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1.
Biotechnol Bioeng ; 121(4): 1271-1283, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38258490

RESUMO

"Giving the cells exactly what they need, when they need it" is the core idea behind the proposed bioprocess control strategy: operating bioprocess based on the physiological behavior of the microbial population rather than exclusive monitoring of environmental parameters. We are envisioning to achieve this through the use of genetically encoded biosensors combined with online flow cytometry (FCM) to obtain a time-dependent "physiological fingerprint" of the population. We developed a biosensor based on the glnA promoter (glnAp) and applied it for monitoring the nitrogen-related nutritional state of Escherichia coli. The functionality of the biosensor was demonstrated through multiple cultivation runs performed at various scales-from microplate to 20 L bioreactor. We also developed a fully automated bioreactor-FCM interface for on-line monitoring of the microbial population. Finally, we validated the proposed strategy by performing a fed-batch experiment where the biosensor signal is used as the actuator for a nitrogen feeding feedback control. This new generation of process control, -based on the specific needs of the cells, -opens the possibility of improving process development on a short timescale and therewith, the robustness and performance of fermentation processes.


Assuntos
Reatores Biológicos , Técnicas Biossensoriais , Fermentação , Escherichia coli , Nitrogênio
2.
Cell Microbiol ; 19(7)2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28114750

RESUMO

The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated 17 mutations in the yeast Golgi-localized Ca2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca2+ -ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos/genética , Antiporters , Canais de Cálcio/genética , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Proc Natl Acad Sci U S A ; 110(17): 6859-64, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23569283

RESUMO

Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Transmembrane protein 165 (TMEM165) belongs to an uncharacterized family of membrane proteins called Uncharacterized Protein Family 0016, which are well conserved throughout evolution and share characteristics reminiscent of the cation/Ca(2+) exchanger superfamily. Gcr1 dependent translation factor 1 (Gdt1p), the budding yeast member of this family, contributes to Ca(2+) homeostasis via an uncharacterized Ca(2+) transport pathway localized in the Golgi apparatus. The gdt1Δ mutant was found to be sensitive to high concentrations of Ca(2+), and interestingly, this sensitivity was suppressed by expression of TMEM165, the human ortholog of Gdt1p, indicating conservation of function among the members of this family. Patch-clamp analyses on human cells indicated that TMEM165 expression is linked to Ca(2+) ion transport. Furthermore, defects in TMEM165 affected both Ca(2+) and pH homeostasis. Based on these results, we propose that Gdt1p and TMEM165 could be members of a unique family of Golgi-localized Ca(2+)/H(+) antiporters and that modification of the Golgi Ca(2+) and pH balance could explain the glycosylation defects observed in TMEM165-deficient patients.


Assuntos
Antiporters/metabolismo , Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexo de Golgi/metabolismo , Homeostase/fisiologia , Proteínas de Membrana/metabolismo , Saccharomycetales/metabolismo , Western Blotting , Proteínas de Transporte de Cátions , Fracionamento Celular , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética
4.
Hum Mol Genet ; 22(14): 2914-28, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23575229

RESUMO

TMEM165 has recently been identified as a novel protein involved in CDG-II. TMEM165 has no biological function described so far. Different mutations were recently found in patients with Golgi glycosylation defects and harboring a peculiar skeletal phenotype. In this study, we examined the effect of naturally occurring mutations on the intracellular localization of TMEM165 and their abilities to complement the TMEM165-deficient yeast, gdt1▵. Wild-type TMEM165 was present within Golgi compartment, plasma membrane and late endosomes/lysosomes, whereas mutated TMEM165 were found differentially localized according to the mutations. We demonstrated that, in the yeast functional assay with TMEM165 ortholog Gdt1, the homozygous point mutation correlating with a mild phenotype restores the yeast functional assay, whereas the truncated mutation, associated with severe disease, failed to restore Gdt1 function. These studies highly suggest that these clinically relevant point mutations do not affect the protein function but critically changes the subcellular protein localization. Moreover, the data point to a critical role of the YNRL motif in TMEM165 subcellular localization.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação Puntual , Antiporters , Proteínas de Transporte de Cátions , Membrana Celular/genética , Membrana Celular/metabolismo , Endossomos/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/química , Sinais Direcionadores de Proteínas , Transporte Proteico
5.
Am J Hum Genet ; 91(1): 15-26, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22683087

RESUMO

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Proteínas de Membrana/genética , Mutação , Adolescente , Antiporters , Proteínas de Transporte de Cátions , Células Cultivadas , Criança , Pré-Escolar , Nanismo/genética , Feminino , Fibroblastos , Complexo de Golgi/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Pele/citologia
6.
Traffic ; 11(7): 931-46, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20406419

RESUMO

SNA (Sensitive to Na(+)) proteins form a membrane protein family, which, in the yeast Saccharomyces cerevisiae, is composed of four members: Sna1p/Pmp3p, Sna2p, Sna3p and Sna4p. In this study, we focused on the 79 residue Sna2p protein. We found that Sna2p is localized in the vacuolar membrane. Directed mutagenesis showed that two functional tyrosine motifs YXXØ are present in the C-terminal region. Each of these is involved in a different Golgi-to-vacuole targeting pathway: the tyrosine 65 motif is involved in adaptor protein (AP-1)-dependent targeting, whereas the tyrosine 75 motif is involved in AP-3-dependent targeting. Moreover, our data suggest that these motifs also play a crucial role in the exit of Sna2p from the endoplasmic reticulum (ER). Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies, probably because of a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were shown to be functional at two different steps of the secretory pathway, ER exit and Golgi-to-vacuole transport.


Assuntos
Retículo Endoplasmático/metabolismo , Tirosina/metabolismo , Vacúolos/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Motivos de Aminoácidos , Transporte Biológico , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição AP-1/metabolismo , Leveduras
7.
NPJ Syst Biol Appl ; 6(1): 6, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170148

RESUMO

In biotechnology, the emergence of high-throughput technologies challenges the interpretation of large datasets. One way to identify meaningful outcomes impacting process and product attributes from large datasets is using systems biology tools such as metabolic models. However, these tools are still not fully exploited for this purpose in industrial context due to gaps in our knowledge and technical limitations. In this paper, key aspects restraining the routine implementation of these tools are highlighted in three research fields: monitoring, network science and hybrid modeling. Advances in these fields could expand the current state of systems biology applications in biopharmaceutical industry to address existing challenges in bioprocess development and improvement.


Assuntos
Bioengenharia/métodos , Produtos Biológicos/metabolismo , Biologia de Sistemas/métodos , Produtos Biológicos/farmacologia , Biotecnologia/métodos , Biotecnologia/tendências , Indústrias/tendências , Modelos Biológicos
8.
Sci Rep ; 6: 24282, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-27075443

RESUMO

Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca(2+) exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca(2+) uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca(2+)/H(+) antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn(2+) to the cells.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Glicosilação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Canais de Cálcio/genética , Expressão Gênica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Pressão Osmótica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
9.
PLoS One ; 9(6): e100851, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24955841

RESUMO

The UPF0016 family is a group of uncharacterized membrane proteins, well conserved through evolution and defined by the presence of one or two copies of an E-Φ-G-D-(KR)-(ST) consensus motif. Our previous results have shown that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and might form a new group of cation/Ca2+ exchangers. Most members of the family are made of two homologous clusters of three transmembrane spans, separated by a central loop and assembled with an opposite orientation in the membrane. However, some bacterial members of the family have only one cluster of transmembrane domains. Among these 'single-domain membrane proteins' some cyanobacterial members were found as pairs of adjacent genes within the genome, but each gene was slightly different. We performed a bioinformatic analysis to propose the molecular evolution of the UPF0016 family and the emergence of the antiparallel topology. Our hypotheses were confirmed experimentally using functional complementation in yeast. This suggests an important and conserved function for UPF0016 proteins in a fundamental cellular process. We also show that members of the UPF0016 family share striking similarities, but no primary sequence homology, with members of the cation/Ca2+ exchangers (CaCA) superfamily. Such similarities could be an example of convergent evolution, supporting the previous hypothesis that members of the UPF0016 family are cation/Ca2+ exchangers.


Assuntos
Cálcio/metabolismo , Evolução Molecular , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Citosol/metabolismo , Genes Fúngicos , Variação Genética , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Filogenia , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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