Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

CIBERER: Spanish national network for research on rare diseases: A highly productive collaborative initiative

13 páginas,1 figura, 3 tablas, 1 apéndice. Se extraen los autores pertenecientes a The CIBERER network que trabajan en Centros del CSIC del Appendix ACIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research.This study has been funded by Instituto de Salud Carlos III (ISCIII) and Spanish Ministry of Science and InnovationPeer reviewe

Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis

15 páginas, 5 figurasHuman carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations.This work was supported by grants from The Fundación Alicia Koplowitz, the Valencian (PrometeoII/2014/029 to V.R) and Spanish Governments (BFU2011-30407 and BFU2014-58229-P to V.R; SAF2010-17933 to J.C; BFU2012-36827 to I.F.). The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement Nº283570), within proposal 7687.Peer reviewe

Structural analysis of the adaptor protein Nck1

Trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Santander (España) del 10 al 13 de septiembre de 2018

Mechanism of autoinhibition of the guanine nucleotide exchange factor C3G

Resumen del trabajo presentado en el 41 Congreso de la SEBBM (Sociedad Española de Bioquímica y Biología Molecular), celebrado en Santander (España) del 10 al 13 de septiembre de 2018

Crystal structures of human carbamoyl phosphate synthetase 1 (CPS1) shed light on domains functions, substrate tunnels and allosteric activation, and allow rationalization of inborn CPS1 deficiency

Comunicación presentada en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM Granada2014, celebrada del 9 al 12 de septiembre de 2014 en Granada (España)ABSTRACT: P10-53 CPS1 is a large six-domain protein that constitutes the first step of the urea cycle: the synthesis of carbamoyl phosphate from bicarbonate, ammonia and two ATP molecules. A paramount feature of this enzyme is its absolute requirement for N-acetyl-L-glutamate (NAG), an allosteric activator without which it is inactive. The report of CPS1 regulation by lysine deacylation by NAD-dependent sirtuin 5 connected the urea cycle with the age-control machinery (Nakagawa et al. Cell 2009; 137:560). CPS1 deficiency (CPS1D) is an inborn disorder that cause severe neonatal hyperammonemia leading to mental retardation or even to death. More than 300 mutations have been reported in CPS1D patients, of which the majority are missense mutations showing little recurrence and having unproven disease-causing potential. The structure of the E. coli homologous CPS has been known for >15 years, but differences with CPS1 (40% identity; use of glutamine instead of ammonia; insensitivity to NAG) rendered essential to obtain the structure of CPS1 for proper understanding of its functioning, and for evaluating disease causation by CPS1D mutations. Using a baculovirus/insect cell system we have finally succeeded in producing recombinant human CPS1 in large amount and pure form, allowing us to experimentally examine the effects of reported mutations and ascertain its disease-causing potential (Díez-Fernández et al. Human Mut 2013; 34:1149). In addition we have determined the structure of CPS1, in both apo and ligand-bound (NAG and ADP/Pi) forms. The liganded structure revealed how NAG binds in a pocket of the C-terminal domain and has identified elements stabilized by ADP binding and conformational changes that lead to define the carbamate tunnel, which in the apo form is heavily branched and open to the environment. Our structures decipher the CPS1 inability to use glutamine and reveal a potential channel for ammonia intake. Furthermore, they help rationalize the disease-causing role of most clinical CPS1 mutations. Supported by Fundación Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish (BFU2011-30407; FPU to CD-F) governments.Supported by Fundación Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish (BFU2011-30407; FPU to CD-F) governmentsPeer Reviewe

Human carbamoyl phosphate synthetase: structure, function and pathology

Comunicación presentada en ICAP2014, 24th International Conference on Arginine and Pyrimidines, organizado del 16 al 18 de julio de 2014 en Oxford (Reino Unido)Abstract publicado en FEBS Journal 281(Supl.1): 570-571 (2014)Abstract: TUE 420; pp: 570. Carbamoyl phosphate synthesis from bicarbonate, ammonia and two molecules of ATP, catalyzed by carbamoyl phosphate synthetase 1 (CPS1), is the first step of of the urea cycle, which detoxifies the ammonia produced in protein catabolism. CPS1, a large (1462-residue) multidomain protein having two ATP-binding phosphorylation sites, is very abundant in liver mitocondria (20% of the matrix protein), and is inactive in the absence of the allosteric activator N-acetyl-L-glutamate (NAG). CPS1 deficiency (CPS1D) is an inborn error causing hyperammonemia leading to death or mental retardation. The report of CPS1 regulation by multiple lysine acylation and by deacylation by sirtuin 5 connected the urea cycle with the age-control machinery (Nakagawa et al. Cell 2009; 137:560). The only structure known for a CPS was that of the Escherichia coli enzyme, which only has 40% sequence identity with CPS1 and differs from it in key traits such as the use of glutamine instead of ammonia as preferred substrate (CPS1 cannot use glutamine), its insensitivity to NAG, and for being active in the absence of effectors. Thus, determination of the CPS1 structure appeared essential for understanding CPS1 function and its control by the NAG switch (an extreme case of allosteric activation) and by acylation, and to judge about the pathogenicity of the >130 CPS1 missense mutations reported in CPS1 deficiency. Exploiting our recent baculovirus/insect cell system for recombinant human CPS1 production (Diez-Fernandez et al., Hum Mutat. 2013; 34:1149¿59), we have crystallized the human enzyme and determined its X-ray structure at up to 2.4 A-resolution, in apo and ligand-bound (NAG and ADP/Pi) forms. The liganded structure revealed how does NAG bind in a pocket of the C-terminal domain and has identified elements that are stabilized by ADP binding, as well as conformational changes induced by NAG and ADP binding that lead to define the carbamate tunnel, which in the apo form is heavily branched and open to the environment. Our structures decipher the CPS1 inability to use glutamine and reveal a potential channel for ammonia intake. Furthermore, they help rationalize the disease-causing role of most clinical CPS1 mutations. Supported by Fundacion Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish (BFU2011-30407; FPU to CD-F) governmentsPeer Reviewe

Looking at Metabolic Regulation and Inborn Errors from a Structural Viewpoint: a Urea Cycle Example

Comunicación presentada en 2nd FEBS Fellows' Meeting, celebrado del 27 al 30 de agosto de 2014 en París (Francia)The urea cycle gets rid of the ammonia derived from protein catabolism, which is highly neurotoxic. Carbamoyl phosphate synthetase 1 (CPS1), a large (1462 residues) six-domain enzyme catalyzing a three-step reaction involving two separate phosphorylation centers, is a key urea cycle catalyst and controller. lt is inactive in the absence of its allosteric activator Nacetyi-L-glutamate (NAG), which is an on/off switch used to prevent amino acid depletion. CPS1 experiences multiple lysine acylation, and deacylation by sirtuin 5, connecting urea cycle control and age-control machinery. >120 CPS1 missense mutations have been reported in patients with CPS1 deficiency (CPS1 D), a urea cycle disorder causing hyperammonemia leading to mental retardation or even to death. Using a baculovirus/insect cell system we have produced pure recombinant human CPS1, which has allowed us to crystallize the enzyme and to determine its structure at 2.4 A-resolution, in ligand-free and NAG and ADP/Pi-bound forms, allowing to understand NAG activation and to place on firm ground our understanding of CPS 1 D, while opening the way for understanding the effects of acylation. Thus, the structure with ligands revealed how does NAG bind in a pocket of the C-terminal domain and has identified elements that are stabilized by ADP binding, as well as conformational changes induced by NAG and ADP binding that lead to define the carbamate tunnel, which in the apo form is heavily branched and open to the environment. Our structures decipher the CPS1 inability to use glutamine and reveal a potential channel for ammonia intake, which account for the lack of usage of glutamine and the utilization of ammonia with high affinity by this enzyme. Furthermore, they help rationalize the disease-causing role of most clinical CPS1 mutations.Supported by Fundación Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish (BFU2011-30407; FPU to CO-F) governmentsPeer Reviewe

Deciphering carbamoyl phosphate synthetase (CPS1) deficiency and urea cycle regulation by determining the structures of human CPS1 in the absence and in the presence of N-acetyl-L-glutamate.

Trabajo presentado en el Annual Symposium of the Society for the Study of Inborn Errors of Metabolism SSIEM, celebrado en Roma, Italia, del 6 al 9 de septiembre de 2016Peer Reviewe