Aurora Martinez

Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH). Over 500 disease-causing mutations have been identified in humans, most of which result in PAH protein misfolding and increased turnover in vivo. The use of pharmacological chaperones to stabilize or promote correct folding of mutant proteins represents a promising new direction in the treatment of misfolding diseases. We performed a high-throughput ligand screen of over 1,000 pharmacological agents and identified 4 compounds (I-IV) that enhanced the thermal stability of PAH and did not show substantial inhibition of PAH activity. In further studies, compounds III (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) and IV (5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno[2,3- d]pyrimidin-4(1H)-one) stabilized the functional tetrameric conformation of recombinant WT-PAH and PKU mutants. These compounds also significantly increased activity and steady-state PAH protein levels in cells transiently transfected with either WT-PAH or PKU mutants. Furthermore, PAH activity in mouse liver increased after a 12-day oral administration of low doses of compounds III and IV. Thus, we have identified 2 small molecules that may represent promising alternatives in the treatment of PKU.

Predicted Effects of Missense Mutations on Native-State Stability Account for Phenotypic Outcome in Phenylketonuria, a Paradigm of Misfolding Diseases

Phenylketonuria (PKU) is a genetic disease caused by mutations in human phenylalanine hydroxylase (PAH). Most missense mutations result in misfolding of PAH, increased protein turnover, and a loss of enzymatic function. We studied the prediction of the energetic impact on PAH native-state stability of 318 PKU-associated missense mutations, using the protein-design algorithm FoldX. For the 80 mutations for which expression analyses have been performed in eukaryote systems, in most cases we found substantial overall correlations between the mutational energetic impact and both in vitro residual activities and patient metabolic phenotype. This finding confirmed that the decrease in protein stability is the main molecular pathogenic mechanism in PKU and the determinant for phenotypic outcome. Metabolic phenotypes have been shown to be better predicted than in vitro residual activities, probably because of greater stringency in the phenotyping process. Finally, all the remaining 238 PKU missense mutations compiled at the PAH locus knowledgebase (PAHdb) were analyzed, and their phenotypic outcomes were predicted on the basis of the energetic impact provided by FoldX. Residues in exons 7-9 and in interdomain regions within the subunit appear to play an important structural role and constitute hotspots for destabilization. FoldX analysis will be useful for predicting the phenotype associated with rare or new mutations detected in patients with PKU. However, additional factors must be considered that may contribute to the patient phenotype, such as possible effects on catalysis and interindividual differences in physiological and metabolic processes.

Epac1 and cAMP-dependent Protein Kinase Holoenzyme Have Similar cAMP Affinity, but Their cAMP Domains Have Distinct Structural Features and Cyclic Nucleotide Recognition

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a Kd value (2.9 μm) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had Kd values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIα led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.

Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus.

1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH4)2SO4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. Both trypsins catalyzed the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. The enzymes had a pH optimum of 8-9 for the hydrolysis of BAPNA and of 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. Trypsins A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca2+ for activity and stability.

Principal component and normal mode analysis of proteins; a quantitative comparison using the GroEL subunit

Principal component analysis (PCA) and normal mode analysis (NMA) have emerged as two invaluable tools for studying conformational changes in proteins. To compare these approaches for studying protein dynamics, we have used a subunit of the GroEL chaperone, whose dynamics is well characterized. We first show that both PCA on trajectories from molecular dynamics (MD) simulations and NMA reveal a general dynamical behavior in agreement with what has previously been described for GroEL. We thus compare the reproducibility of PCA on independent MD runs and subsequently investigate the influence of the length of the MD simulations. We show that there is a relatively poor one‐to‐one correspondence between eigenvectors obtained from two independent runs and conclude that caution should be taken when analyzing principal components individually. We also observe that increasing the simulation length does not improve the agreement with the experimental structural difference. In fact, relatively short MD simulations are sufficient for this purpose. We observe a rapid convergence of the eigenvectors (after ca. 6 ns). Although there is not always a clear one‐to‐one correspondence, there is a qualitatively good agreement between the movements described by the first five modes obtained with the three different approaches; PCA, all‐atoms NMA, and coarse‐grained NMA. It is particularly interesting to relate this to the computational cost of the three methods. The results we obtain on the GroEL subunit contribute to the generalization of robust and reproducible strategies for the study of protein dynamics, using either NMA or PCA of trajectories from MD simulations. Proteins 2010.

Conformational Properties and Stability of Tyrosine Hydroxylase Studied by Infrared Spectroscopy EFFECT OF IRON/CATECHOLAMINE BINDING AND PHOSPHORYLATION

The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% α-helix, 35% β-extended structures (including β-sheet), 14% β-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in α-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.

The 14-3-3 proteins in regulation of cellular metabolism

Thirty years ago, it was discovered that 14-3-3 proteins could activate enzymes involved in amino acid metabolism. In the following decades, 14-3-3s have been shown to be involved in many different signaling pathways that modulate cellular and whole body energy and nutrient homeostasis. Large scale screening for cellular binding partners of 14-3-3 has identified numerous proteins that participate in regulation of metabolic pathways, although only a minority of these targets have yet been subject to detailed studies. Because of the wide distribution of potential 14-3-3 targets and the resurging interest in metabolic pathway control in diseases like cancer, diabetes, obesity and cardiovascular disease, we review the role of 14-3-3 proteins in the regulation of core and specialized cellular metabolic functions. We cite illustrative examples of 14-3-3 action through their direct modulation of individual enzymes and through regulation of master switches in cellular pathways, such as insulin signaling, mTOR- and AMP dependent kinase signaling pathways, as well as regulation of autophagy. We further illustrate the quantitative impact of 14-3-3 association on signal response at the target protein level and we discuss implications of recent findings showing 14-3-3 protein membrane binding of target proteins.

A Structural Approach into Human Tryptophan Hydroxylase and its Implications for the Regulation of Serotonin Biosynthesis

Tryptophan hydroxylase (TPH) catalyzes the 5-hydroxylation of tryptophan, which is the first step in the biosynthesis of indoleamines (serotonin and melatonin). Serotonin functions mainly as a neurotransmitter, whereas melatonin is the principal hormone secreted by the pineal gland. TPH belongs to the family of the aromatic amino acid hydroxylases, including phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH), which all have a strict requirement for dioxygen, non-heme iron (II) and tetrahydrobiopterin (BH4). During the last three years there has been a formidable increase in the amount of structural information about PAH and TH, which has provided new insights into the active site structure, the binding of substrates, inhibitors and pterins, as well as on the effect of disease-causing mutations in these hydroxylases. Although structural information about TPH is not yet available, the high sequence homology between the three mammalian hydroxylases, notably at the catalytic domains, and the similarity of the reactions that they catalyze, indicate that they share a similar 3D-structure and a common catalytic mechanism. Thus, we have prepared a model of the structure of TPH based on the crystal structures of TH and PAH. This structural model provides a frame for understanding the specific interactions of TPH with L-tryptophan and substrate analogues, BH4 and cofactor analogues, L-DOPA and catecholamines. The interactions of these ligands with the enzyme are discussed focusing on the physiological and pharmacological regulation of serotonin biosynthesis, notably by tryptophan supplementation therapy and substitution therapy with tetrahydrobiopterin analogues (positive effects), as well as the effect of catecholamines on TPH activity in L-DOPA treated Parkinsons disease patients (enzyme inhibition).

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

Protein folding barriers, which range from zero to the tens of RT that result in classical two-state kinetics, are primarily determined by protein size and structural topology [Plaxco KW, Simons KT, Baker D (1998) J Mol Biol 277:985–994]. Here, we investigate the thermodynamic folding barriers of two relatively large proteins of the same size and topology: bovine α-lactalbumin (BLA) and hen-egg-white lysozyme (HEWL). From the analysis of differential scanning calorimetry experiments with the variable-barrier model [Muñoz V, Sanchez-Ruiz JM (2004) Proc Natl Acad Sci USA 101:17646–17651] we obtain a high barrier for HEWL and a marginal folding barrier for BLA. These results demonstrate a remarkable tuning range of at least 30 kJ/mol (i.e., five to six orders of magnitude in population) within a unique protein scaffold. Experimental and theoretical analyses on these proteins indicate that the surprisingly small thermodynamic folding barrier of BLA arises from the stabilization of partially unfolded conformations by electrostatic interactions. Interestingly, there is clear reciprocity between the barrier height and the biological function of the two proteins, suggesting that the marginal barrier of BLA is a product of natural selection. Electrostatic surface interactions thus emerge as a mechanism for the modulation of folding barriers in response to special functional requirements within a given structural fold.

The Membrane-bound Conformation of α-Lactalbumin Studied by NMR-monitored 1H Exchange

Abstract The interaction of bovine α-lactalbumin (BLA) with negatively charged phospholipid bilayers was studied by NMR monitored 1H exchange to characterize the conformational transition that enables a water-soluble protein to associate with and partially insert into a membrane. BLA was allowed to exchange in deuterated buffer in the absence (reference) and the presence (membrane-bound) of acidic liposomes at pH 4.5, experimental conditions that allow efficient protein–membrane interaction. After adjusting the pH to 6.0, to dissociate the protein from the membrane, reference and membrane-released samples of BLA were analysed by (F1) band-selective homonuclear decoupled total correlation spectroscopy in the αH–NH region. The overall exchange behaviour of the membrane-bound state is molten globule-like, suggesting an overall destabilization of the polypeptide. Nevertheless, the backbone amide protons of residues R10, L12, C77, K94, K98, V99 and W104 show significant protection against solvent exchange in the membrane-bound protein. We propose a mechanism for the association of BLA with negatively charged membranes that includes initial protonation of acidic side-chains at the membrane interface, and formation of an interacting site with the membrane which involves helixes A and C. In the next step these helices would slide away from each other, adopting a parallel orientation to the membrane, and would rotate to maximize the interaction between their hydrophobic residues and the lipid bilayer.