strength plays a crucial role in tuning the nanowire structure. Under relatively weak electric fields such as E = 0.1 V Å-1, square and pentagonal prism-like structures are preferred; when intermediate strength electric fields are applied (E = 1.0 V Å-1), water nanowires featuring a disordered mixture of four-, five- and six-membered rings are formed; and an open ordered structure which is reminiscent of two-dimensional (2D) ice is observed when the field strength becomes very high (E > 3.0 V Å-1). Bond parameter analysis based on density-functional theory calculations shows that the electric field affects anisotropically the conformation of water molecules as well as the hydrogen-bond properties. Along the electric field, the H-O bond is stretched and the hydrogen bond shrinks with field strength in contrast to the changes perpendicular to the electric field. As a result, the hydrogen bonding is enhanced along the electric field. Under very high electric fields, the anisotropic hydrogen-bond network opens up via breaking of the bonds perpendicular to the electric field and ultimately relaxes into a loose quasi-2D ordered network.

6.
Protein Sci ; 28(9): 1640-1651, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299125

RESUMO

Collagen fibrils represent a unique case of protein folding and self-association. We have recently successfully developed triple-helical peptides that can further self-assemble into collagen-mimetic mini-fibrils. The 35 nm axially repeating structure of the mini-fibrils, which is designated the d-period, is highly reminiscent of the well-known 67 nm D-period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo-identical repeating sequence units in the primary structure of the designed peptides that give rise to the d-period of the quaternary structure of the mini-fibrils. In this work, we characterize the self-assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple-helix domain of Col877 consists of three pseudo-identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self-associate laterally under fibril forming conditions to form mini-fibrils having the predicted d-period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self-assembly of collagen mini-fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen-mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.

7.
Acta Pharmacol Sin ; 40(12): 1568-1577, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31197245

RESUMO

c-Maf is a critical oncogenic transcription factor that contributes to myelomagenesis. Our previous studies demonstrated that the deubiquitinase USP5 stabilizes c-Maf and promotes myeloma cell proliferation and survival; therefore, the USP5/c-Maf axis could be a potential target for myeloma therapy. As a concept of principle, the present study established a USP5/c-Maf-based luciferase system that was used to screen an FDA-approved drug library. It was found that mebendazole, a typical anthelmintic drug, preferentially induced apoptosis in c-Maf-expressing myeloma cells. Moreover, oral administration of mebendazole delayed the growth of human myeloma xenografts in nude mice but did not show overt toxicity. Further studies showed that the selective antimyeloma activity of mebendazole was associated with the inhibition of the USP5/c-Maf axis. Mebendazole downregulated USP5 expression and disrupted the interaction between USP5 and c-Maf, thus leading to increased levels of c-Maf ubiquitination and subsequent c-Maf degradation. Mebendazole inhibited c-Maf transcriptional activity, as confirmed by both luciferase assays and expression measurements of c-Maf downstream genes. In summary, this study identified mebendazole as a USP5/c-Maf inhibitor that could be developed as a novel antimyeloma agent.

8.
Spectrochim Acta A Mol Biomol Spectrosc ; 217: 215-222, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30939368

RESUMO

The interaction between plant protein and polyphenol is a topic of considerable interest. However, there is relatively little understanding about the interaction between rice protein and epigallocatechin-3-gallate (EGCG). The spectroscopy and computational docking program were used to investigate the potential interaction between rice glutelin (RG) and EGCG. It was found that the intrinsic fluorescence of RG could be quenched by EGCG, which indicated interaction occurred between them. Thermodynamic analysis elucidated that the interaction process between RG and EGCG happened spontaneously with hydrogen bond as the primary driving force. The ANS-fluorescence indicated that the surface hydrophobicity of RG reduced with the increasing of EGCG. Circular dichroism spectra and synchronous fluorescence gave further information for the conformational and microenvironmental changes of RG. Particularly, the α-helix structure reduced and random coil structure increased after the binding interaction. Furthermore, the computational docking program exhibited target sites in which the amino acid residues of RG and EGCG might be bound together.


Assuntos
Catequina/análogos & derivados , Glutens/metabolismo , Simulação de Acoplamento Molecular , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Catequina/química , Catequina/metabolismo , Glutens/química , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Plantas/química , Ligação Proteica , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Termodinâmica
9.
Cell Biol Int ; 43(3): 279-289, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30632646

RESUMO

Long-standing untreated hyperuricemia could lead to gout. Several recent studies have demonstrated a significant decrease of serum urate during acute gout attack, which is an aseptic inflammation process focusing on IL-1ß. However, how IL-1ß, by itself, alters the expression and the functional activity of urate transporters in renal tubular epithelial cells is still unclear. Herein, we revealed that IL-1ß could attenuate the mRNA and protein levels of ABCG2, a major urate efflux pump, in HK-2 cells by real-time PCR and Western-blot assays. Moreover, using an ABCG2 specific inhibitor and a new sensitive and specific detection system, it was found that IL-1ß also reduced the ABCG2 transporter activities. Incubation with specific inhibitors of the NF-κB pathway partly dampened the inhibitory effect of IL-1ß on ABCG2, indicating that IL-1ß reduced the ABCG2 expression partially through the NF-ĸB pathway. Furthermore, the decreased expression of PDZK1 induced by IL-1ß, which is dependent on the NF-κB pathway, could account for the imbalance between the functions and expressions of ABCG2 on this status. These findings demonstrated a new role for IL-1ß, whereby it leads to the inhibition of ABCG2 in renal tubular epithelial cells; this new role probably does not encompass its involvement in the process of renal urate excretion mediated by inflammation. Therefore, other regulation mechanisms of urate reabsorption in renal tubular epithelial cells deserve to be examined in further studies.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/metabolismo , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Bases , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Humanos , Proteínas de Membrana , Proteínas de Neoplasias/genética , Nitrilos/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Prolina/análogos & derivados , Prolina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfonas/farmacologia , Tiocarbamatos/farmacologia , Fatores de Tempo , Ácido Úrico/metabolismo
10.
Biopolymers ; 109(7): e23226, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30133697

RESUMO

It has proven challenging to obtain collagen-mimetic fibrils by protein design. We recently reported the self-assembly of a mini-fibril showing a 35 nm, D-period like, axially repeating structure using the designed triple helix Col108. Peptide Col108 was made by bacterial expression using a synthetic gene; its triple helix domain consists of three pseudo-identical units of amino acid sequence arranged in tandem. It was postulated that the 35 nm d-period of Col108 mini-fibrils originates from the periodicity of the Col108 primary structure. A mutual staggering of one sequence unit of the associating Col108 triple helices can maximize the inter-helical interactions and produce the observed 35 nm d-period. Based on this unit-staggered model, a triple helix consisting of only two sequence units is expected to have the potential to form the same d-periodic mini-fibrils. Indeed, when such a peptide, peptide 2U108, was made it was found to self-assemble into mini-fibrils having the same d-period of 35 nm. In contrast, no d-periodic mini-fibrils were observed for peptide 1U108, which does not have long-range repeating sequences in its primary structure. The findings of the periodic mini-fibrils of Col108 and 2U108 suggest a way forward to create collagen-mimetic fibrils for biomedical and industrial applications.


Assuntos
Biomimética/métodos , Colágeno/química , Peptídeos/química
11.
Anatol J Cardiol ; 19(5): 296-302, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29724983

RESUMO

OBJECTIVE: To investigate the effects and mechanisms of catalpol on cardiac function in rats with isoproterenol (ISO)-induced myocardial infarction (MI). METHODS: Adult male Wistar rats were divided into four groups: control group, ISO group, catalpol (L, low dose) group, and catalpol (H, high dose) group. Isoproterenol (85 mg/kg) was injected subcutaneously for 2 consecutive days to induce experimental MI. At the end of experiment, the effects of catalpol on cardiac function; apelin levels; apoptosis index; apelin, APJ, Bcl-2, and Bax protein expression; and caspase-3/9 activities were investigated. RESULTS: The rats in the ISO group showed lower left ventricular maximum rate of positive or negative pressure development (±LVdp/dtmax) and left ventricular end-systolic pressure (LVSP) and higher left ventricular end-diastolic pressure (LVEDP) than those in the control group, suggesting severe cardiac dysfunction. Interestingly, catalpol administration significantly ameliorated the ISO-induced cardiac dysfunction. The groups administered low and high dosages catalpol (5 and 10 mg/kg/day, respectively) showed higher ±LVdp/dtmax and LVSP and lower LVEDP than the group administered ISO alone. Catalpol markedly upregulated apelin levels in the plasma and myocardium. Further, catalpol increased the apelin and APJ expression levels in the myocardium of the ISO-treated rats. In addition, catalpol pretreatment inhibited cardiomyocyte apoptosis as indicated by a decrease in the TUNEL-positive cell percentage, alterations in the Bax and Bcl-2 expression levels, and a decline in caspase-3 and caspase-9 activities. CONCLUSION: Our results revealed that catalpol can improve cardiac function. Its protective effects may be linked to the enhancement of myocardium contractility, regulation of the apelin/APJ pathway, and inhibition of cardiomyocyte apoptosis.


Assuntos
Cardiotônicos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Glucosídeos Iridoides/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Cardiotônicos/farmacologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Glucosídeos Iridoides/farmacologia , Isoproterenol , Masculino , Ratos , Ratos Wistar
12.
J Biosci Bioeng ; 126(1): 88-95, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29534943

RESUMO

In this study, the nitrogen removal performance and microbial community structure were investigated during the start-up, instability, and recovery stages of an anaerobic ammonium oxidation (anammox) reactor loaded with compound carriers (shale ceramsite and suspended ball carrier). The results indicated that the anammox reactor successfully started up on 116th d when the nitrogen loading rate (NLR) reached 0.72 ± 0.05 kg N m-3 d-1. The anammox reactor ran well with free ammonia (FA) at 13.65 ± 2.69 mg/L and free nitrous acid (FNA) at 39.49 ± 10.95 µg/L, indicating that its tolerance for FA and FNA was higher than that of granular sludge anammox reactors. The anammox system was inhibited when FA and FNA reached 29.65 mg/L and 77.02 µg/L, respectively. The tolerance of anammox bacteria towards FA and FNA decreased after this inhibition. The nitrogen removal performance could be efficiently recovered by decreasing the influent substrate concentration and increasing the hydraulic retention time (HRT). Candidatus Brocadia and Candidatus Jettenia, two genus-level anammox bacteria, were detected in this reactor using a high-throughput sequencing technique. After high substrate shock, the abundance of Candidatus Brocadia decreased while that of Candidatus Jettenia increased, which might be due to the competition between Candidatus Jettenia and Candidatus Brocadia. The relationships between anammox communities and operational factors were investigated via redundancy analysis (RDA), which showed that FA was the principal factor affecting the microbial community structure during the operation stage.


Assuntos
Amônia/química , Reatores Biológicos/microbiologia , Biota/fisiologia , Nitrogênio/isolamento & purificação , Amônia/isolamento & purificação , Amônia/farmacocinética , Anaerobiose , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Desnitrificação/fisiologia , Nitrogênio/química , Nitrogênio/farmacocinética , Oxirredução , Esgotos/microbiologia , Fatores de Tempo
13.
J Biol Chem ; 293(16): 5847-5859, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29467225

RESUMO

TMEPAI (transmembrane prostate androgen-induced protein, also called prostate transmembrane protein, androgen-induced 1 (PMEPA1)) is a type I transmembrane (TM) protein, but its cellular function is largely unknown. Here, studying factors influencing the stability of c-Maf, a critical transcription factor in multiple myeloma (MM), we found that TMEPAI induced c-Maf degradation. We observed that TMEPAI recruited NEDD4 (neural precursor cell expressed, developmentally down-regulated 4), a WW domain-containing ubiquitin ligase, to c-Maf, leading to its degradation through the proteasomal pathway. Further investigation revealed that TMEPAI interacts with NEDD4 via its conserved PY motifs. Alanine substitution or deletion of these motifs abrogated the TMEPAI complex formation with NEDD4, resulting in failed c-Maf degradation. Functionally, TMEPAI suppressed the transcriptional activity of c-Maf. Of note, increased TMEPAI expression was positively associated with the overall survival of MM patients. Moreover, TMEPAI was down-regulated in MM cells, and re-expression of TMEPAI induced MM cell apoptosis. In conclusion, this study highlights that TMEPAI decreases c-Maf stability by recruiting the ubiquitin ligase NEDD4 to c-Maf for proteasomal degradation. Our findings suggest that the restoration of functional TMEPA1 expression may represent a promising complementary therapeutic strategy for treating patients with MM.


Assuntos
Apoptose , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-maf/metabolismo , Humanos , Mieloma Múltiplo/patologia , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Mapas de Interação de Proteínas , Ubiquitinação
14.
Cell Death Dis ; 8(9): e3058, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28933784

RESUMO

The deubiquitinase USP5 stabilizes c-Maf, a key transcription factor in multiple myeloma (MM), but the mechanisms and significance are unclear. In the present study, USP5 was found to interact with c-Maf and prevented it from degradation by decreasing its polyubiquitination level. Specifically, the 308th and 347th lysine residues in c-Maf were critical for USP5-mediated deubiquitination and stability. There are five key domains in the USP5 protein and subsequent studies revealed that the cryptic ZnF domain and the C-box domain interacted with c-Maf but the UBA1/UBA2 domain partly increased its stability. Notably, MafA and MafB are also members of the c-Maf family, however, USP5 failed to deubiquitinate MafA, suggesting its substrate specificity. In the functional studies, USP5 was found to promoted the transcriptional activity of c-Maf. Consistent with the high level of c-Maf protein in MM cells, USP5 was also highly expressed. When USP5 was knocked down, c-Maf underwent degradation. Interestingly, USP5 silence led to apoptosis of MM cells expressing c-Maf but not MM cells lacking c-Maf, indicating c-Maf is a key factor in USP5-mediated MM cell proliferation and survival. Consistent with this finding, WP1130, an inhibitor of several Dubs including USP5, suppressed the transcriptional activity of c-Maf and induced MM cell apoptosis. When c-Maf was overexpressed, WP1130-induced MM cell apoptosis was abolished. Taken together, these findings suggest that USP5 regulates c-Maf stability and MM cell survival. Targeting the USP5/c-Maf axis could be a potential strategy for MM treatment.


Assuntos
Apoptose , Endopeptidases/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteólise , Proteínas Proto-Oncogênicas c-maf/metabolismo , Apoptose/genética , Endopeptidases/química , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lisina/metabolismo , Terapia de Alvo Molecular , Mieloma Múltiplo/genética , Poliubiquitina/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Relação Estrutura-Atividade , Transcrição Genética , Ubiquitinação
15.
J Pharmacol Sci ; 134(4): 197-202, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28779993

RESUMO

S14161 is a pan-Class I PI3K inhibitor that induces blood cancer cell death, but its mechanism is largely unknown. In the present study, we evaluated the role of S14161 in autophagy, an emerging event in cell destination. Multiple myeloma cell lines RPMI-8226, OPM2, KMS11 and leukemia cell line K562 were treated with S14161. The results showed that S14161 induced autophagy as demonstrated by increased LC3-II and decreased p62, which were prevented by autophagy inhibitors including 3-methyladenine and bafilomycin A1. Mechanistic studies showed that S14161 had no effects on Vps34 expression, but increased Beclin 1 and decreased Bcl-2, two major regulators of autophagy. Furthermore, S14161 dissociated the Beclin 1/Bcl-2 complex and enhanced the formation of Beclin 1/Vps34 complex. Moreover, S14161 inhibited the mTORC1 signaling transduction. S14161 downregulated activation of mTOR and its two critical targets 4E-BP1 and p70S6K, suggesting S14161 inhibited protein synthesis. Taken together, these results demonstrated that Class I PI3K regulates autophagy by modulating protein synthesis and the Beclin 1 signaling pathway. This finding helps understanding the roles of PI3K in autophagy and cancer treatment.


Assuntos
Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Benzopiranos/farmacologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Hematológicas/patologia , Leucemia/patologia , Mieloma Múltiplo/patologia , Proteína Beclina-1/genética , Linhagem Celular Tumoral , Classe III de Fosfatidilinositol 3-Quinases/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
J Hematol Oncol ; 10(1): 132, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28673317

RESUMO

BACKGROUND: UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown. METHODS: Mass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively. RESULTS: UBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway. CONCLUSIONS: UBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.


Assuntos
Apoptose , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Humanos , Camundongos Nus , Mieloma Múltiplo/patologia , Mapas de Interação de Proteínas , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-maf/análise , Enzimas de Conjugação de Ubiquitina/análise
17.
Acta Pharmacol Sin ; 38(5): 651-659, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28260800

RESUMO

The signal transducer and activator of transcription 3 (STAT3) plays a critical role in platelet functions. This study sought to understand the effects of the STAT3 inhibitor SC99 on platelet activation and aggregation. Immunoblotting assays were applied to measure the effects of SC99 on the STAT3 signaling pathway. A ChronoLog aggregometer was used to evaluate platelet aggregation. A flow cytometer was used to evaluate P-selectin expression in the presence of SC99. AlamarBlue and Annexin-V staining were used to evaluate platelet viability and apoptosis, respectively. A fluorescence microscope was applied to analyze platelet spreading. SC99 inhibited the phosphorylation of JAK2 and STAT3 in human platelets but had no effects on the phosphorylation of AKT, p65 or Src, all of which are involved in platelet activation. Further studies revealed that SC99 inhibited human platelet aggregation induced by collagen and thrombin in a dose-dependent manner. SC99 inhibited thrombin-induced P-selectin expression and fibrinogen binding to single platelets. Moreover, SC99 inhibited platelet spreading on fibrinogen and clot retraction mediated by outside-in signaling. SC99 inhibited platelet aggregation in mice but it did not significantly prolong the bleeding time. Taken together, the present study revealed that SC99 inhibited platelet activation and aggregation as a STAT3 inhibitor. This agent can be developed as a promising treatment for thrombotic disorders.


Assuntos
Hidrazonas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Tempo de Sangramento , Retração do Coágulo/efeitos dos fármacos , Humanos , Hidrazonas/toxicidade , Camundongos Endogâmicos C57BL , Inibidores da Agregação de Plaquetas/toxicidade , Transdução de Sinais
18.
Oncotarget ; 7(8): 9296-308, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26814430

RESUMO

The oncogenic STAT3 signaling pathway is emerging as a promising target for the treatment of multiple myeloma (MM). In the present study, we identified a novel STAT3 inhibitor SC99 in a target-based high throughput screen. SC99 inhibited JAK2-STAT3 activation but had no effects on other transcription factors such as NF-κB, and kinases such as AKT, ERK, and c-Src that are in association with STAT3 signaling pathway. Furthermore, SC99 downregulated the expression of STAT3-modulated genes, including Bcl-2, Bcl-xL, VEGF, cyclin D2, and E2F-1. By inhibiting the STAT3 signaling, SC99 induced MM cell apoptosis which could be partly abolished by the ectopic expression of STAT3. Furthermore, SC99 displayed potent anti-MM activity in two independent MM xenograft models in nude mice. Oral administration of SC99 led to marked decrease of tumor growth within 10 days at a daily dosage of 30 mg/kg, but did not raise toxic effects. Taken together, this study identified a novel oral JAK2/STAT3 inhibitor that could be developed as an anti-myeloma agent.


Assuntos
Antineoplásicos/uso terapêutico , Hidrazonas/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina D2/biossíntese , Fator de Transcrição E2F1/biossíntese , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Nus , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/biossíntese
19.
Food Chem ; 199: 387-92, 2016 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-26775986

RESUMO

ß-Lactoglobulin (ß-LG) was conjugated with monomethoxy polyethylene glycol-succinimidyl carbonates (mPEG-SC, 20 kDa) to investigate the relationship between the antigenicity and conformational changes of ß-LG. The effect of molar ratio of protein to mPEG-SC (1:3-1:6), pH (6-8) and time (4-24h) on the antigenicity of ß-LG was investigated. The lowest antigenicity of ß-LG was observed at the molar ratio of 1:3, pH 7.0, and reaction time for 8h, which was 70% lower than that of control ß-LG. At the optimal modification conditions, it was indicated that two fractions obtained after purification showed the tense and single band on the SDS-PAGE at the position of approximate 78 kDa and 58 kDa, which corresponded to the tri- and di-PEGylated conjugate, respectively. As conjugated number of mPEG-SC with ß-LG increased, the quenching of fluorescence and the content of ß-strands were increased gradually, which may contribute to the decrease of antigenicity from two aspects.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Lactoglobulinas/química , Polietilenoglicóis/química , Animais , Bovinos
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