Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 60
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Am J Med Genet A ; 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913576

RESUMO

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.

2.
Elife ; 92020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31958057

RESUMO

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here we characterize RAS in live human and mouse cells using single molecule tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b we used complementary experimental and molecular dynamic simulation approaches to reveal a detailed mechanism.

3.
Cancer Discov ; 10(1): 104-123, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31649109

RESUMO

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. SIGNIFICANCE: We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1.

4.
J Biol Chem ; 295(4): 1105-1119, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-31836666

RESUMO

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

5.
Mol Cell ; 76(6): 872-884.e5, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31606273

RESUMO

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.

6.
Sci Rep ; 9(1): 10512, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324887

RESUMO

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

7.
Methods Mol Biol ; 2009: 259-277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31152410

RESUMO

Protein prenylation is a common posttranslational modification that enhances the ability of proteins to interact with membrane components within the cell. In many cases, these prenylated proteins are involved in important human diseases, including aging-related disorders and cancer. To effectively study these proteins or develop therapeutics, large quantities of properly modified proteins are required. Historically, production of fully modified farnesylated and methylated proteins at high yield has been challenging. Recently, an engineered insect cell system which is capable of producing authentically modified KRAS protein was used to generate material for structural studies and assay development. Here we describe protocols for extending this work to other farnesylated and methylated substrates.

8.
Drug Metab Dispos ; 47(7): 715-723, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31048454

RESUMO

Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how coexpression of multiple transporters affected resistance to chemotherapeutic drugs. We therefore examined how coexpression of ABCB1 (P-glycoprotein) and ABCG2 contributes to drug resistance in a cell line model. HEK293 cells were transfected with vector-encoding full-length ABCB1, ABCG2, or a bicistronic vector containing both genes, each under the control of a separate promoter. Cells transfected with both transporters (B1/G2 cells) demonstrated high levels of both transporters, and uptake of both the ABCB1-specific substrate rhodamine 123 and the ABCG2-specific substrate pheophorbide a was reduced when examined by flow cytometry. B1/G2 cells were also cross-resistant to the ABCB1 substrate doxorubicin, the ABCG2 substrate topotecan, as well as mitoxantrone and the cell cycle checkpoint kinase 1 inhibitor prexasertib, both of which were found to be substrates of both ABCB1 and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that ABCB1 and ABCG2, when coexpressed, can function independently to transport substrates. ABCB1 and ABCG2 also functioned additively to transport the common fluorescent substrates mitoxantrone and BODIPY-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. ABCG2 expression was also found to decrease the efficacy of the ABCB1 inhibitor tariquidar in B1/G2 cells. Thus, ABCB1 and ABCG2 can independently and additively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed.

9.
Gene Ther ; 26(6): 277-286, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31127187

RESUMO

Neurofibromatosis type 1, including the highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), is featured by the loss of functional neurofibromin 1 (NF1) protein resulting from genetic alterations. A major function of NF1 is suppressing Ras activities, which is conveyed by an intrinsic GTPase-activating protein-related domain (GRD). In this study, we explored the feasibility of restoring Ras GTPase via exogenous expression of various GRD constructs, via gene delivery using a panel of adeno-associated virus (AAV) vectors in MPNST and human Schwann cells (HSCs). We demonstrated that several AAV serotypes achieved favorable transduction efficacies in those cells and a membrane-targeting GRD fused with an H-Ras C-terminal motif (C10) dramatically inhibited the Ras pathway and MPNST cells in a NF1-specific manner. Our results opened up a venue of gene replacement therapy in NF1-related tumors.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Neurofibromatose 1/terapia , Neurofibromina 1/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Estudos de Viabilidade , Vetores Genéticos/genética , Humanos , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Domínios Proteicos , Células de Schwann/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
10.
Genes (Basel) ; 10(2)2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30678108

RESUMO

BACKGROUND: Trichoplusia ni derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusia ni-derived cell line Tni-FNL. METHODS: By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL. RESULTS: Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly. CONCLUSIONS: This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.


Assuntos
Genoma de Inseto , Lepidópteros/genética , Anotação de Sequência Molecular , Animais , Linhagem Celular , Mapeamento de Sequências Contíguas , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Insetos/química , Proteínas de Insetos/genética , Lepidópteros/citologia , Domínios Proteicos , Análise de Sequência de DNA
11.
J Biol Chem ; 294(6): 2193-2207, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30559287

RESUMO

The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers. The KRAS fraction in the plasma membrane (PM) correlates with activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent cellular proliferation. Understanding KRAS's interaction with the PM is challenging given the complexity of the cellular environment. To gain insight into key components necessary for KRAS signal transduction at the PM, we used synthetic membranes such as liposomes and giant unilamellar vesicles. Using surface plasmon resonance (SPR) spectroscopy, we demonstrated that KRAS and Raf-1 proto-oncogene Ser/Thr kinase (RAF1) domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. We found that the RAF1 region spanning RBD through CRD (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS partitioning to the membrane. We also found that calmodulin and phosphodiesterase 6 delta (PDE6δ), but not galectin3 previously proposed to directly interact with KRAS, passively sequester KRAS and prevent it from partitioning into the PM. RAF1 RBDCRD interacted with membranes preferentially at nonraft lipid domains. Moreover, a C-terminal O-methylation was crucial for KRAS membrane localization. These results contribute to a better understanding of how the KRAS-membrane interaction is tuned by multiple factors whose identification could inform drug discovery efforts to disrupt this critical interaction in diseases such as cancer.


Assuntos
Membrana Celular/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Calmodulina/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Membranas Artificiais , Domínios Proteicos , Proteínas Proto-Oncogênicas c-raf , Transdução de Sinais , Eletricidade Estática
12.
Semin Cancer Biol ; 54: 174-182, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29432816

RESUMO

Development of therapeutic strategies against RAS-driven cancers has been challenging due in part to a lack of understanding of the biology of the system and the ability to design appropriate assays and reagents for targeted drug discovery efforts. Recent developments in the field have opened up new avenues for exploration both through advances in the number and quality of reagents as well as the introduction of novel biochemical and cell-based assay technologies which can be used for high-throughput screening of compound libraries. The reagents and assays developed at the NCI RAS Initiative offer a suite of new weapons that could potentially be used to enable the next generation of RAS drug discovery efforts with the hope of finding novel therapeutics for a target once deemed undruggable.


Assuntos
Descoberta de Drogas , Proteínas ras/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Descoberta de Drogas/métodos , Descoberta de Drogas/normas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Seleção de Medicamentos Antitumorais/normas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Controle de Qualidade , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismo
13.
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
14.
Oncotarget ; 9(24): 16634-16647, 2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-29682174

RESUMO

Esophageal squamous cell carcinoma (ESCC) presents poor prognosis, and patients diagnosed with this tumor currently lack target treatments. Therefore, in order to identify potential targets for ESCC treatment, we carried out a transcriptome analysis with ESCC and paired nonmalignant surrounding mucosa samples, followed by a master regulator analysis, and further explored the role of the identified central regulatory genes through in vivo and in vitro assays. Among the transcription factors deregulated/enriched in ESCC, we focused on FOXM1 because of its involvement in the regulation of critical biological processes. A new transcriptome analysis performed with ESCC cell lineage TE-1 showed that the modulation of FOXM1 expression resulted in PIK3R3 expression changes, whereas chromatin immunoprecipitation assay revealed that FOXM1 was capable of binding onto PIK3R3 promoter, thus demonstrating that PIK3R3 is a new FOXM1 target. Furthermore, FOXM1 overexpression resulted in the activation of PIK3/AKT signaling pathway through PIK3R3-mediated AKT phosphorylation. Finally, the analysis of the clinic-pathological data of ESCC patients revealed that overexpression of both FOXM1 and PIK3R3 was associated with poor prognosis, but only the latter was an independent prognosis factor for ESCC patients. In conclusion, our results show that FOXM1 seems to play a central role in ESCC carcinogenesis by upregulating many oncogenes found overexpressed in this tumor. Furthermore, PIK3R3 is a novel FOXM1 target that triggers the activation of the PI3K/AKT pathway in ESCC cells.

16.
Methods Mol Biol ; 1586: 65-82, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28470599

RESUMO

The major goal of any protein expression experiment is to combine the maximum production per cell of soluble protein with the highest possible cell density to most efficiently obtain high yields of protein. A large number of parameters can be optimized in these experiments, but one of the most interesting parameters that have a strong effect on both per cell productivity and cell density is the cellular growth media coupled to the expression induction process. Using specialized media and testing multiple induction conditions, it is possible to significantly enhance the production of heterologous proteins from E. coli.


Assuntos
Técnicas de Cultura de Células/métodos , Clonagem Molecular/métodos , Meios de Cultura/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Escherichia coli/citologia , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade
17.
Proc Natl Acad Sci U S A ; 113(44): E6766-E6775, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27791178

RESUMO

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/química , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Prenilação de Proteína/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/genética , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X , Genes ras , Humanos , Metilação , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo
18.
J Biotechnol ; 238: 1-8, 2016 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-27616621

RESUMO

One of the most common methods for producing recombinant baculovirus for insect cell protein production involves a transposition mediated system invented over 2 decades ago. This Tn7-mediated system, commercially sold as Bac-to-Bac, has proven highly useful for construction of high quality baculovirus, but suffers from a number of drawbacks which reduce the efficiency of the process and limit its utility for high throughput protein production processes. We describe here the creation of Bac-2-the-Future, a 2nd generation Tn7-based system for recombinant baculovirus production which uses optimized expression vectors, new E. coli strains, and enhanced protocols to dramatically enhance the efficiency of the baculovirus production process. The new system which we describe eliminates the need for additional screening of positive clones, improves the efficiency of transposition, and reduces the cost and time required for high throughput baculovirus production. The system is compatible with multiple cloning methodologies, and has been demonstrated to produce baculovirus with equal or better titer and protein productivity than the currently available systems.


Assuntos
Baculoviridae/genética , Clonagem Molecular/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Animais , Escherichia coli/genética , Plasmídeos , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Transfecção
19.
Clin Cancer Res ; 22(4): 1000-10, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26446940

RESUMO

PURPOSE: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. EXPERIMENTAL DESIGN: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax-Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL-Bak heterodimer, Mcl1-Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. RESULTS: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. CONCLUSIONS: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials.


Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Dipeptídeos/farmacologia , Indóis/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunoensaio , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos Nus , Proteínas Mitocondriais/química , Mimetismo Molecular , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Sci Rep ; 5: 15916, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26522388

RESUMO

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.


Assuntos
Lipídeos/fisiologia , Prenilação de Proteína/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Biofísica/métodos , Membrana Celular/metabolismo , Células Cultivadas , Guanosina Trifosfato/metabolismo , Humanos , Insetos/metabolismo , Metilação , Ligação Proteica/fisiologia , Quinases raf/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA