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1.
Protein Expr Purif ; 151: 99-105, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29936133

RESUMO

Protein prenylation is a vital eukaryotic post-translational modification which permits interaction of proteins with cellular membranes. Prenylated proteins are involved in a number of human diseases, and play a major role in cancers driven by the oncogene KRAS, which is normally farnesylated. In cases where the farnesylation machinery is inhibited, however, KRAS eludes inactivation by using an alternative prenylation pathway in which the protein is geranylgeranylated. In order to study this alternative prenylation, large quantities of accurately processed protein are required. We have developed a system to permit high-yield production of geranylgeranylated KRAS which utilizes an engineered baculovirus system. The development of this system helped to elucidate a potential metabolic bottleneck in insect cell production that should enable better production of any geranylgeranylated proteins using this system.


Assuntos
Baculoviridae/genética , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/química , Animais , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Humanos , Insetos/citologia , Engenharia de Proteínas , Prenilação de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química
2.
Nat Commun ; 15(1): 1703, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402212

RESUMO

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.


Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/genética , Linhagem Celular Tumoral , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases/metabolismo
3.
Pharm Res ; 29(3): 722-38, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22009587

RESUMO

PURPOSE: The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. METHODS: The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. RESULTS: Adding 20% ethanol to buffers or heating at ≥32°C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. CONCLUSIONS: The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.


Assuntos
Substituição de Aminoácidos , Asparagina/química , Interleucina-15/química , Interleucina-15/genética , Sequência de Aminoácidos , Animais , Asparagina/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Interleucina-15/farmacologia , Camundongos , Dados de Sequência Molecular , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Linfócitos T/efeitos dos fármacos
4.
Cell Rep ; 22(7): 1889-1902, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29444439

RESUMO

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.


Assuntos
Oncogenes , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adenilato Quinase/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
5.
PLoS One ; 10(8): e0136396, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26309032

RESUMO

The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , DNA Complementar/genética , Escherichia coli/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Sítios de Ligação , Western Blotting , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Plasmídeos/genética , Conformação Proteica , Fator sigma/metabolismo , Espectrometria de Fluorescência
6.
Methods Mol Biol ; 1116: 193-208, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24395366

RESUMO

Generation of DNA clones for use in proteomic and genomic research often requires a significant level of parallel production, as the number of downstream options for these experiments increases. Where a single fluorescently tagged construct may have sufficed before, there is now the need for multiple types of labels for different readouts and different assays. Protein expression, which once utilized a very small set of vectors because of low throughput expression and purification, has now rapidly matured into a high throughput system in which dozens of conditions can be tested in parallel to identify the best candidate clones. This has returned the bottleneck in many of these technologies to the generation of DNA clones, and standard cloning techniques often dramatically limit the throughput and success of such processes. In order to overcome this bottleneck, higher-throughput and more parallel cloning processes need to be developed which would allow rapid, inexpensive production of final clones. In addition, there is a strong need to utilize standardized elements to avoid unnecessarily remaking fragments of clones that could be used in multiple constructs. The advent of recombinational cloning helped to increase the parallel processing of DNA clones, but was still limited by the need to generate different vector backbones for each specific need. The solution to this problem emerged with the introduction of combinatorial approaches to clone construction, based on either homologous or site-specific recombination processes. In particular, the Gateway Multisite system provides all of the necessary components for a highly parallel, inexpensive, rapid, and diverse platform for clone construction in many areas of proteomic and genomic research. Here we describe our optimized system for combinatorial cloning, including improvements in cloning protocols and construct design that permit users to easily generate libraries of clones which can be combined in parallel to create an unlimited number of final constructs. The system is capable of utilizing the tens of thousands of commercially available Gateway clones already in existence, and allows easy adaptation of most DNA vectors to the system.


Assuntos
Clonagem Molecular/métodos , Recombinação Genética , Vetores Genéticos/genética , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase
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