The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence

Funding: This work was funded by the European Research Council [http://erc.europa.eu/], AJPB (STRIFE Advanced Grant; C-2009-AdG-249793). The work was also supported by: the Wellcome Trust [www.wellcome.ac.uk], AJPB (080088, 097377); the UK Biotechnology and Biological Research Council [www.bbsrc.ac.uk], AJPB (BB/F00513X/1, BB/K017365/1); the CNPq-Brazil [http://cnpq.br], GMA (Science without Borders fellowship 202976/2014-9); and the National Centre for the Replacement, Refinement and Reduction of Animals in Research [www.nc3rs.org.uk], DMM (NC/K000306/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments We thank Dr. Elizabeth Johnson (Mycology Reference Laboratory, Bristol) for providing strains, and the Aberdeen Proteomics facility for the biotyping of S. cerevisiae clinical isolates, and to Euroscarf for providing S. cerevisiae strains and plasmids. We are grateful to our Microscopy Facility in the Institute of Medical Sciences for their expert help with the electron microscopy, and to our friends in the Aberdeen Fungal Group for insightful discussions.Peer reviewedPublisher PD

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

Background The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). Results The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. Conclusions E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Rovitto, Bruno David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

Robust Metabolic Responses to Varied Carbon Sources in Natural and Laboratory Strains of Saccharomyces cerevisiae

Understanding factors that regulate the metabolism and growth of an organism is of fundamental biologic interest. This study compared the influence of two different carbon substrates, dextrose and galactose, on the metabolic and growth rates of the yeast Saccharomyces cerevisiae. Yeast metabolic and growth rates varied widely depending on the metabolic substrate supplied. The metabolic and growth rates of a yeast strain maintained under long-term laboratory conditions was compared to strain isolated from natural condition when grown on different substrates. Previous studies had determined that there are numerous genetic differences between these two strains. However, the overall metabolic and growth rates of a wild isolate of yeast was very similar to that of a strain that had been maintained under laboratory conditions for many decades. This indicates that, at in least this case, metabolism and growth appear to be well buffered against genetic differences. Metabolic rate and cell number did not co-vary in a simple linear manner. When grown in either dextrose or galactose, both strains showed a growth pattern in which the number of cells continued to increase well after the metabolic rate began a sharp decline. Previous studied have reported that O2 consumption in S. cerevisiae grown in reduced dextrose levels were elevated compared to higher levels. Low dextrose levels have been proposed to induce caloric restriction and increase life span in yeast. However, there was no evidence that reduced levels of dextrose increased metabolic rates, measured by either O2 consumption or CO2 production, in the strains used in this study

Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations

A Linear Framework for Time-Scale Separation in Nonlinear Biochemical Systems

Cellular physiology is implemented by formidably complex biochemical systems with highly nonlinear dynamics, presenting a challenge for both experiment and theory. Time-scale separation has been one of the few theoretical methods for distilling general principles from such complexity. It has provided essential insights in areas such as enzyme kinetics, allosteric enzymes, G-protein coupled receptors, ion channels, gene regulation and post-translational modification. In each case, internal molecular complexity has been eliminated, leading to rational algebraic expressions among the remaining components. This has yielded familiar formulas such as those of Michaelis-Menten in enzyme kinetics, Monod-Wyman-Changeux in allostery and Ackers-Johnson-Shea in gene regulation. Here we show that these calculations are all instances of a single graph-theoretic framework. Despite the biochemical nonlinearity to which it is applied, this framework is entirely linear, yet requires no approximation. We show that elimination of internal complexity is feasible when the relevant graph is strongly connected. The framework provides a new methodology with the potential to subdue combinatorial explosion at the molecular level

Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1

Protein tyrosine phosphatases in glioma biology

Gliomas are a diverse group of brain tumors of glial origin. Most are characterized by diffuse infiltrative growth in the surrounding brain. In combination with their refractive nature to chemotherapy this makes it almost impossible to cure patients using combinations of conventional therapeutic strategies. The drastically increased knowledge about the molecular underpinnings of gliomas during the last decade has elicited high expectations for a more rational and effective therapy for these tumors. Most studies on the molecular pathways involved in glioma biology thus far had a strong focus on growth factor receptor protein tyrosine kinase (PTK) and phosphatidylinositol phosphatase signaling pathways. Except for the tumor suppressor PTEN, much less attention has been paid to the PTK counterparts, the protein tyrosine phosphatase (PTP) superfamily, in gliomas. PTPs are instrumental in the reversible phosphorylation of tyrosine residues and have emerged as important regulators of signaling pathways that are linked to various developmental and disease-related processes. Here, we provide an overview of the current knowledge on PTP involvement in gliomagenesis. So far, the data point to the potential implication of receptor-type (RPTPδ, DEP1, RPTPμ, RPTPζ) and intracellular (PTP1B, TCPTP, SHP2, PTPN13) classical PTPs, dual-specific PTPs (MKP-1, VHP, PRL-3, KAP, PTEN) and the CDC25B and CDC25C PTPs in glioma biology. Like PTKs, these PTPs may represent promising targets for the development of novel diagnostic and therapeutic strategies in the treatment of high-grade gliomas