Ming Ying

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.

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding.

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca(2+)/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser(19), but PKA also phosphorylated Ser(104), as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser(19), Ser(99), Ser(104) and Ser(306). On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser(19). This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins gamma, epsilon and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1–43)) and Ser19-phosphorylated (THp-(1–43)) states by surface plasmon resonance. THp-(1–43) showed high affinity for 14-3-3 proteins (Kd ∼ 0.5 μm for 14-3-3γ and -ζ and 7 μm for 14-3-3η). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S0.5 = 25–58 μm (TH-(1–43)) and S0.5 = 135–475 μm (THp-(1–43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3γ showed a preferential binding to membranes, compared with 14-3-3ζ, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3γ for negatively charged membranes (S0.5 = 1–9 μm) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser19-phosphorylated TH, 14-3-3γ, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of l-DOPA and dopamine synthesis.

Binding of the viral immunogenic octapeptide VSV8 to native glucose-regulated protein Grp94 (gp96) and its inhibition by the physiological ligands ATP and Ca2+

The molecular chaperone Grp94 (gp96) of the endoplasmic reticulum (ER) lumen plays an essential role in the structural maturation and/or secretion of proteins destined for transport to the cell surface. Its proposed role in binding and transferring peptides for immune recognition is, however, controversial. Using SPR spectroscopy, we studied the interaction of native glycosylated Grp94 at neutral pH and 25 and 37 °C with the viral immunogenic octapeptide RGYVYQGL (VSV8), derived from vesicular stomatitis virus nucleoprotein (52–59). The peptide binds reversibly with low affinity ([A]0.5 ≈ 640 µm) and a hyperbolic binding isotherm, and the binding is partially inhibited by ATP and Ca2+ at concentrations that are present in the ER lumen, and the effects are explained by conformational changes in the native chaperone induced by these ligands. Our data present experimental support for the recent proposal that, under native conditions, VSV8 binds to Grp94 by an adsorptive, rather than a bioselective, mechanism, and thus further challenge the proposed in vivo peptide acceptor–donor function of the chaperone in the context of antigen‐presenting cell activation.

Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans

In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah‐1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S0.5(L‐Phe)~150 μΜ; Κm for tetrahydrobiopterin (BH4)~35 μΜ and comparable Vmax], but cePAH is devoid of positive cooperativity for L‐Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L‐Phe. Pah‐1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah‐1 knockouts lack a yelloworange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah‐1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution.—Calvo, A. C., Pey, A. L., Ying, M., Loer, C. M., Martinez, A. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans. FASEB J. 22, 3046–3058 (2008)

HAMLET Forms Annular Oligomers When Deposited with Phospholipid Monolayers

Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLETs bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLETs tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.

Effect of pharmacological chaperones on brain tyrosine hydroxylase and tryptophan hydroxylase 2

J. Neurochem. (2010) 114, 853–863.

Colocalization of Ca2+-ATPase and GRP94 with p58 and the effects of thapsigargin on protein recycling suggest the participation of the pre-Golgi intermediate compartment in intracellular Ca2+ storage.

We have studied the localization of functional components of cellular Ca2+ transport and storage and the effects of thapsigargin (TG), a specific inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), with respect to the p58-containing pre-Golgi intermediate compartment (IC). The depletion of Ca2+ stores in normal rat kidney (NRK) cells by TG abolished the retention of the KDEL-containing, Ca2+-binding, luminal ER chaperones GRP94/endoplasmin and GRP78/BiP, and resulted in the appearance of the proteins in the culture medium before inducing their synthesis. Immunolocalization of GRP94 in TG-treated cells showed that the protein was transported to the Golgi complex and, in parallel, the KDEL receptor was redistributed from the Golgi to p58-positive IC structures, but was not transported further to the ER. Similarly, p58 that normally cycles between the ER, IC, and cis-Golgi, was largely depleted from the cell periphery and arrested in large-sized IC elements and numerous vesicles or buds in the Golgi region, showing that TG selectively blocks its recycling from the IC back to the ER. Importantly, cell fractionation analyses and confocal fluorescence microscopy provided evidence that the IC elements in unperturbed cells contain SERCA and a considerable pool of GRP94. Thus, the observed effects of TG on protein retention and recycling can be explained by a change in the luminal Ca2+ concentration of the IC. Moreover, the compositional properties of the IC elements suggest that they participate in intracellular Ca2+ storage.

The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency

Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum.