RESUMEN
The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca(2+) influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca(2+) after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Azoles/farmacología , Canales de Calcio/genética , Candida/genética , Candida/metabolismo , Membrana Celular/genética , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/fisiología , Estudio de Asociación del Genoma Completo/métodos , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Glicoproteínas de Membrana/genética , Estabilidad Proteica/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Animales , Células CHO , Colina , Ciclo del Ácido Cítrico , Cricetinae , Cricetulus , Histidina , Hígado/metabolismo , Teoría Cuántica , Proteínas Recombinantes/biosíntesisRESUMEN
The Saccharomyces cerevisiae open reading frame YKR090w encodes a predicted protein displaying similarity in organization to paxillin, a scaffolding protein that organizes signaling and actin cytoskeletal regulating activities in many higher eucaryotic cell types. We found that YKR090w functions in a manner analogous to paxillin as a mediator of polarized cell growth; thus, we have named this gene PXL1 (Paxillin-like protein 1). Analyses of pxl1Delta strains show that PXL1 is required for the selection and maintenance of polarized growth sites during vegetative growth and mating. Genetic analyses of strains lacking both PXL1 and the Rho GAP BEM2 demonstrate that such cells display pronounced growth defects in response to different conditions causing Rho1 pathway activation. PXL1 also displays genetic interactions with the Rho1 effector FKS1. Pxl1p may therefore function as a modulator of Rho-GTPase signaling. A GFP::Pxl1 fusion protein localizes to sites of polarized cell growth. Experiments mapping the localization determinants of Pxl1p demonstrate the existence of localization mechanisms conserved between paxillin and Pxl1p and indicate an evolutionarily ancient and conserved role for LIM domain proteins in acting to modulate cell signaling and cytoskeletal organization during polarized growth.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas del Citoesqueleto/química , Fosfoproteínas/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , División Celular , Supervivencia Celular , Citoesqueleto/metabolismo , Genoma Fúngico , Genotipo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Paxillin , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal , TemperaturaRESUMEN
Air-liquid interface rings were observed on the side walls of stainless steel buffer vessels after certain downstream buffer preparations. Those rings were resistant to regular cleaning-in-place procedures but could be removed by manual means. To investigate the root cause of this issue, multiple analytical techniques, including liquid chromatography with tandem mass spectrometry detection (LC-MS/MS), high-resolution accurate mass liquid chromatography with mass spectrometry, nuclear magnetic resonance, Fourier transform infrared spectroscopy, and scanning electron microscopy with energy-dispersive X-ray spectroscopy have been employed to characterize the chemical composition of air-liquid interface rings. The main component of air-liquid interface rings was determined to be slip agents, and the origin of the slip agents can be traced back to their presence on raw material packaging liners. Slip agents are commonly used in plastic industry as additives to reduce the coefficient of friction during the manufacturing process of thin films. To mitigate this air-liquid interface ring issue, an alternate liner with low slip agent was identified and implemented with minimal additional cost. We have also proactively tested the packaging liners of other raw materials currently used in our downstream buffer preparation to ensure slip agent levels are appropriate. LAY ABSTRACT: Air-liquid interface rings were observed on the side walls of stainless steel buffer vessels after certain downstream buffer preparations. To investigate the root cause of this issue, multiple analytical techniques have been employed to characterize the chemical composition of air-liquid interface rings. The main components of air-liquid interface rings were determined to be slip agents, which are common additives used in the manufacturing process of thin films. The origin of the slip agents can be traced back to their presence on certain raw material packaging liners. To mitigate this air-liquid interface ring issue, an alternate liner with low slip agent was identified and implemented.
Asunto(s)
Química Farmacéutica/métodos , Embalaje de Medicamentos/métodos , Preparaciones Farmacéuticas , Acero Inoxidable , Tampones (Química) , Química Farmacéutica/instrumentación , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Embalaje de Medicamentos/instrumentación , Espectroscopía de Resonancia Magnética/instrumentación , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Preparaciones Farmacéuticas/síntesis química , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Propiedades de SuperficieRESUMEN
Shake flasks and bench-top bioreactors are widely used for cell culture process development, however, culture performances significantly differ between them. In order to apply the results received from small-scale cultures to production scale, it is important to understand the mechanisms underlying the differences between various culture systems. This study analyzes the expression patterns of Chinese hamster ovary (CHO) cells producing IgG-fusion protein B0 cultured in shake flasks and 5-L bench-top bioreactors by CHO-specific DNA microarrays. The data show that hypoxia was present in shake flask cultures but not in controlled, bench-top bioreactors. Hypoxic conditions appeared to be associated with epigenetic repression resulting in decreased cell culture performance and protein productivity, which is also present during large-scale bioreactor operations due to oxygen gradients. High protein productivity was associated with increased cellular machinery for protein transport and secretion in conjunction with decreased epigenetic repression in bench-top bioreactor cultivation. Metal ions could improve cell growth and protein production under hypoxia and this condition could be mimicked in small-scale bioreactors to facilitate cell culture process scale-up.
Asunto(s)
Reactores Biológicos , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Epigénesis Genética/efectos de los fármacos , Oxígeno/farmacología , Transporte de Proteínas/efectos de los fármacos , Animales , Células CHO , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
During the mating process of yeast cells, two Ca2+ influx pathways become activated. The resulting elevation of cytosolic free Ca2+ activates downstream signaling factors that promote long term survival of unmated cells, but the roles of Ca2+ in conjugation have not been described. The high affinity Ca2+ influx system is composed of Cch1p and Mid1p and sensitive to feedback inhibition by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. To identify components and regulators of the low affinity Ca2+ influx system (LACS), we screened a collection of pheromone-responsive genes that when deleted lead to defects in LACS activity but not high affinity Ca2+ influx system activity. Numerous factors implicated in polarized morphogenesis and cell fusion (Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p) were found to be necessary for LACS activity. Each of these factors was also required for activation of the cell integrity mitogen-activated protein kinase cascade during the response to alpha-factor. Interestingly a polytopic plasma membrane protein, Fig1p, was required for LACS activity but not required for activation of Mpk1p mitogen-activated protein kinase. Mpk1p was not required for LACS activity, suggesting Mpk1p and Fig1p define two independent branches in the pheromone response pathways. Fig1p-deficient mutants exhibit defects in the cell-cell fusion step of mating, but unlike other fus1 and fus2 mutants the fusion defect of fig1 mutants can be largely suppressed by high Ca2+ conditions, which bypass the requirement for LACS. These findings suggest Fig1p is an important component or regulator of LACS and provide the first evidence for a role of Ca2+ signals in the cell fusion step of mating.