RESUMO
Saccharomyces cerevisiae, the budding yeast, must remodel initial cell shape and cell wall integrity during vegetative growth and pheromone-induced morphogenesis. The cell wall remodeling is monitored and regulated by the cell wall integrity (CWI) signaling pathway. Wsc1p, together with Wsc2p and Wsc3p, belongs to a family of highly O-glycosylated cell surface proteins that function as stress sensors of the cell wall in S. cerevisiae. These cell surface proteins have the main role of activating the CWI signaling pathway by stimulating the small G-protein Rho1p, which subsequently activates protein kinase C (Pkc1p) and a mitogen activated protein (MAP) kinase cascade that activates downstream transcription factors of stress-response genes. Wsc1p, Wsc2p, and Wsc3p possess a cytoplasmic domain where two conserved regions of the sequence have been assessed to be important for Rom2p interaction. Meanwhile, other research groups have also proposed that these transmembrane proteins could support protein-protein interactions with Ras2p. Molecular structures of the cytoplasmic domain of Wsc1p, Wsc2p and Wsc3p were generated using the standard and fully-automated ORCHESTAR procedures provided by the Sybyl-X 2.1.1 program. The tridimensional structure of full length Ras2p was also generated with Phyre2. These protein models were validated with Procheck-PDBsum and ProSA-web tools and subsequently used in docking-based modeling of protein-protein and protein-compound interfaces for extensive structural and functional characterization of their interaction. The results retrieved from STRING 10.5 suggest that the Wsc-family is involved in protein-protein interactions with each other and with Ras2p. Docking-based studies also validated the existence of protein-protein interactions mainly between Motif I (Wsc3p > Wsc1p > Wsc2p) and Ras2p, in agreement with the data provided by STRING 10.5. Additionally, it has shown that Calcofluor White preferably binds to Wsc1p (-9.5 kcal/mol), meanwhile Caspofungin binds to Wsc3p (-9.1 kcal/mol), Wsc1p (-9.1 kcal/mol) and more weakly Wsc2p (-6.9 kcal/mol). Thus, these data suggests Caspofungin as a common inhibitor for the Wsc-family. MTiOpenScreen database has provided a list of new compounds with energy scores higher than those compounds used in our docking studies, thus suggesting these new compounds have a better affinity towards the cytoplasmic domains and Ras2p. Based on these data, there are new and possibly more effective compounds that should be considered as therapeutic agents against yeast infection.
Assuntos
Antifúngicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Simulação de Acoplamento Molecular , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas ras/antagonistas & inibidores , Antifúngicos/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas ras/metabolismoRESUMO
Azoles are the most widely used drugs in antifungal therapy. They have a wide spectrum of activity against pathogenic fungi that are clinically relevant. However, they have been associated with adverse reactions and toxicity, both of which can be significant in patients. Compared to diazoles, triazoles discriminate better between their intended molecular target, the fungal CYP51 enzyme, and several enzymes of the human CYP450 system. Over the years, this superior discrimination has led to the favoring of triazoles over diazoles in the treatment of systemic mycoses. Nevertheless, despite their being better able to discriminate between the fungal CYP51 and host CYP450 enzymes, they are still capable of inducing significant toxicity and adverse reactions in the host, especially when taken concomitantly with other therapeutic drugs by patients with compromised immune systems. In this writing, we review some of the fundamental concepts regarding the chemistry and mechanisms of action of azole compounds, as well as the spectrum of activity, pharmacokinetics, and adverse effects of triazole antifungals. In addition, we discuss some of the mechanisms that pathogenic fungi have developed to overcome the cytotoxic effects of therapeutic drugs, with an emphasis on triazoles.
Assuntos
Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Azóis/farmacologia , Azóis/uso terapêutico , Micoses/tratamento farmacológico , Triazóis/farmacologia , Triazóis/uso terapêutico , Antifúngicos/metabolismo , Azóis/metabolismo , Interações Medicamentosas , Farmacorresistência Fúngica , Fungos/efeitos dos fármacos , Humanos , Triazóis/metabolismoRESUMO
The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50 values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2-M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10× more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805-18. ©2017 AACR.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carbazóis/administração & dosagem , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Pirimidinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
OBJECTIVE: To describe the risk factors for infection, complications, treatment received and response in Puerto Ricans with HCV attending gastroenterology clinics at UPR-MSC, and the prevalence of single nucleotide polymorphisms (SNPs) in IFNL3 and IFNL4 in this population. METHODS: After consent, demographic and medical data were obtained and blood samples were drawn from each patient. The QIAamp Blood-Maxi Kit was employed for DNA extraction. The TaqMan allelic discrimination assay was employed for SNP genotyping. HCV-RNA was measured by branched-chain DNA assay. Frequency distributions were used to describe the study population and the prevalence of SNPs. The UPR Medical Sciences Campus IRB approved the study. RESULTS: Of 259 patients recruited, 64% were men. Genotype 1was found in 112/136 (82%). Of 150 subjects treated, 19% had sustained virological response (SVR), 40% received treatment with pegylated interferon plus ribavirin. The SNP frequencies (n = 239) of IFNL3 locus rs12979860 were 27% (C/C), 50% (C/T), and 23% (T/T), and for rs8099917 were 46% (T/T), 47% (T/G), and 7% (G/G). SNP frequencies of IFNL4 locus ss469415590 were 26% (TT/TT), 48% (TT/ΔG), and 26% (ΔG/ΔG). CONCLUSION: HCV-infected Hispanics in our sample (all of which were Puerto Rican) were shown to have a low SVR rate of 19%. The demographic characteristics were similar to those of other study groups in the US, except for the annual income. Genotype-1 was the most prevalent in those patients with known HCV genotypes. This study group showed significant differences with frequencies observed in other populations. Lower frequencies of the favorable genotypes were found in our group compared with the populations having European and Asian ancestry.
Assuntos
Hepatite C/genética , Interleucinas/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Alelos , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Etnicidade/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Hepatite C/tratamento farmacológico , Hepatite C/epidemiologia , Humanos , Interferons/administração & dosagem , Interferons/uso terapêutico , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/uso terapêutico , Porto Rico/epidemiologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Sistema de Registros , Ribavirina/administração & dosagem , Ribavirina/uso terapêutico , Fatores SocioeconômicosRESUMO
BACKGROUND: Myosin II-dependent contraction of the cytokinetic ring and primary septum formation by chitin synthase II are interdependent processes during cytokinesis in Saccharomyces cerevisiae. Hence, null mutants of myosin II (myo1Delta) and chitin synthase II (chs2Delta) share multiple morphological and molecular phenotypes. To understand the nature of their interdependent functions, we will seek to identify genes undergoing transcriptional regulation in chs2Delta strains and to establish a transcription signature profile for comparison with myo1Delta strains. RESULTS: A total of 467 genes were commonly regulated between myo1Delta and chs2Delta mutant strains (p
RESUMO
Myosin II is important for normal cytokinesis and cell wall maintenance in yeast cells. Myosin II-deficient (myo1) strains of the budding yeast Saccharomyces cerevisiae are hypersensitive to nikkomycin Z (NZ), a competitive inhibitor of chitin synthase III (Chs3p), a phenotype that is consistent with compromised cell wall integrity in this mutant. To explain this observation, we hypothesized that the absence of myosin type II will alter the normal levels of proteins that regulate cell wall integrity and that this deficiency can be overcome by the overexpression of their corresponding genes. We further hypothesized that such genes would restore normal (wild-type) NZ resistance. A haploid myo1 strain was transformed with a yeast pRS316-GAL1-cDNA expression library and the cells were positively selected with an inhibitory dose of NZ. We found that high expression of the ubiquitin-conjugating protein cDNA, UBC4, restores NZ resistance to myo1 cells. Downregulation of the cell wall stress pathway and changes in cell wall properties in these cells suggested that changes in cell wall architecture were induced by overexpression of UBC4. UBC4-dependent resistance to NZ in myo1 cells was not prevented by the proteasome inhibitor clasto-lactacystin-beta-lactone and required the expression of the vacuolar protein sorting gene VPS4, suggesting that rescue of cell wall integrity involves sorting of ubiquitinated proteins to the PVC/LE-vacuole pathway. These results point to Ubc4p as an important enzyme in the process of cell wall remodelling in myo1 cells.
Assuntos
Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Cadeias Pesadas de Miosina/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/metabolismo , Aminoglicosídeos/farmacologia , Parede Celular/química , Parede Celular/metabolismo , Farmacorresistência Fúngica , Inibidores Enzimáticos/farmacologia , Testes de Sensibilidade Microbiana , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
The natural history of HIV infection has been dramatically changed by the highly active antiretroviral therapies, reducing complications, morbidity and mortality of the disease. Approximately 25% of persons infected with HIV are co-infected with hepatitis C, and some high risk populations have a prevalence of HCV of more than 75%. Liver disease has become one of the principal causes of morbidity and mortality in this population. Co-infection increases viremia of hepatitis C, with increase in fibrosis progression, cirrhosis and death related to hepatitis C. The permanent state of chronic immune activation related to the persistent hepatitis C virus favors transcription of HIV in infected cells and causes a more rapid destruction of T4 and absolute lymphocytes. In addition, the immunologic response after the start of highly active antiretroviral therapy for HIV is less than in mono-infected patients. The role of liver biopsy in the management of co-infected patients is controversial. Many of these patients, even with normal transaminases, show fibrosis in liver biopsy. Predictive factors for advanced fibrosis include male sex, alcohol consumption in excess of 50 grams per day, age over 35, and HIV infection of more than 15 years with CD4 lymphocytes less than 400/ mm3. The treatment of hepatitis C is limited and sustained viral response is less than 30% for genotypes 1 and 4. This response is even less in the more advanced stages of HIV and hepatitis C. The determination of when to start treatment and the increased toxicity when combining pegylated interferon plus ribavirin and antiretroviral medications makes the management of these patients more difficult. The development of more potent, safe and tolerated medications is required. Management strategies for patients unresponsive to conventional therapy are geared towards improving liver histology and delaying progression to cirrhosis, hepatocellular cancer and liver transplantation.
Assuntos
Infecções por HIV/complicações , Infecções por HIV/terapia , Hepatite C/complicações , Hepatite C/terapia , Biópsia , Hepatite C/patologia , HumanosRESUMO
OBJECTIVE: To determine whether cell cycle changes can be detected in myosin II-deficient cells using flow cytometry techniques. BACKGROUND: Although the primary role of myosin II (Myo1p) in the yeast Saccharomyces cerevisiae is in cytokinesis we have reported that this conventional myosin also appears to inuence the regulation of cell wall metabolism as indicated by increases in the expression of chitin metabolizing enzymes in a null mutant of the MYO1 gene. The expression of these enzymes is known to be regulated in the cell cycle suggesting that cell cycle changes may alter their expression. METHODS: Flow cytometry was employed to assess the nuclear DNA content of logarithmic yeast cell cultures as a means of determining changes in the cell cycle of Myo1p-deficient cells. RESULTS: Significant changes were observed in the Myo1p-deficient strain suggesting that these cells are arrested in G2/M-phase of the cell cycle. CONCLUSIONS: Based on the results of this preliminary study, we propose a model in which the increased activity of chitin metabolizing enzymes may be explained by a mitotic arrest in these cells.
Assuntos
Cadeias Pesadas de Miosina/metabolismo , Leveduras/citologia , Leveduras/metabolismo , Técnicas de Cultura de Células , Ciclo Celular , Divisão Celular , Parede Celular/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Quitina/metabolismo , Citometria de Fluxo , Expressão Gênica , Haploidia , Mitose , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Leveduras/genéticaRESUMO
Serum from patients which tested positive for hepatitis C virus (HCV) by Enzyme Linked Immunosorbent Assay (ELISA) were analyzed for the presence of HCV RNA by nested Polymerase Chain Reaction (PCR) and for anti-HCV antibodies by Recombinant Immunoblot Assay (RIBA II). Total RNA was extracted from whole blood by a new procedure and subjected to reverse transcription of HCV RNA employing primers to the conserved 5' non-coding region of the HCV genome. PCR performed on these samples uncovered several false positive ELISAs. Reciprocal confirmation between PCR and RIBA II results was observed. These results substantiate this variation of the HCV PCR assay as a reliable alternative for routine confirmation of HCV serological tests