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1.
Br J Cancer ; 128(8): 1491-1502, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36759727

RESUMO

BACKGROUND: Chaperon-mediated autophagy (CMA) has taken on a new emphasis in cancer biology. However, the roles of CMA in hypoxic tumours are poorly understood. We investigated the anti-tumour effects of the natural product ManA through the activation of CMA in tumour progression under hypoxia. METHODS: The effect of ManA on CMA activation was assessed in mouse xenograft models and cells. The gene expressions of HIF-1α, HSP90AA1, and transcription factor EB (TFEB) were analysed using The Cancer Genome Atlas (TCGA) datasets to assess the clinical relevance of CMA. RESULTS: ManA activates photoswitchable CMA reporter activity and inhibits Hsp90 chaperone function by disrupting the Hsp90/F1F0-ATP synthase complex. Hsp90 inhibition enhances the interaction between CMA substrates and LAMP-2A and TFEB nuclear localisation, suggesting CMA activation by ManA. ManA-activated CMA retards tumour growth and displays cooperative anti-tumour activity with anti-PD-1 antibody. TCGA datasets show that a combined expression of HSP90AA1High/HIF1AHigh or TFEBLow/HIF1AHigh is strongly correlated with poor prognosis in patients with lung cancer. CONCLUSIONS: ManA-induced CMA activation by modulating Hsp90 under hypoxia induces HIF-1α degradation and reduces tumour growth. Thus, inducing CMA activity by targeting Hsp90 may be a promising therapeutic strategy against hypoxic tumours.


Assuntos
Autofagia Mediada por Chaperonas , Neoplasias Pulmonares , Camundongos , Animais , Humanos , Hipóxia , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares , Autofagia/genética
2.
Biochim Biophys Acta ; 1854(5): 449-59, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25707357

RESUMO

The Hox DNA binding domain, the homeodomain, plays critical roles in genetic control of development and cell fate determination. The variable regulatory functions of Hox proteins are accomplished by binding to target DNA sequences and collaborating protein partners that includes human high mobility group B1 (HMGB1). To better understand the interaction between Hox and HMGB1 and the facilitation of Hox-DNA binding by HMGB1, we solved the solution structure of the homeodomain of Hox including the N-terminal arm region (Hoxc9DBD hereafter). In addition, the details of the interaction between these two proteins, as well as DNA binding of the Hox-HMGB1 complex, were investigated by NMR, ITC, and EMSA. The results suggest that binding of the HMGB1 A-box to Hoxc9DBD makes the loop-1 (loop preceding helix-2 of Hoxc9DBD) more access to DNA backbone, which facilitate Hox-DNA binding with enhanced affinity.


Assuntos
DNA/metabolismo , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína
3.
J Dent Educ ; 87(2): 198-207, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36176031

RESUMO

PURPOSE/OBJECTIVES: This study aimed to evaluate a nonface-to-face crown designing module in a preclinical dental course. METHODS: Free dental planning software (Blue Sky Plan) was installed on the personal computers of dental college students, and a #46 full veneer crown designing practice was performed individually. An online survey was conducted on the computers' specification and main usage of the students, the practice process, and results. Statistical analysis was conducted to analyze the association between variables, such as "operating system," "central processing unit ," "number of cores," "random-access memory (RAM)," "graphic card," and task performance. RESULTS: Of the D2 students, 75.4% (52 of 69) responded to the survey. Overall, 96% of the respondents used their computers, and all respondents had no problem running the program. Most of the students marked their level of computer literacy as intermediate and had purchased the computers for the purpose of performing light work. The most common specifications of the computer were Intel i5, quad core, 8 GB RAM, and Windows 10. Students had little experience with computer-aided design/computer-aided manufacturing before the class. The relationship between computer specifications and task performance was not statistically significant. CONCLUSIONS: Overall, students with intermediate-level computer literacy used computers with less than the recommended specifications of the program; however, they were able to run the program and individually proceed with modules to submit results. Using an individually available crown designing program can provide an opportunity to diversify curricula and broaden students' perspectives even under circumstances like the COVID-19 pandemic that limits intimate face-to face classes.


Assuntos
COVID-19 , Pandemias , Humanos , Prostodontia/educação , Estudantes de Odontologia , Desenho Assistido por Computador
4.
Blood Adv ; 7(1): 92-105, 2023 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-36269842

RESUMO

Bruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade. BCR activation can contribute to the growth and survival of B-cell lymphoma or leukemia. The inhibition of the BCR signaling pathway is critical for blocking downstream events and treating B-cell lymphomas. Herein, we report potent and orally available proteolysis-targeting chimeras (PROTACs) that target BTK to inactivate BCR signaling. Of the PROTACs tested, UBX-382 showed superior degradation activity for wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration in diffuse large B-cell lymphoma cell line. UBX-382 was effective on 7 out of 8 known BTK mutants in in vitro experiments and was highly effective in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells over ibrutinib, ARQ-531, and MT-802. Remarkably, oral dosing of UBX-382 for <2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. UBX-382 also provoked the cell type-dependent and selective degradation of cereblon neosubstrates in various hematological cancer cells. These results suggest that UBX-382 treatment is a promising therapeutic strategy for B-cell-related blood cancers with improved efficacy and diverse applicability.


Assuntos
Linfoma Difuso de Grandes Células B , Pirimidinas , Humanos , Animais , Camundongos , Tirosina Quinase da Agamaglobulinemia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais , Modelos Animais de Doenças , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética
5.
Sci Rep ; 9(1): 1077, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30705347

RESUMO

Guillain-Barré syndrome (GBS) is an acute fatal progressive disease caused by autoimmune mechanism mainly affecting peripheral nervous system. Although the syndrome is clinically sub-classified into several variants, specific biomarker and exact pathomechanism of each subtypes are not well elucidated yet. In current study, integrative metabolomic and lipidomic profiles were acquisitioned from cerebrospinal fluid samples of 86 GBS from three variants and 20 disease controls. And the data were systematically compared to our previous result on inflammatory demyelination disorders of central nervous system (IDDs) and healthy controls. Primary metabolite profiles revealed unique metabolic traits in which 9 and 7 compounds were specifically changed in GBS and IDD, respectively. Next, the biomarker panel with 10 primary metabolites showed a fairly good discrimination power among 3 GBS subtypes, healthy controls, and disease controls (AUCs ranged 0.849-0.999). The robustness of the biomarker panel was vigorously validated by multi-step statistical evaluation. Subsequent lipidomics revealed GBS variant-specific alteration where the significant elevations of lyso-phosphatidylcholines and sphingomyelins were unique to AIDP (acute inflammatory demyelinating polyneuropathy) and AMAN (acute motor axonal neuropathy), respectively. And metabolome-wide multivariate correlation analysis identified potential clinical association between GBS disability scale (Hughes score) and CSF lipids (monoacylglycerols, and sphingomyelins). Finally, Bayesian network analysis of covarianced structures of primary metabolites and lipids proposed metabolic hub and potential biochemical linkage associated with the pathology.


Assuntos
Síndrome de Guillain-Barré/metabolismo , Metaboloma , Metabolômica , Adulto , Idoso , Biomarcadores/metabolismo , Feminino , Síndrome de Guillain-Barré/patologia , Humanos , Masculino , Pessoa de Meia-Idade
6.
Sci Rep ; 8(1): 368, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29321504

RESUMO

Heat shock protein 90 (Hsp90) is one of the most abundant cellular proteins and plays a substantial role in the folding of client proteins. The inhibition of Hsp90 has been regarded as an attractive therapeutic strategy for treating cancer because many oncogenic kinases are Hsp90 client proteins. In this study, we report new inhibitors that directly bind to N-terminal ATP-binding pocket of Hsp90. Optimized structure-based virtual screening predicted candidate molecules, which was followed by confirmation using biophysical and cell-based assays. Among the reported crystal structures, we chose the two structures that show the most favourable early enrichments of true-positives in the receiver operating characteristic curve. Four molecules showed significant changes in the signals of 2D [1H, 15N] correlation NMR spectroscopy. Differential scanning calorimetry analysis supported the results indicating direct binding. Quantified dissociation constant values of the molecules, determined by a series of 2D NMR experiments, lie in the range of 0.1-33 µM. Growth inhibition assay with breast and lung cancer cells confirmed the cellular activities of the molecules. Cheminformatics revealed that the molecules share limited chemical similarities with known inhibitors. Molecular dynamics simulations detailed the putative binding modes of the inhibitors.


Assuntos
Proteínas de Choque Térmico HSP90/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Algoritmos , Sítios de Ligação , Biologia Computacional/métodos , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Relação Estrutura-Atividade
7.
PLoS One ; 12(7): e0181758, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28746356

RESUMO

Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are inflammatory diseases of the central nervous system. Although several studies have characterized the metabolome in the cerebrospinal fluid (CSF) from MS and NMOSD patients, comparative analyses between them and between the relapse and the remission of each disease have not been performed. Both univariate and multivariate analyses were used to compare 1H-NMR spectra of CSF from MS, NMOSD, and healthy controls (HCs). The statistical analysis showed alterations of eight metabolites that were dependent on the disease. Levels of 2-hydroxybutyrate, acetone, formate, and pyroglutamate were higher and levels of acetate and glucose were lower in both MS and NMOSD. Citrate was lower in MS patients, whereas lactate was higher in only NMOSD specifically. The shared feature of metabolic changes between MS and NMOSD may be related to altered energy metabolism and fatty acid biosynthesis in the brain. Another analysis to characterize relapse and remission status showed that isoleucine and valine were down-regulated in MS relapse compared to MS remission. The other metabolites identified in the disease comparison showed the same alterations regardless of disease activity. These findings would be helpful in understanding the biological background of these diseases, and distinguishing between MS and NMOSD, as well as determining the disease activity.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metaboloma , Metabolômica/métodos , Esclerose Múltipla/líquido cefalorraquidiano , Neuromielite Óptica/líquido cefalorraquidiano , Acetatos/líquido cefalorraquidiano , Acetona/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Criança , Ácido Cítrico/líquido cefalorraquidiano , Feminino , Formiatos/líquido cefalorraquidiano , Glucose/líquido cefalorraquidiano , Humanos , Hidroxibutiratos/líquido cefalorraquidiano , Ácido Láctico/líquido cefalorraquidiano , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/metabolismo , Análise Multivariada , Neuromielite Óptica/diagnóstico por imagem , Neuromielite Óptica/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/métodos , Ácido Pirrolidonocarboxílico/líquido cefalorraquidiano , Adulto Jovem
8.
Biomol NMR Assign ; 6(1): 109-13, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21904986

RESUMO

Bub1 is an evolutionarily conserved mitotic checkpoint control protein that is present in diverse organisms including yeast and humans. Bub1 is a serine/threonine protein kinase and is required for recruitment of Mad1, Mad2, Bub3, and CENP-E to kinetochores (Sharp-Baker and Chen in J Cell Biol 153:1239-1250, 2001). The evolutionarily conserved amino acid region in the N-terminus has been called as the CD1 domain. To clarify the action mechanism of Bub1 in controlling check point signals, we initiated an NMR structure determination of the Bub1 CD1 domain. Here, we report the sequence-specific backbone resonance assignments of CD1 domain of human Bub1 (hBub1CD1).


Assuntos
Sequência Conservada , Ressonância Magnética Nuclear Biomolecular , Proteínas Serina-Treonina Quinases/química , Humanos , Peso Molecular , Estrutura Terciária de Proteína
9.
Neoplasia ; 13(2): 98-107, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21403836

RESUMO

Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.


Assuntos
Neoplasias da Mama/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Genes ras , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Invasividade Neoplásica , Prolina/genética , Prolina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Relação Estrutura-Atividade
10.
Arch Pharm Res ; 33(8): 1285-8, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20803133

RESUMO

SpoVG, originally identified in spore-forming Bacillus subtilis was known to be involved in spore formation of B. subtilis stationary phase cells at stage V. Later, close homologues of SpoVG of B. subtilis are shown to be present in the genomes of several nonsporulating bacteria as well. Especially in Staphylococcus aureus, SpoVG is speculated to be the major factor of the yabJ-spoVG operon required for capsule formation and methicillin and glycopeptides resistance. The putative SpoVG from S. aureus, a homodimeric protein consisting of two identical 100-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 293 K using a precipitant solution consisting of 1.9 M (NH(4))(2)SO(4), 100 mM Tris-HCl, pH 7.5. X-ray diffraction data were collected to 3.10 A at 100 K. The crystals belong to the primitive tetragonal space group P41 (or P4(3)), with unit cell parameters of a = b = 92.239, c = 98.588 A, alpha = beta = gamma = 90 degrees. Two dimers are present in the crystallographic asymmetric unit, with a calculated crystal volume per protein weight (V(M)) of 4.37 A(3)Da(-1) and a solvent content of 71.9%.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Staphylococcus aureus/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/metabolismo , Difração de Raios X
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