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1.
PLoS One ; 16(8): e0256238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34411141

RESUMO

S100B is frequently elevated in malignant melanoma. A regulatory mechanism was uncovered here in which elevated S100B lowers mRNA and secreted protein levels of interleukin-6 (IL6) and inhibits an autocrine loop whereby IL6 activates STAT3 signaling. Our results showed that S100B affects IL6 expression transcriptionally. S100B was shown to form a calcium-dependent protein complex with the p90 ribosomal S6 kinase (RSK), which in turn sequesters RSK into the cytoplasm. Consistently, S100B inhibition was found to restore phosphorylation of a nuclear located RSK substrate, CREB, which is a potent transcription factor for IL6 expression. Thus, elevated S100B reduces IL6-STAT3 signaling via RSK signaling pathway in malignant melanoma. Indeed, the elevated S100B levels in malignant melanoma cell lines correspond to low levels of IL6 and p-STAT3.


Assuntos
Interleucina-6/genética , Melanoma/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Fator de Transcrição STAT3/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Citoplasma/genética , Doxiciclina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Transdução de Sinais/efeitos dos fármacos
2.
Biochemistry ; 56(17): 2328-2337, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28409622

RESUMO

Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.


Assuntos
Sinalização do Cálcio , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Histona Desacetilases/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas S100/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Ativação Enzimática , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas S100/genética
3.
Acta Crystallogr D Struct Biol ; 72(Pt 6): 753-60, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27303795

RESUMO

Structure-based drug discovery is under way to identify and develop small-molecule S100B inhibitors (SBiXs). Such inhibitors have therapeutic potential for treating malignant melanoma, since high levels of S100B downregulate wild-type p53 tumor suppressor function in this cancer. Computational and X-ray crystallographic studies of two S100B-SBiX complexes are described, and both compounds (apomorphine hydrochloride and ethidium bromide) occupy an area of the S100B hydrophobic cleft which is termed site 3. These data also reveal novel protein-inhibitor interactions which can be used in future drug-design studies to improve SBiX affinity and specificity. Of particular interest, apomorphine hydrochloride showed S100B-dependent killing in melanoma cell assays, although the efficacy exceeds its affinity for S100B and implicates possible off-target contributions. Because there are no structural data available for compounds occupying site 3 alone, these studies contribute towards the structure-based approach to targeting S100B by including interactions with residues in site 3 of S100B.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas S100/antagonistas & inibidores , Proteínas S100/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Melanoma/tratamento farmacológico , Simulação de Acoplamento Molecular , Proteínas S100/química
4.
J Med Chem ; 59(2): 592-608, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26727270

RESUMO

The drug pentamidine inhibits calcium-dependent complex formation with p53 ((Ca)S100B·p53) in malignant melanoma (MM) and restores p53 tumor suppressor activity in vivo. However, off-target effects associated with this drug were problematic in MM patients. Structure-activity relationship (SAR) studies were therefore completed here with 23 pentamidine analogues, and X-ray structures of (Ca)S100B·inhibitor complexes revealed that the C-terminus of S100B adopts two different conformations, with location of Phe87 and Phe88 being the distinguishing feature and termed the "FF-gate". For symmetric pentamidine analogues ((Ca)S100B·5a, (Ca)S100B·6b) a channel between sites 1 and 2 on S100B was occluded by residue Phe88, but for an asymmetric pentamidine analogue ((Ca)S100B·17), this same channel was open. The (Ca)S100B·17 structure illustrates, for the first time, a pentamidine analog capable of binding the "open" form of the "FF-gate" and provides a means to block all three "hot spots" on (Ca)S100B, which will impact next generation (Ca)S100B·p53 inhibitor design.


Assuntos
Subunidade beta da Proteína Ligante de Cálcio S100/antagonistas & inibidores , Subunidade beta da Proteína Ligante de Cálcio S100/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Bovinos , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Pentamidina/análogos & derivados , Pentamidina/química , Pentamidina/farmacologia , Conformação Proteica , Ratos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/efeitos dos fármacos
5.
Microvasc Res ; 97: 55-64, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25283348

RESUMO

Angiogenesis is regulated by hyperglycemic conditions, which can induce cellular stress responses, reactive oxygen species (ROS), and anti-oxidant defenses that modulate intracellular signaling to prevent oxidative damage. The RUNX2 DNA-binding transcription factor is activated by a glucose-mediated intracellular pathway, plays an important role in endothelial cell (EC) function and angiogenesis, and is a target of oxidative stress. RUNX2 DNA-binding and EC differentiation in response to glucose were conserved in ECs from different tissues and inhibited by hyperglycemia, which stimulated ROS production through the aldose reductase glucose-utilization pathway. Furthermore, the redox status of cysteine and methionine residues regulated RUNX2 DNA-binding and reversal of oxidative inhibition was consistent with an endogenous Methionine sulfoxide reductase-A (MsrA) activity. Low molecular weight MsrA substrates and sulfoxide scavengers were potent inhibitors of RUNX2 DNA binding in the absence of oxidative stress, but acted as antioxidants to increase DNA binding in the presence of oxidants. MsrA was associated with RUNX2:DNA complexes, as measured by a sensitive, quantitative DNA-binding ELISA. The related RUNX2 protein family member, RUNX1, which contains an identical DNA-binding domain, was a catalytic substrate of recombinant MsrA. These findings define novel redox pathways involving aldose reductase and MsrA that regulate RUNX2 transcription factor activity and biological function in ECs. Targeting of these pathways could result in more effective strategies to alleviate the vascular dysfunction associated with diabetes or cancer.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , DNA/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , Hiperglicemia/metabolismo , Neovascularização Patológica , Aldeído Redutase/metabolismo , Inibidores da Angiogênese/farmacologia , Antioxidantes/farmacologia , Sítios de Ligação , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Metionina Sulfóxido Redutases/metabolismo , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Fenótipo , Transdução de Sinais , Especificidade por Substrato , Fatores de Tempo
6.
Biochemistry ; 53(42): 6628-40, 2014 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25268459

RESUMO

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca(2+)-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNA(S100B)) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B-p53 complex and restore active p53 in this deadly cancer. Using a structure-activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1-3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX-S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity.


Assuntos
Cálcio/química , Subunidade beta da Proteína Ligante de Cálcio S100/antagonistas & inibidores , Subunidade beta da Proteína Ligante de Cálcio S100/química , Animais , Benzofenantridinas/química , Benzofenantridinas/farmacologia , Benzoquinonas/química , Benzoquinonas/farmacologia , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Dissulfiram/química , Dissulfiram/farmacologia , Diterpenos/química , Diterpenos/farmacologia , Humanos , Melanoma , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo
7.
J Biol Chem ; 289(18): 12886-95, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24627490

RESUMO

S100B is a prognostic marker for malignant melanoma. Increasing S100B levels are predictive of advancing disease stage, increased recurrence, and low overall survival in malignant melanoma patients. Using S100B overexpression and shRNA(S100B) knockdown studies in melanoma cell lines, elevated S100B was found to enhance cell viability and modulate MAPK signaling by binding directly to the p90 ribosomal S6 kinase (RSK). S100B-RSK complex formation was shown to be Ca(2+)-dependent and to block ERK-dependent phosphorylation of RSK, at Thr-573, in its C-terminal kinase domain. Additionally, the overexpression of S100B sequesters RSK into the cytosol and prevents it from acting on nuclear targets. Thus, elevated S100B contributes to abnormal ERK/RSK signaling and increased cell survival in malignant melanoma.


Assuntos
Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Citosol/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Microscopia Confocal , Complexos Multiproteicos/metabolismo , Mutação , Fosforilação , Ligação Proteica , Interferência de RNA , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Treonina/metabolismo
8.
J Cell Biochem ; 113(1): 282-92, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21913213

RESUMO

The runt-related protein-2 (RUNX2) is a DNA-binding transcription factor that regulates bone formation, tumor cell metastasis, endothelial cell (EC) proliferation, and angiogenesis. RUNX2 DNA binding is glucose and cell cycle regulated. We propose that glucose may activate RUNX2 through changes in post-translational phosphorylation that are cell cycle-specific and will regulate EC function. Glucose increased cell cycle progression in EC through both G2/M and G1 phases with entry into S-phase occurring only in subconfluent cells. In the absence of nutrients and growth factors (starvation), subconfluent EC were delayed in G1 when RUNX2 expression was reduced. RUNX2 phosphorylation, activation of DNA binding, and pRb phosphorylation were stimulated by glucose and were necessary to promote cell cycle progression. Glucose increased RUNX2 localization at focal subnuclear sites, which co-incided with RUNX2 occupancy of the cyclin-dependent kinase (cdk) inhibitor p21(Cip1) promoter, a gene normally repressed by RUNX2. Mutation of the RUNX2 cdk phosphorylation site in the C-terminal domain (S451A.RUNX2) reduced RUNX2 phosphorylation and DNA binding. Expression of this cdk site mutant in EC inhibited glucose-stimulated differentiation (in vitro tube formation), monolayer wound healing, and proliferation. These results define a novel relationship between glucose-activated RUNX2 phosphorylation, cell cycle progression, and EC differentiation. These data suggest that inhibition of RUNX2 expression or DNA binding may be a useful strategy to inhibit EC proliferation in tumor angiogenesis.


Assuntos
Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células Endoteliais/fisiologia , Glucose/metabolismo , Neovascularização Fisiológica , Ciclo Celular/fisiologia , Divisão Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Neoplasias/metabolismo , Fosforilação , Regiões Promotoras Genéticas
9.
J Bone Miner Res ; 27(4): 913-25, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22189971

RESUMO

The fat-soluble prohormone cholecalciferol (Vitamin D3) is a precursor of the circulating 25-OH Vitamin D3, which is converted by 1α-hydroxylase to the biologically active 1,25-OH Vitamin D3. Active Vitamin D3 interacts with the Vitamin D receptor (VDR), a transcription factor that plays an important role in calcium mobilization and bone formation. RUNX2 is a DNA-binding transcription factor that regulates target genes important in bone formation, angiogenesis, and cancer metastasis. Using computer-assisted drug design (CADD) and a microtiter plate-based DNA-binding enzyme-linked immunosorbent assay (D-ELISA) to measure nuclear RUNX2 DNA binding, we have found that Vitamin D3 prohormones can modulate RUNX2 DNA binding, which was dose-dependent and sensitive to trypsin, salt, and phosphatase treatment. Unlabeled oligonucleotide or truncated, dominant negative RUNX2 proteins were competitive inhibitors of RUNX2 DNA binding. The RUNX2 heterodimeric partner, Cbfß, was detected in the binding complexes with specific antibodies. Evaluation of several RUNX2:DNA targeted small molecules predicted by CADD screening revealed a previously unknown biological activity of the inactive Vitamin D3 precursor, cholecalciferol. Cholecalciferol modulated RUNX2:DNA binding at nanomolar concentrations even in cells with low VDR. Cholecalciferol and 25-OH Vitamin D3 prohormones were selective inhibitors of RUNX2-positive endothelial, bone, and breast cancer cell proliferation, but not of cells lacking RUNX2 expression. These compounds may have application in modulating RUNX2 activity in an angiogenic setting, in metastatic cells, and to promote bone formation in disease-mediated osteoporosis. The combination CADD discovery and D-ELISA screening approaches allows the testing of other novel derivatives of Vitamin D and/or transcriptional inhibitors with the potential to regulate DNA binding and biological function.


Assuntos
Colecalciferol/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , DNA/metabolismo , Calcifediol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Subunidade beta de Fator de Ligação ao Core/metabolismo , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Receptores de Calcitriol/metabolismo
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