309 research outputs found
Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants
The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria
type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele
AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal
segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1
is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation
in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was
studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability
effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and
P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide
by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation
had no effect on the overall AGT function or conformation, but enhanced aggregation of
the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings
indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with
PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation
should be considered a novel regulatory mechanism in PH1 pathogenesis.Comunidad Valenciana CIAICO/2021/135
AULA FUNCANIS-UGRERDF/Spanish Ministry of Science, Innovation, and Universities-State Research Agency RTI2018-096246-B-I00Junta de Andalucia P18-RT-2413
ERDF/ Counseling of Economic transformation, Industry, Knowledge, and Universities B-BIO-84-UGR2
A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation
ALP thanks Professors Jose Manuel Sanchez-Ruiz and Beatriz Ibarra-Molero (both from the University of Granada) for providing access and advice on their home-built software for electrostatic calculations. BR acknowledges kind hospitality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy. This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-BI00 to JLN and RTI2018-096246-B-I00 to ALP]; FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades [Grant P18-RT-2413 to ALP]. Financial support from EU Horizon 2020 project EU FT-ICR MS (731077) as well as institutional (CZ.1.05/1.1.00/02.0109) and MS facility support (LM2018127 CIISB) are gratefully acknowledged. Funding for open charge: Universidad de Granada/CBUA.The phosphomimetic mutation S82D in the cancer-associated, FADdependent
human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a
decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability.
We test in this work whether the evolutionarily recent neutral mutation
R80H in the vicinity of S82 may alter the strong functional effects of S82
phosphorylation through electrostatic interactions. We show using biophysical
and bioinformatic analyses that the reverse mutation H80R prevents the
effects of S82D phosphorylation on hNQO1 by modulating the local stability.
Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation
were milder, resembling the behaviour found in hNQO1 when this
residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently
neutral and evolutionarily divergent mutations may determine the functional
response of mammalian orthologues towards phosphorylation.Spanish GovernmentEuropean ERDF Funds (MCIU/AEI/FEDER, EU) RTI2018-097991-BI00
RTI2018-096246-B-I00FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades P18-RT-2413EU Horizon 2020 project EU FT-ICR MS 731077Universidad de Granada/CBUA
CZ.1.05/1.1.00/02.0109
LM2018127 CIIS
NQO1:A target for the treatment of cancer and neurological diseases, and a model to understand loss of function disease mechanisms
Molecular Recognition of PTS-1 Cargo Proteins by Pex5p: Implications for Protein Mistargeting in Primary Hyperoxaluria
Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3–4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state
Caenorhabditis elegans AGXT-1 is a mitochondrial and temperature-adapted ortholog of peroxisomal human AGT1: New insights into between-species divergence in glyoxylate metabolism
In humans, glyoxylate is an intermediary product of metabolism, whose concentration is finely balanced. Mutations in peroxisomal alanine:glyoxylate aminotransferase (hAGT1) cause primary hyperoxaluria type 1 (PH1), which results in glyoxylate accumulation that is converted to toxic oxalate. In contrast, glyoxylate is used by the nematode Caenorhabditis elegans through a glyoxylate cycle to by-pass the decarboxylation steps of the tricarboxylic acid cycle and thus contributing to energy production and gluconeogenesis from stored lipids. To investigate the differences in glyoxylate metabolism between humans and C. elegans and to determine whether the nematode might be a suitable model for PH1, we have characterized here the predicted nematode ortholog of hAGT1 (AGXT-1) and compared its molecular properties with those of the human enzyme. Both enzymes form active PLP-dependent dimers with high specificity towards alanine and glyoxylate, and display similar three-dimensional structures. Interestingly, AGXT-1 shows 5-fold higher activity towards the alanine/glyoxylate pair than hAGT1. Thermal and chemical stability of AGXT-1 is lower than that of hAGT1, suggesting temperature-adaptation of the nematode enzyme linked to the lower optimal growth temperature of C. elegans. Remarkably, in vivo experiments demonstrate the mitochondrial localization of AGXT-1 in contrast to the peroxisomal compartmentalization of hAGT1. Our results support the view that the different glyoxylate metabolism in the nematode is associated with the divergent molecular properties and subcellular localization of the alanine:glyoxylate aminotransferase activity.This work was supported by the Spanish Ministry of Science and Innovation (CSD2009-00088, BIO2012-34937 and SAF2011-23933), the Junta de Andalucia (P11-CTS-7187), and the Oxalosis and Hyperoxaluria Foundation (OHF2012 to B.C.). A.L.P. acknowledges a Ramon y Cajal research contract (RyC2009-04147) from the Spanish Ministry of Science and Innovation and the University of Granada. N. M-T acknowledges a FPI predoctoral fellowship from the Spanish Ministry of Science and Innovation. A.C.C. and N.T. were supported by the grant IOS-1353845 from the National Science Foundation (NSF). N.T. acknowledges the Tetelman Fellowship for International Research on the Sciences awarded by Yale University.Peer Reviewe
Determination of Ligand Profiles for Pseudomonas aeruginosa Solute Binding Proteins
Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all
kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing
the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed
of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant
ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine,
or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have
purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches
followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of
them. Experimentation revealed that PA0222 was specific for
y-aminobutyrate (GABA), DppA2 for
tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine.
Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate,
whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands
were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be
predicted with confidence using bioinformatic methods, but experimental work is necessary to
identify the precise ligand profile.This work was supported by FEDER funds and Fondo Social Europeo through a grant from the Spanish
Ministry for Economy and Competitiveness to T. Krell (BIO2016-76779-P). This funding source was not involved
in the design and conduct of this stud
Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution
Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine: glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases.Spanish Ministry of Economy and Competitiveness, MINECO (BIO 2015 66426-R to JMSR, CTQ 2015-64445-R to JLN, ‘Factoría Española de Cristalización’, Consolider-Ingenio 2010 to JAG and SAF2015-69796 to ES), Junta de Andalucia (P11-CTS-07187 to ALP) and FEDER fun
High-affinity chemotaxis to histamine of the human pathogen Pseudomonas aeruginosa
FEMS 2019: 8th congress of European Microbiologists, in Glasgow, Scotland, 7-11 July 201
Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands
Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function
Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution
Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases
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