RESUMEN
Multiple genes in Mycobacterium tuberculosis (Mtb) are regulated by copper including socAB (small orf induced by copper A and B), which is induced by copper and repressed by RicR (regulated in copper repressor). socA and socB encode hypothetical proteins of 61 and 54 amino acids, respectively. Here, we use biophysical and computational methods to evaluate the SocB structure. We find that SocB lacks evidence for secondary structure, with no thermal cooperative unfolding event, according to circular dichroism measurements. 2D NMR spectra similarly exhibit hallmarks of a disordered structural state, which is also supported by analyzing SocB diffusion. Altogether, these findings suggest that by itself SocB is intrinsically disordered. Interestingly, SocB interacts with a synthetic phospholipid bilayer and becomes helical, which suggests that it may be membrane-associated.
Asunto(s)
Proteínas Bacterianas/química , Cobre/química , Proteínas Intrínsecamente Desordenadas/química , Proteínas de la Membrana/química , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia MolecularRESUMEN
STAT3 N-terminal domain is a promising molecular target for cancer treatment and modulation of immune responses. However, STAT3 is localized in the cytoplasm, mitochondria, and nuclei, and thus, is inaccessible to therapeutic antibodies. Its N-terminal domain lacks deep pockets on the surface and represents a typical "non-druggable" protein. In order to successfully identify potent and selective inhibitors of the domain, we have used virtual screening of billion structure-sized virtual libraries of make-on-demand screening samples. The results suggest that the expansion of accessible chemical space by cutting-edge ultra-large virtual compound databases can lead to successful development of small molecule drugs for hard-to-target intracellular proteins.
RESUMEN
Here we demonstrate that aerosols of host directed therapies [HDT] administered during a chronic Mycobacterium tuberculosis (Mtb) infection have bactericidal effect. The pulmonary bacterial load of C57BL/6 mice chronically infected with Mtb was reduced by 1.7 and 0.6 log10CFU after two weeks of treatment via aerosol delivery with ST3-H2A2, [a selective peptide inhibitor of the STAT3 N-terminal domain] or IL10R1-7 [selective peptide inhibitor for the IL-10Ra] respectively and when compared to control mice treated with IL10R1-14 [peptide inhibitor used as negative control] or untreated mice infected with Mtb. Accordingly, when compared to control mice, the bactericidal capacity in mice was enhanced upon treatment with peptide inhibitors ST3-H2A2 and IL10R1-7 as evidenced by higher pulmonary activities of nitric oxide synthase, NADPH oxidase and lysozyme enzymes and decreased arginase enzyme activity. This therapy also modulated important checkpoints [Bcl2, Beclin-1, Atg 5, bax] in the apoptosis-autophagy pathways. Thus, even in the absence of antibiotics, targeting of the host pulmonary IL-10-STAT3 pathway can significantly reduce the Mtb bacilli load in the lungs, modulate the host own bactericidal capacity and apoptosis and autophagy pathways. Our approach here also allows targeting checkpoints of the lungs to determine their specific contribution in pulmonary immunity or pathogenesis.
Asunto(s)
Sistemas de Liberación de Medicamentos , Interleucina-10/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Fragmentos de Péptidos/administración & dosificación , Factor de Transcripción STAT3/antagonistas & inhibidores , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis/tratamiento farmacológico , Administración por Inhalación , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/farmacología , Tuberculosis/microbiología , Tuberculosis Pulmonar/microbiologíaRESUMEN
p205 is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter in vitro in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb- and p53-mediated pathways of growth inhibition.
Asunto(s)
Ciclo Celular , Proliferación Celular , Proteínas Nucleares/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Fase G2 , Proteínas de Homeodominio/aislamiento & purificación , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Unión Proteica , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
To understand long terminal repeat (LTR)-retrotransposon copy number dynamics, Ty1 elements were reintroduced into a "Ty-less" Saccharomyces strain where elements had been lost by LTR-LTR recombination. Repopulated strains exhibited alterations in chromosome size that were associated with Ty1 insertions, but did not become genetically isolated. The rates of element gain and loss under genetic and environmental conditions known to affect Ty1 retrotransposition were determined using genetically tagged reference elements. The results show that Ty1 retrotransposition varies with copy number, temperature, and cell type. In contrast to retrotransposition, Ty1 loss by LTR-LTR recombination was more constant and not markedly influenced by copy number. Endogenous Ty1 cDNA was poorly utilized for recombination when compared with LTR-LTR recombination or ectopic gene conversion. Ty1 elements also appear to be more susceptible to copy number fluctuation in haploid cells. Ty1 gain/loss ratios obtained under different conditions suggest that copy number oscillates over time by altering the rate of retrotransposition, resulting in the diverse copy numbers observed in Saccharomyces.
Asunto(s)
Retroelementos/genética , Retroelementos/fisiología , Saccharomyces cerevisiae/genética , Southern Blotting , Cromosomas Fúngicos , ADN Complementario/metabolismo , ADN de Hongos/genética , Conversión Génica , Genes Fúngicos , Técnicas Genéticas , Vectores Genéticos , Genoma Fúngico , Haploidia , Cariotipificación , Modelos Genéticos , Reacción en Cadena de la Polimerasa , Recombinación Genética , Temperatura , Secuencias Repetidas TerminalesRESUMEN
Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been suspected that dimeric Ras may promote dimerization and activation of Raf. Here, we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated ß-sheet dimer interface is at the Switch I and effector binding regions, overlapping the binding surfaces of Raf, PI3K, RalGDS, and additional effectors. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps the binding sites of some effectors. This interface may promote activation of Raf. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf signaling in cancer.
Asunto(s)
Guanosina Trifosfato/química , Proteínas Proto-Oncogénicas p21(ras)/química , Dominio Catalítico , Humanos , Cinética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.
Asunto(s)
Técnicas de Química Analítica/métodos , Proteínas/química , Proteínas/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Cinética , Proteínas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , TemperaturaRESUMEN
Despite their evolutionary distance, the Saccharomyces cerevisiae retrotransposon Ty1 and retroviruses use similar strategies for replication, integration, and interactions with their hosts. Here we examine the formation of circular Ty1 DNA, which is comparable to the dead-end circular products that arise during retroviral infection. Appreciable levels of circular Ty1 DNA are present with one-long terminal repeat (LTR) circles and deleted circles comprising major classes, while two-LTR circles are enriched when integration is defective. One-LTR circles persist when homologous recombination pathways are blocked by mutation, suggesting that they result from reverse transcription. Ty1 autointegration events readily occur, and many are coincident with and dependent upon DNA flap structures that result from DNA synthesis initiated at the central polypurine tract. These results suggest that Ty1-specific mechanisms minimize copy number and raise the possibility that special DNA structures are a targeting determinant.
Asunto(s)
ADN Circular/genética , Retroelementos/genética , Retroviridae/genética , Transcripción Reversa/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Southern Blotting , Cartilla de ADN , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Secuencias Repetidas Terminales/genéticaRESUMEN
Because Ty elements transpose through an RNA intermediate, element accumulation through retrotransposition must be regulated or offset by element loss to avoid uncontrolled genome expansion. Here we examine the fate of Ty sequences in Saccharomyces strain 337, a strain that is reported to lack Ty1 and Ty2 elements, but contains remnant solo long terminal repeats (LTRs). Although strain 337 was initially classified as Saccharomyces cerevisiae, our work indicates that this strain is more closely related to S. paradoxus. Several degenerate Ty1 and Ty2 LTRs were mapped to the same insertion sites as full-length Ty1 and Ty2 elements in S. cerevisiae, suggesting that this strain lost Ty elements by LTR-LTR recombination. Southern analysis indicates that strain 337 also lacks Ty4 and Ty5 elements. We estimated the rates of element gain and loss in this strain by introducing a single transposition-competent Ty1 element. The results indicate that Ty1 retrotransposition occurs at a much higher rate than elimination, suggesting that copy-number-dependent co-factors or environmental conditions contribute to the loss of Ty elements in this genome.