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Background: Neddylation (NAE) inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report the cytotoxic vulnerability to NAE inhibitors in a subset of glioblastoma (GBM) preclinical models and identify genetic alterations and biological processes underlying differential response. Methods: GBM DNA sequencing and transcriptomic data were queried for genes associated with response to NAE inhibition; candidates were validated by molecular techniques. Multi-omics and functional assays revealed processes implicated in NAE inhibition response. Results: Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and DNA repair pathways as significant differentiators between sensitive and resistant models. Vulnerability to MLN4924, a NAE inhibitor, is associated with elevated S-phase populations, DNA re-replication, and DNA damage. In a panel of GBM models, loss of WT PTEN is associated with resistance to different NAE inhibitors. A NAE inhibition response gene set could segregate the GBM cell lines that are most resistant to MLN4924. Conclusions: Loss of WT PTEN is associated with non-sensitivity to 3 different compounds that inhibit NAE in GBM. A NAE inhibition response gene set largely consisting of DNA replication genes could segregate GBM cell lines most resistant to NAEi and may be the basis for future development of NAE inhibition signatures of vulnerability and clinical trial enrollment within a precision medicine paradigm.
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Glioblastoma (GB) is the most lethal brain cancer in adults, with a 5-year survival rate of 5%. The standard of care for GB includes maximally safe surgical resection, radiation, and temozolomide (TMZ) therapy, but tumor recurrence is inevitable in most GB patients. Here, we describe the development of a blood-brain barrier (BBB)-penetrant tubulin destabilizer, RGN3067, for the treatment of GB. RGN3067 shows good oral bioavailability and achieves high concentrations in rodent brains after oral dosing (Cmax of 7807 ng/mL (20 µM), Tmax at 2 h). RGN3067 binds the colchicine binding site of tubulin and inhibits tubulin polymerization. The compound also suppresses the proliferation of the GB cell lines U87 and LN-18, with IC50s of 117 and 560 nM, respectively. In four patient-derived GB cell lines, the IC50 values for RGN3067 range from 148 to 616 nM. Finally, in a patient-derived xenograft (PDX) mouse model, RGN3067 reduces the rate of tumor growth compared to the control. Collectively, we show that RGN3067 is a BBB-penetrant small molecule that shows in vitro and in vivo efficacy and that its design addresses many of the physicochemical properties that prevent the use of microtubule destabilizers as treatments for GB and other brain cancers.
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BACKGROUND: Neddylation inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report vulnerability to MLN4924 or pevonedistat (a neddylation inhibitor) in a subset of glioblastoma (GBM) preclinical models and identify biomarkers, mechanisms, and signatures of differential response. METHODS: GBM sequencing data were queried for genes associated with MLN4924 response status; candidates were validated by molecular techniques. Time-course transcriptomics and proteomics revealed processes implicated in MLN4924 response. RESULTS: Vulnerability to MLN4924 is associated with elevated S-phase populations, re-replication, and DNA damage. Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and chromatin instability pathways as significant differentiators between sensitive and resistant models. Loss of PTEN and its nuclear functions is associated with resistance to MLN4924. Time-course proteomics identified elevated TOP2A in resistant models through treatment. TOP2A inhibitors combined with MLN4924 prove synergistic. CONCLUSIONS: We show that PTEN status serves as both a novel biomarker for MLN4924 response in GBM and reveals a vulnerability to TOP2A inhibitors in combination with MLN4924.
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Glioblastoma , PTEN Fosfo-Hidrolase , Inibidores da Topoisomerase II , Humanos , Apoptose , Linhagem Celular Tumoral , Ciclopentanos/farmacologia , Ciclopentanos/uso terapêutico , Glioblastoma/tratamento farmacológico , Proteína NEDD8/metabolismo , PTEN Fosfo-Hidrolase/genética , Pirimidinas/farmacologia , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/uso terapêutico , Resistencia a Medicamentos AntineoplásicosRESUMO
There is substantial evidence that in addition to nicotine, other compounds found in tobacco smoke significantly influence smoking behavior. Further, recent years have seen an explosion in the availability of non-combusted products that deliver nicotine, such as e-cigarettes and "home-brew" vaping devices that are essentially unregulated. There are many thousands of compounds in tobacco smoke alone, and new products are constantly introducing new compounds. Uncovering which of these compounds are active, across multiple smoking-relevant subtypes of the nicotinic acetylcholine receptor (nAChR) that influence tobacco/nicotine addiction, requires a high-throughput screening (HTS) approach. Accordingly, we developed a panel of HTS-friendly cell-based assays, all performed in the same cellular background and using the same membrane potential dye readout, to measure the function of the α3ß4-, α4ß2-, and α6ß2-nAChR subtypes. These subtypes have each been prominently and consistently associated with human smoking behavior. We validated our assays by performing pilot screening of an expanded set of the Prestwick FDA-approved drug library. The screens displayed excellent performance parameters, and moderate hit rates (mean of 1.2% across all three assays) were achieved when identifying antagonists (chosen since effects of endogenous antagonists on consumption of nicotine/tobacco products are under-studied). Validation rates using an orthogonal assay (86Rb+ efflux) averaged 73% across the three assays. The resulting panel of assays represents a valuable new platform with which to screen and identify nAChR subtype-selective compounds. This provides a resource for identifying smoking-related compounds in both combusted and non-combusted tobacco products, with potential relevance in the search for additional smoking-cessation therapies.
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Sistemas Eletrônicos de Liberação de Nicotina , Receptores Nicotínicos , Poluição por Fumaça de Tabaco , Ensaios de Triagem em Larga Escala , Humanos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/uso terapêutico , Fumar/tratamento farmacológicoRESUMO
To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.
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Proteína Substrato Associada a Crk/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Microtúbulos/metabolismo , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Acetamidas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Calpaína/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Humanos , Microtúbulos/efeitos dos fármacos , Morfolinas/farmacologia , Mutação/genética , Organoides/efeitos dos fármacos , Organoides/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridinas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932-43. ©2018 AACR.
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Antineoplásicos/farmacologia , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Imidazóis/farmacologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Animais , Carcinoma de Células Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA interference (RNAi) is a readily available research tool that can be used to accelerate the identification and functional validation of a multitude of new candidate drug targets by experimentally perturbing gene expression and function. High-throughput RNAi technology using libraries of short-interfering RNA (siRNA) makes it possible to rapidly identify genes and biomarkers associated with biological processes such as diseases or a cellular response to therapy. Thus, RNAi-based screening is an extremely powerful technology that can provide tremendous insights into the mechanisms of action and contexts of vulnerability of a particular drug treatment. This chapter describes the infrastructure requirements needed to successfully perform HT-RNAi screening. Information on the methodology, instrumentation, experimental design, and workflow aspects is provided, as well as insights on how to successfully implement a high-throughput RNAi screen.
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Ensaios de Triagem em Larga Escala/métodos , RNA Interferente Pequeno , Linhagem Celular , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Interferência de RNA , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer characterized by complex aberrant genomes. A fundamental goal of current studies is to identify those somatic events arising in the variable landscape of PDA genomes that can be exploited for improved clinical outcomes. METHODS: We used DNA content flow sorting to identify and purify tumor nuclei of PDA samples from 50 patients. The genome of each sorted sample was profiled by oligonucleotide comparative genomic hybridization and targeted resequencing of STAG2. Transposon insertions within STAG2 in a KRAS (G12D)-driven genetically engineered mouse model of PDA were screened by RT-PCR. We then used a tissue microarray to survey STAG2 protein expression levels in 344 human PDA tumor samples and adjacent tissues. Univariate Kaplan Meier analysis and multivariate Cox Regression analysis were used to assess the association of STAG2 expression relative to overall survival and response to adjuvant therapy. Finally, RNAi-based assays with PDA cell lines were used to assess the potential therapeutic consequence of STAG2 expression in response to 18 therapeutic agents. RESULTS: STAG2 is targeted by somatic aberrations in a subset (4%) of human PDAs. Transposon-mediated disruption of STAG2 in a KRAS (G12D) genetically engineered mouse model promotes the development of PDA and its progression to metastatic disease. There was a statistically significant loss of STAG2 protein expression in human tumor tissue (Wilcoxon-Rank test) with complete absence of STAG2 staining observed in 15 (4.3%) patients. In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (>95% of nuclei positive) was associated with a median survival benefit of 6.41 months (P = 0.031). The survival benefit of adjuvant chemotherapy was only seen in patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; P = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in pancreatic cancer patients. Finally, we show that RNAi-mediated knockdown of STAG2 selectively sensitizes human PDA cell lines to platinum-based therapy. CONCLUSIONS: Based on these iterative findings we propose that STAG2 is a clinically significant tumor suppressor in PDA.
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We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
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MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinases/genética , Melanoma/genética , Mutação , Neoplasias Cutâneas/genética , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Exoma , Humanos , Perda de Heterozigosidade , Melanoma/tratamento farmacológico , Melanoma/secundário , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Temozolomida , Células Tumorais CultivadasRESUMO
Myo1c is a member of the myosin-I family that binds phosphoinositides and links the actin cytoskeleton to cellular membranes. Recent investigations suggest that targeting of myo1c to some subcellular regions requires the binding of an unknown protein to the IQ motifs in the myo1c regulatory domain. We identify two myristoylated proteins that bind the myo1c regulatory domain: calcium-binding protein 1 (CaBP1) and calcium- and integrin-binding-protein-1 (CIB1). CIB1 and CaBP1 interact with myo1c in vivo as determined by pull-down experiments and fluorescence microscopy where the endogenously expressed proteins show extensive cellular colocalization with myo1c. CIB1 and CaBP1 bind to the myo1c IQ motifs in the regulatory domain where they compete with calmodulin for binding. CaBP1 has a higher apparent affinity for myo1c than CIB1, and both proteins better compete with calmodulin in the presence of calcium. We propose that these proteins may play a role in specifying subcellular localization of myo1c.
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Proteínas de Ligação ao Cálcio/metabolismo , Miosina Tipo I/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Calmodulina/metabolismo , Chlorocebus aethiops , Camundongos , Miosina Tipo I/química , Células PC12 , Estrutura Terciária de Proteína , Ratos , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. Myo1b is alternatively spliced within the regulatory domain of the molecule, yielding isoforms with six (myo1b(a)), five (myo1b(b)), or four (myo1b(c)) non-identical IQ motifs. The calmodulin binding properties of the myo1b IQ motifs have not been investigated, and the mechanical and cell biological consequences of alternative splicing are not known. Therefore, we expressed the alternatively spliced myo1b isoforms truncated after the final IQ motif and included a sequence at their C termini that is a substrate for bacterial biotin ligase. Site-specific biotinylation allows us to specifically attach the myosin to motility surfaces via a biotin-streptavidin linkage. We measured the ATPase and motile properties of the recombinant myo1b splice isoforms, and we correlated these properties with calmodulin binding. We confirmed that calcium-dependent changes in the ATPase activity are due to calcium binding to the calmodulin closest to the motor. We found that calmodulin binds tightly to some of the IQ motifs (Kd < 0.2 microM) and very weakly to the others (Kd > 5 microM), suggesting that a subset of the IQ motifs are not calmodulin bound under physiological conditions. Finally, we found the in vitro motility rate to be dependent on the myo1b isoform and the calmodulin concentration and that the myo1b regulatory domain acts as a rigid lever arm upon calmodulin binding to the high affinity and low affinity IQ motifs.
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Miosina Tipo I/química , Actinas/química , Difosfato de Adenosina/química , Adenosina Trifosfatases/química , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biotina/química , Biotinilação , Calmodulina/química , Relação Dose-Resposta a Droga , Cinética , Dados de Sequência Molecular , Miosina Tipo I/metabolismo , Miosinas/química , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de TempoRESUMO
The ATPase activity of myosin-Is from lower eukaryotes is activated by phosphorylation by the p21-activated kinase family at the TEDS site on an actin-binding surface-loop. This actin-binding loop is the site of a cardiac myosin-II mutation responsible for some forms of familial hypertrophic cardiomyopathy. To determine the mechanism of myosin-I regulation by heavy-chain phosphorylation (HCP) and to better understand the importance of this loop in the function of all myosin isoforms, we performed a kinetic investigation of the regulatory mechanism of the Acanthamoeba myosin-IC motor domain (MIC(IQ)). Phosphorylated and dephosphorylated MIC(IQ) show actin-activated ATPase activity; however, HCP increases the ATPase activity >20-fold. HCP does not greatly affect the rate of phosphate release from MIC in the absence of actin, as determined by single turnover experiments. Additionally, HCP does not significantly affect the affinity of myosin for actin in the absence or presence of ATP, the rate of ATP-induced dissociation of actoMIC(IQ), the affinity of ADP, or the rate of ADP release. Sequential-mix single-turnover experiments show that HCP regulates the rate of phosphate release from actin-bound MIC(IQ). We propose that the TEDS-containing actin-binding loop plays a direct role in regulating phosphate release and the force-generating (A-to-R) transition of myosin-IC.
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Acanthamoeba/química , Cadeias Pesadas de Miosina/química , Miosina Tipo I/química , Acanthamoeba/enzimologia , Acanthamoeba/genética , Actinas/química , Difosfato de Adenosina/química , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Animais , Vetores Genéticos/química , Cinética , Modelos Químicos , Cadeias Pesadas de Miosina/genética , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Miosina Tipo I/genética , Proteínas de Ligação a Fosfato/química , Fosfatos/química , Fosforilação , Ligação Proteica , CoelhosRESUMO
Myosin-I is the single-headed member of the myosin superfamily that associates with lipid membranes. Biochemical experiments have shown that myosin-I membrane binding is the result of electrostatic interactions between the basic tail domain and acidic phospholipids. To better understand the dynamics of myosin-I membrane association, we measured the rates of association and dissociation of a recombinant myo1c tail domain (which includes three IQ domains and bound calmodulins) to and from large unilamellar vesicles using fluorescence resonance energy transfer. The apparent second-order rate constant for lipid-tail association in the absence of calcium is fast with nearly every lipid-tail collision resulting in binding. The rate of binding is decreased in the presence of calcium. Time courses of myo1c-tail dissociation are best fit by two exponential rates: a fast component that has a rate that depends on the ratio of acidic phospholipid to myo1c-tail (phosphatidylserine (PS)/tail) and a slow component that predominates at high PS/tail ratios. The dissociation rate of the slow component is slower than the myo1c ATPase rate, suggesting that myo1c is able to stay associated with the lipid membrane during multiple catalytic cycles of the motor. Calcium significantly increases the lifetimes of the membrane-bound state, resulting in dissociation rates 0.001 s(-1).
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Metabolismo dos Lipídeos , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Animais , Cálcio/farmacologia , Clonagem Molecular , Ditiotreitol , Cinética , Lipossomos , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , VertebradosRESUMO
Myo1e is the widely expressed subclass-1 member of the myosin-I family. We performed a kinetic analysis of a truncated myo1e that consists of the motor and the single IQ motif with a bound calmodulin. We determined the rates and equilibrium constants for the key steps in the ATPase cycle. The maximum actin activated ATPase rate (V(max)) and the actin concentration at half-maximum of V(max) (K(ATPase)) of myo1e are similar to those of the native protein. The K(ATPase) is low (approximately 1 microm), however the affinity of myo1e for actin in the presence of ATP is very weak. A weak actin affinity and a rapid rate of phosphate release result in a pathway under in vitro assay conditions in which phosphate is released while myo1e is dissociated from actin. Actin activation of the ATPase activity and the low K(ATPase) are the result of actin activation of ADP release. We propose that myo1e is tuned to function in regions of high concentrations of cross-linked actin filaments. Additionally, we found that ADP release from actomyo1e is > 10-fold faster than other vertebrate myosin-I isoforms. We propose that subclass-1 myosin-Is are tuned for rapid sliding, whereas subclass-2 isoforms are tuned for tension maintenance or stress sensing.
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Miosinas/química , Miosinas/metabolismo , Miosinas/fisiologia , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Hidrólise , Cinética , Modelos Químicos , Dados de Sequência Molecular , Miosina Tipo I , Ligação Proteica , Isoformas de Proteínas , Coelhos , Fatores de TempoRESUMO
The K. fragilis CBS 397 gene library was screened with a GAP probe, which was designed according to the homology with S. cerevisiae, K. lactis, K. marxianus GAP gene. One positive clone pG1 containing GAP1 gene was isolated and confirmed by Southern hybridization. The GAP1 gene was partially sequenced. By using a fragment of the clone as a probe, another positive clone pG2 was acquired and also confirmed by Southern hybridization. The GAP2 gene from pG2 was completely sequenced. The upstream sequences of both genes were shown to have promoter activity. The fragment of pG1 could hybridize with three chromosomes of K. fragilis.
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In order to investigate the relationship between the structure of the mRNA translation initiation region (TIR) and gene expression, We mutated multiple sites of the 5' end of IFN-alpha8 and GM-CSF genes by site-directed mutagenesis without changing their amino acid sequences. SDS-PAGE showed that the protein products of mutated genes increased greatly in recombinant clones, as compared with their native genes. RNA dot blot revealed that the difference of their corresponding amount of mRNA transcribed between the native and the mutated genes was negligible. These results imply that the elevated expressions are attributed mainly to increased translation level. The prediction of mRNA secondary structure suggests that the delta G of TIR may have close relations to the expression level.
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The K. fragilis CBS 397 gene library was screened by the PDI probe, which was designed according to the homology with the S. cerevisiae PDI gene. One positive clone was found and confirmed. In order to learn its physical map,Southern hybridization was done on this positive clone. The K. fragilis PDI gene was found to locate in chromosome V genome. Its sequence was also made partly known by DNA sequencing. From the method of the turbidimetric assay of insulin disulfide reduction, we have also demonstrated that the cloned PDI gene can exhibit PDI activity.
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The vector pIRK was constructed by using a 2.2 kb EcoRI fragment of rDNA from Kluyveromyces lactis for targeted homologous recombination, with the URA3 gene from Saccharomyces cerevisiae acting as a selection marker. By the examination of the copy number, stability and chromosomal location of the vector in K. lactis transformants the results demonstrated that: (1) of the different transformants, the average copy number of the plasmid pIRK was 120 per cell; (2) after 50 generations of growth in rich medium, the vector displayed high stability. (3) all integration events occurred in the chromosome IV where genomic rDNA located. Using this vector, the LAC4 gene cloned from K. fragilis was expressed. The yield of beta-galactosidase related directly to the vector's copy number. The highest activity of beta-galactosidase produced by transformants was 8.6 times higher than that produced by the wild type strain of K. fragilis under the same conditions.