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1.
Angew Chem Int Ed Engl ; 63(28): e202404360, 2024 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-38676693

RESUMEN

Despite great progress in the construction of non-equilibrium systems, most approaches do not consider the structure of the fuel as a critical element to control the processes. Herein, we show that the amino acid side chains (A, F, Nal) in the structure of abiotic phosphates can direct assembly and reactivity during transient structure formation. The fuels bind covalently to substrates and subsequently influence the structures in the assembly process. We focus on the ways in which the phosphate esters guide structure formation and how structures and reactivity cross regulate when constructing assemblies. Through the chemical functionalization of energy-rich aminoacyl phosphate esters, we are able to control the yield of esters and thioesters upon adding dipeptides containing tyrosine or cysteine residues. The structural elements around the phosphate esters guide the lifetime of the structures formed and their supramolecular assemblies. These properties can be further influenced by the peptide sequence of substrates, incorporating anionic, aliphatic and aromatic residues. Furthermore, we illustrate that oligomerization of esters can be initiated from a single aminoacyl phosphate ester incorporating a tyrosine residue (Y). These findings suggest that activated amino acids with varying reactivity and energy contents can pave the way for designing and fabricating structured fuels.


Asunto(s)
Péptidos , Fosfatos , Fosfatos/química , Péptidos/química , Ésteres/química , Estructura Molecular
2.
J Am Chem Soc ; 145(48): 26086-26094, 2023 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-37992133

RESUMEN

Nature chose phosphates to activate amino acids, where reactive intermediates and complex machinery drive the construction of polyamides. Outside of biology, the pathways and mechanisms that allow spontaneous and selective peptide elongation in aqueous abiotic systems remain unclear. Herein we work to uncover those pathways by following the systems chemistry of aminoacyl phosphate esters, synthetic counterparts of aminoacyl adenylates. The phosphate esters act as solubility tags, making hydrophobic amino acids and their oligomers soluble in water and enabling selective elongation and different pathways to emerge. Thus, oligomers up to dodecamers were synthesized in one flask and on the minute time scale, where consecutive additions activated autonomous phase changes. Depending on the pathway, the resulting phases initially carry nonpolar peptides and amphiphilic oligomers containing phosphate esters. During elongation and phosphate release, shorter oligomers dominate in solution, while the aggregated phase favors the presence of longer oligomers due to their self-assembly propensity. Furthermore we demonstrated that the solution phases can be isolated and act as a new environment for continuous elongation, by adding various phosphate esters. These findings suggest that the systems chemistry of aminoacyl phosphate esters can activate a selection mechanism for peptide bond formation by merging aqueous synthesis and self-assembly.


Asunto(s)
Péptidos , Agua , Agua/química , Péptidos/química , Organofosfatos , Aminoácidos/química , Fosfatos/química , Ésteres
3.
Beilstein J Org Chem ; 18: 1278-1288, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36225726

RESUMEN

Polyphosphate kinases (PPKs) have become popular biocatalysts for nucleotide 5'-triphosphate (NTP) synthesis and regeneration. Two unrelated families are described: PPK1 and PPK2. They are structurally unrelated and use different catalytic mechanisms. PPK1 enzymes prefer the usage of adenosine 5'-triphosphate (ATP) for polyphosphate (polyP) synthesis while PPK2 enzymes favour the reverse reaction. With the emerging use of PPK enzymes in biosynthesis, a deeper understanding of the enzymes and their thermodynamic reaction course is of need, especially in comparison to other kinases. Here, we tested four PPKs from different organisms under the same conditions without any coupling reactions. In comparison to other kinases using phosphate donors with comparably higher phosphate transfer potentials that are characterised by reaction yields close to full conversion, the PPK-catalysed reaction reaches an equilibrium in which about 30% ADP is left. These results were obtained for PPK1 and PPK2 enzymes, and are supported by theoretical data on the basic reaction. At high concentrations of substrate, the different kinetic preferences of PPK1 and PPK2 can be observed. The implications of these results for the application of PPKs in chemical synthesis and as enzymes for ATP regeneration systems are discussed.

4.
Pflugers Arch ; 473(1): 79-93, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33200256

RESUMEN

The renal distal convoluted tubule (DCT) is critical for the fine-tuning of urinary ion excretion and the control of blood pressure. Ion transport along the DCT is tightly controlled by posttranscriptional mechanisms including a complex interplay of kinases, phosphatases, and ubiquitin ligases. Previous work identified the transcription factor Prox-1 as a gene significantly enriched in the DCT of adult mice. To test if Prox-1 contributes to the transcriptional regulation of DCT function and structure, we developed a novel mouse model (NCCcre:Prox-1flox/flox) for an inducible deletion of Prox-1 specifically in the DCT. The deletion of Prox-1 had no obvious impact on DCT structure and growth independent whether the deletion was achieved in newborn or adult mice. Furthermore, DCT-specific Prox-1 deficiency did not alter DCT-proliferation in response to loop diuretic treatment. Likewise, the DCT-specific deletion of Prox-1 did not cause other gross phenotypic abnormalities. Body weight, urinary volume, Na+ and K+ excretion as well as plasma Na+, K+, and aldosterone levels were similar in Prox-1DCTKO and Prox-1DCTCtrl mice. However, Prox-1DCTKO mice exhibited a significant hypomagnesemia with a profound downregulation of the DCT-specific apical Mg2+ channel TRPM6 and the NaCl cotransporter (NCC) at both mRNA and protein levels. The expression of other proteins involved in distal tubule Mg2+ and Na+ handling was not affected. Thus, Prox-1 is a DCT-enriched transcription factor that does not control DCT growth but contributes to the molecular control of DCT-dependent Mg2+ homeostasis in the adult kidney.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Túbulos Renales Distales/efectos de los fármacos , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Canales Catiónicos TRPM/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Eliminación de Gen , Proteínas de Homeodominio/genética , Túbulos Renales Distales/citología , Magnesio/metabolismo , Ratones , Potasio/metabolismo , Sodio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Canales Catiónicos TRPM/genética , Proteínas Supresoras de Tumor/genética
5.
Kidney Int ; 100(4): 850-869, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34252449

RESUMEN

Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria. Consistent with the hypomagnesemia, CnB1-KO mice showed a downregulation of proteins implicated in DCT magnesium transport, including TRPM6, CNNM2, SLC41A3 and parvalbumin but expression of calcium channel TRPV5 in the kidney was unchanged. The abundance of the chloride/bicarbonate exchanger pendrin was increased, likely explaining the acidosis. Plasma aldosterone levels, kidney renin expression, abundance of phosphorylated sodium chloride-cotransporter and abundance of the epithelial sodium channel were similar in control and CnB1-KO mice, consistent with a normal sodium balance. Long-term potassium homeostasis was maintained in CnB1-KO mice, but in-vivo and ex-vivo experiments indicated that CnB1 contributes to acute regulation of potassium balance and sodium chloride-cotransporter. Tacrolimus treatment of control and CnB1-KO mice demonstrated that CNI-related hypomagnesemia is linked to impaired calcineurin-signaling in DCT, while hypocalciuria and hyponatremia occur independently of CnB1 in DCT. Transcriptome and proteome analyses of isolated DCTs demonstrated that CnB1 deletion impacts the expression of several DCT-specific proteins and signaling pathways. Thus, our data support a critical role of calcineurin for DCT function and provide novel insights into the pathophysiology of CNI side effects and involved molecular players in the DCT.


Asunto(s)
Acidosis , Magnesio , Animales , Calcineurina/genética , Túbulos Renales Distales , Ratones , Proteoma/genética , Transcriptoma
6.
Yeast ; 37(1): 163-172, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31606910

RESUMEN

The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (-)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-ß-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.


Asunto(s)
Ingeniería Metabólica/métodos , Naftoles/metabolismo , Saccharomyces cerevisiae/metabolismo , Biocatálisis , Furanos , Microorganismos Modificados Genéticamente , Naftalenos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Escualeno/metabolismo , Escualeno-Monooxigenasa/metabolismo , Terbinafina/metabolismo , Triterpenos/metabolismo
7.
J Am Soc Nephrol ; 30(5): 737-750, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30902838

RESUMEN

BACKGROUND: A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT. METHODS: We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. RESULTS: Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)-dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. CONCLUSIONS: Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.


Asunto(s)
Transporte Biológico/efectos de los fármacos , Colforsina/farmacología , Túbulos Renales Distales/metabolismo , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteínas/farmacología , Análisis de Varianza , Animales , Transporte Biológico/genética , Humanos , Immunoblotting , Técnicas In Vitro , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , Transducción de Señal/genética , Cloruro de Sodio/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo
8.
Appl Microbiol Biotechnol ; 103(14): 5501-5516, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31129740

RESUMEN

More than 70,000 different terpenoid structures are known so far; many of them offer highly interesting applications as pharmaceuticals, flavors and fragrances, or biofuels. Extraction of these compounds from their natural sources or chemical synthesis is-in many cases-technically challenging with low or moderate yields while wasting valuable resources. Microbial production of terpenoids offers a sustainable and environment-friendly alternative starting from simple carbon sources and, frequently, safeguards high product specificity. Here, we provide an overview on employing recombinant bacteria and yeasts for heterologous de novo production of terpenoids. Currently, Escherichia coli and Saccharomyces cerevisiae are the two best-established production hosts for terpenoids. An increasing number of studies have been successful in engineering alternative microorganisms for terpenoid biosynthesis, which we intend to highlight in this review. Moreover, we discuss the specific engineering challenges as well as recent advances for microbial production of different classes of terpenoids. Rationalizing the current stages of development for different terpenoid production hosts as well as future prospects shall provide a valuable decision basis for the selection and engineering of the cell factory(ies) for industrial production of terpenoid target molecules.


Asunto(s)
Bacterias/metabolismo , Escherichia coli/metabolismo , Ingeniería Metabólica , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Bacterias/genética , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Levaduras/genética , Levaduras/metabolismo
9.
J Cell Sci ; 129(1): 191-205, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26644182

RESUMEN

PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.


Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Fosfoserina/metabolismo , Secuencia de Aminoácidos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Semivida , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Interfase/efectos de los fármacos , Mitógenos/farmacología , Datos de Secuencia Molecular , Oncogenes , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
10.
Fungal Genet Biol ; 89: 114-125, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26898115

RESUMEN

Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.


Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Pichia/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Biotransformación , Medios de Cultivo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Perfilación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Mentha piperita/enzimología , Oxidación-Reducción , Pichia/enzimología , Pichia/crecimiento & desarrollo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Sesquiterpenos/metabolismo , Terpenos/metabolismo , Regulación hacia Arriba
11.
Chromosome Res ; 19(3): 307-19, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21461697

RESUMEN

Proper mitotic chromosome structure is essential for faithful chromosome segregation. Mounting evidence suggests that mitotic chromosome assembly is a progressive, dynamic process that requires topoisomerase II, condensins and cohesin and the activity of several signalling molecules. Current results suggest how these different activities might interact to achieve the familiar form of the mitotic chromosome.


Asunto(s)
Cromosomas/química , Cromosomas/metabolismo , Mitosis/genética , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Humanos , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Cohesinas
12.
PLoS Genet ; 5(4): e1000451, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19360121

RESUMEN

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2-null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Ciclo Celular , Proteínas de Unión a Telómeros/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proliferación Celular , Desarrollo Embrionario , Células Germinativas/citología , Células Germinativas/crecimiento & desarrollo , Células Germinativas/metabolismo , Proteínas de Unión a Telómeros/genética
13.
Proc Natl Acad Sci U S A ; 105(35): 12879-84, 2008 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-18728180

RESUMEN

Since cdc48 mutants were isolated by the first genetic screens for cell division cycle (cdc) mutants in yeast, the requirement of the chaperone-like ATPase Cdc48/p97 during cell division has remained unclear. Here, we discover an unanticipated function for Caenorhabditis elegans CDC-48 in DNA replication linked to cell cycle control. Our analysis of the CDC-48(UFD-1/NPL-4) complex identified a general role in S phase progression of mitotic cells essential for embryonic cell division and germline development of adult worms. These developmental defects result from activation of the DNA replication checkpoint caused by replication stress. Similar to loss of replication licensing factors, DNA content is strongly reduced in worms depleted for CDC-48, UFD-1, and NPL-4. In addition, these worms show decreased DNA synthesis and hypersensitivity toward replication blocking agents. Our findings identified a role for CDC-48(UFD-1/NPL-4) in DNA replication, which is important for cell cycle progression and genome stability.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Replicación del ADN , Proteínas Nucleares/metabolismo , Animales , Caenorhabditis elegans/embriología , Cromatina/patología , Regulación hacia Abajo , Embrión no Mamífero/citología , Fase S , Proteína que Contiene Valosina
14.
Acta Physiol (Oxf) ; 233(1): e13705, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34114742

RESUMEN

AIM: The phosphorylation level of the furosemide-sensitive Na+ -K+ -2Cl- cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti-pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform-specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice. METHODS: Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouse NKCC2 antibody was developed and characterized. RESULTS: Amino acids sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acids deletion (ΔF97-T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross-react with the phosphorylated thiazide-sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross-reactivity with phosphorylated NCC. CONCLUSION: Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat.


Asunto(s)
Aminoácidos , Simportadores de Cloruro de Sodio-Potasio , Aminoácidos/metabolismo , Animales , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo
15.
Metab Eng Commun ; 7: e00077, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30197866

RESUMEN

The triterpenoid (+)-ambrein is a natural precursor for (-)-ambrox, which constitutes one of the most sought-after fragrances and fixatives for the perfume industry. (+)-Ambrein is a major component of ambergris, an intestinal excretion of sperm whales that is found only serendipitously. Thus, the demand for (-)-ambrox is currently mainly met by chemical synthesis. A recent study described for the first time the applicability of an enzyme cascade consisting of two terpene cyclases, namely squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AaSHC D377C) and tetraprenyl-ß-curcumene cyclase from Bacillus megaterium (BmeTC) for in vitro (+)-ambrein production starting from squalene. Yeasts, such as Pichia pastoris, are natural producers of squalene and have already been shown in the past to be excellent hosts for the biosynthesis of hydrophobic compounds such as terpenoids. By targeting a central enzyme in the sterol biosynthesis pathway, squalene epoxidase Erg1, intracellular squalene levels in P. pastoris could be strongly enhanced. Heterologous expression of AaSHC D377C and BmeTC and, particularly, development of suitable methods to analyze all products of the engineered strain provided conclusive evidence of whole-cell (+)-ambrein production. Engineering of BmeTC led to a remarkable one-enzyme system that was by far superior to the cascade, thereby increasing (+)-ambrein levels approximately 7-fold in shake flask cultivation. Finally, upscaling to 5 L bioreactor yielded more than 100 mg L-1 of (+)-ambrein, demonstrating that metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-ambrein.

16.
J Biotechnol ; 235: 112-20, 2016 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-27046070

RESUMEN

The production of isoprenoids in recombinant microbes for flavor & fragrance, pharmaceutical, agricultural or fuel applications is a booming research field. Isoprenoid extraction from natural resources and chemical synthesis is frequently neither ecological nor commercially profitable. However, recombinant microbes also show severe limitations in specific isoprenoid synthesis. Therefore, diverse directed evolution strategies have been developed for recombinant microbes. The focus has been laid either on the overall engineering of recombinant hosts or on the improvement of isoprenoid synthases. Currently, the most prominent and advanced approaches are based on carotenoid-producing strains, which can be screened by simple colorimetric readout. Other screening strategies are based on spectrophotometric analyses of colored by-products, fluorescence applications, growth selection and, to a minor extent, the use of biosensors indicating the pool of isoprenoid precursors. Although the number of approaches is still small, we observe a trend towards rigorous and highly creative assays that, however, often rely on the indirect detection of the evolved enzyme activities or host strains. We conclude that the use of whole-cellular systems is clearly favored over cell extracts and predict that next-generation screening assays need to be developed towards broader applicability and more direct assessment of isoprenoid production levels.


Asunto(s)
Bacterias , Ingeniería Metabólica/métodos , Terpenos/metabolismo , Transferasas Alquil y Aril , Bacterias/química , Bacterias/metabolismo , Carotenoides , Hongos/química , Hongos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Ácido Mevalónico , Espectrometría de Fluorescencia
17.
Sci Rep ; 6: 26178, 2016 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-27185577

RESUMEN

SUMO and ubiquitin play important roles in the response of cells to DNA damage. These pathways are linked by the SUMO Targeted ubiquitin Ligase Rnf4 that catalyses transfer of ubiquitin from a ubiquitin loaded E2 conjugating enzyme to a polySUMO modified substrate. Rnf4 can functionally interact with multiple E2s, including Ube2w, in vitro. Chicken cells lacking Rnf4 are hypersensitive to hyroxyurea, DNA alkylating drugs and DNA crosslinking agents, but this sensitivity is suppressed by simultaneous depletion of Ube2w. Cells depleted of Ube2w alone are not hypersensitive to the same DNA damaging agents. Similar results were also obtained in human cells. These data indicate that Ube2w does not have an essential role in the DNA damage response, but is deleterious in the absence of Rnf4. Thus, although Rnf4 and Ube2w functionally interact in vitro, our genetic experiments indicate that in response to DNA damage Ube2w and Rnf4 function in distinct pathways.


Asunto(s)
Daño del ADN , Proteínas Nucleares/deficiencia , Factores de Transcripción/deficiencia , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Células Cultivadas , Pollos , Humanos
18.
Dev Cell ; 26(4): 381-92, 2013 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-23932902

RESUMEN

PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell's metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.


Asunto(s)
Ciclo Celular/efectos de los fármacos , Centrosoma/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Oxígeno/farmacología , Secuencia de Aminoácidos , Centriolos/efectos de los fármacos , Centriolos/metabolismo , Proteínas Cromosómicas no Histona/química , Células HeLa , Humanos , Hidroxilación/efectos de los fármacos , Mitosis/efectos de los fármacos , Modelos Biológicos , Datos de Secuencia Molecular , Prolina/metabolismo , Unión Proteica , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Ubiquitinación/efectos de los fármacos
19.
Methods Cell Biol ; 107: 321-52, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22226529

RESUMEN

In response to genotoxic insults, cells activate DNA damage response pathways that either stimulate transient cell cycle arrest and DNA repair or induce apoptosis. The Caenorhabditis elegans germ line is now well established as a model system to study these processes in a genetically tractable, multicellular organism. Upon treatment with genotoxic agents, premeiotic C. elegans germ cells transiently halt cell cycle progression, whereas meiotic prophase germ cells in the late-pachytene stage undergo apoptosis. Further, accumulation of unrepaired meiotic recombination intermediates can also lead to apoptosis of affected pachytene cells. DNA damage-induced cell death requires key components of the evolutionarily conserved apoptotic machinery. Moreover, both cell cycle arrest and pachytene apoptosis responses depend on conserved DNA damage checkpoint proteins. Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways. In this chapter, we briefly review the C. elegans DNA damage response field, discuss in detail methods currently used to assay DNA damage responses in C. elegans, and describe the development of new experimental tools that will facilitate a more comprehensive understanding of the DNA damage response.


Asunto(s)
Bioensayo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Reparación del ADN , Células Germinativas/metabolismo , Larva/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Biomarcadores/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de la radiación , Proteínas de Caenorhabditis elegans/genética , Daño del ADN , Rayos gamma , Células Germinativas/efectos de los fármacos , Células Germinativas/efectos de la radiación , Hidroxiurea/farmacología , Larva/efectos de los fármacos , Larva/efectos de la radiación , Meiosis/efectos de los fármacos , Meiosis/genética , Meiosis/efectos de la radiación , Mitosis/efectos de los fármacos , Mitosis/genética , Mitosis/efectos de la radiación , Interferencia de ARN , Transducción de Señal/genética
20.
J Biol Chem ; 281(16): 11301-11, 2006 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-16461354

RESUMEN

The molecular chaperone Hsp90 is required for the folding and activation of a large number of substrate proteins. These are involved in essential cellular processes ranging from signal transduction to viral replication. For the activation of its substrates, Hsp90 binds and hydrolyzes ATP, which is the key driving force for conformational conversions within the dimeric chaperone. Dimerization of Hsp90 is mediated by a C-terminal dimerization site. In addition, there is a transient ATP-induced dimerization of the two N-terminal ATP-binding domains. The resulting ring-like structure is thought to be the ATPase-active conformation. Hsp90 is a slow ATPase with a turnover number of 1 ATP/min for the yeast protein. A key question for understanding the molecular mechanism of Hsp90 is how ATP hydrolysis is regulated and linked to conformational changes. In this study, we analyzed the activation process structurally and biochemically with a view to identify the conformational limitations of the ATPase reaction cycle. We showed that the first 24 amino acids stabilize the N-terminal domain in a rigid state. Their removal confers flexibility specifically to the region between amino acids 98 and 120. Most surprisingly, the deletion of this structure results in the complete loss of ATPase activity and in increased N-terminal dimerization. Complementation assays using heterodimeric Hsp90 show that this rigid lid acts as an intrinsic kinetic inhibitor of the Hsp90 ATPase cycle preventing N-terminal dimerization in the ground state. On the other hand, this structure acts, in concert with the 24 N-terminal amino acids of the other N-terminal domain, to form an activated ATPase and thus regulates the turnover number of Hsp90.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Dicroismo Circular , Clonación Molecular , Cristalografía por Rayos X , Difusión , Dimerización , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Hidrólisis , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Transducción de Señal , Especificidad por Sustrato , Factores de Tiempo , Urea/farmacología
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