RESUMO
The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.
Assuntos
Metaloendopeptidases/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/metabolismo , Complexo do Signalossomo COP9 , Simulação de Dinâmica MolecularRESUMO
PURPOSE: Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and survival, and are targets of drugs in clinical development. We assessed expression of PI3K in melanomas and nevi, and studied associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002. EXPERIMENTAL DESIGN: Using Automated Quantitative Analysis, we quantified expression of p85 and p110alpha subunits in 540 nevi and 523 melanomas. We determined the IC(50) for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002. RESULTS: p85 and p110alpha tend to be coexpressed (P < 0.0001); expression was higher in melanomas than nevi (P < 0.0001) for both subunits, and higher in metastatic than primary melanomas for p85 (P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable. We found no association by t tests between baseline p85, p110alpha, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser(235) and Ser(240) were lower in the more resistant cell lines (P = 0.01 and P = 0.004, respectively). CONCLUSIONS: Expression of p85 and p110alpha subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials.
Assuntos
Neoplasias Encefálicas/enzimologia , Melanoma/enzimologia , Nevo Pigmentado/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Cutâneas/enzimologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Proliferação de Células , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Melanoma/tratamento farmacológico , Melanoma/patologia , Morfolinas/farmacologia , Nevo Pigmentado/tratamento farmacológico , Nevo Pigmentado/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Análise Serial de Proteínas , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/secundário , Análise Serial de Tecidos , Células Tumorais CultivadasRESUMO
Apoptotic markers include proteins from the intrinsic and extrinsic pathways. These cascades include both pro-apoptotic and anti-apoptotic elements. The expression levels of these elements can be assessed by immunohistochemistry (IHC) and can indicate general trends in prov-ersus anti-apoptotic tendencies of the cells. IHC is particularly useful when studying large cohorts of paraffin-embedded specimens. Advances in tissue microarray (TMA) technology have facilitated evaluation of large cohorts of specimens, as cores from hundreds of patients can be represented on a single glass slide and stained in a uniform fashion. In this chapter, we discuss construction and staining methods of TMAs and present examples of assessment of apoptotic marker expression in malignant and benign cells using a novel method of automated, quantitative analysis of in situ protein expression.
Assuntos
Apoptose/genética , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica/métodos , Análise Serial de Tecidos/métodos , Biomarcadores Tumorais/análise , Estudos de Coortes , Interpretação Estatística de Dados , Processamento Eletrônico de Dados , Humanos , Imuno-Histoquímica/métodos , Modelos Biológicos , Tamanho da AmostraRESUMO
The heat shock protein HSP90 chaperones proteins implicated in breast cancer progression, including Her2/neu. HSP90-targeting agents are in clinical trials for breast cancer. HSP90 expression is high in breast cancer cell lines, yet no large studies have been conducted on expression in human tumors and the association with clinical/pathologic variables. Tissue microarrays containing 10 cell lines and primary specimens from 655 patients with 10-year follow-up were assessed using our automated quantitative analysis (AQUA) method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured HSP90 expression within the mask using Cy5-conjugated antibodies. We similarly assessed estrogen receptor, progesterone receptor, and Her2/neu expression. HSP90 expression was more variable in human tumors than in cell lines (P < 0.0001). High HSP90 expression was associated with decreased survival (P = 0.0024). On multivariable analysis, high HSP90 expression remained an independent prognostic marker. High HSP90 expression was associated with high Her2/neu and estrogen receptor, large tumors, high nuclear grade, and lymph node involvement. Although HSP90 levels were high in all our cell lines, expression in tumors was more variable. High HSP90 expression in primary breast cancer defines a population of patients with decreased survival. Evaluation of HSP90 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/metabolismo , Proteínas de Choque Térmico HSP90/biossíntese , Animais , Western Blotting , Neoplasias da Mama/patologia , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Receptor ErbB-2/biossíntese , Análise Serial de TecidosRESUMO
Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8-RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.
Assuntos
Endopeptidases/fisiologia , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Peptídeo Hidrolases/química , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Carboxiliases/genética , Carboxiliases/fisiologia , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Endopeptidases/deficiência , Endopeptidases/genética , Teste de Complementação Genética , Peptídeo Hidrolases/fisiologia , Mapeamento de Peptídeos/métodos , Peptídeos/genética , Peptídeos/fisiologia , Fenótipo , Complexo de Endopeptidases do Proteassoma/deficiência , Complexo de Endopeptidases do Proteassoma/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismoRESUMO
The COP9 signalosome (CSN), the lid subcomplex of the proteasome and translational initiation factor 3 (eIF3) share structural similarities and are often referred to as the PCI family of complexes. In multicellular eukaryotes, the CSN is highly conserved as an 8-subunit complex but in Saccharomyces cerevisiae the complex is rather divergent. We further characterize the composition and properties of the CSN in budding yeast and its interactions with these related complexes. Using the generalized profile method we identified CSN candidates, four with PCI domains: Csn9, Csn10, Pci8/Csn11, and Csn12, and one with an MPN domain, Csn5/Rri1. These proteins and an additional interactor, Csi1, were tested for pairwise interactions by yeast two-hybrid and were found to form a cluster surrounding Csn12. Csn5 and Csn12 cofractionate in a complexed form with an apparent molecular weight of roughly 250kDa. However, Csn5 migrates as a monomer in Deltacsn12 supporting the pivotal role of Csn12 in stabilizing the complex. Confocal fluorescence microscopy detects GFP-tagged Csn5 preferentially in the nucleus, whereas in absence of Csn12, Csn10, Pci8/Csn11, or Csi1, Csn5 is delocalized throughout the cell, indicating that multiple subunits are required for nuclear localization of Csn5. Two CSN subunits, Csn9 and Csi1, interact with the proteasome lid subunit Rpn5. Pci8/Csn11 has previously been shown to interact with eIF3. Together, these results point to a network of interactions between these three structurally similar, yet functionally diverse, complexes.