Cysteine Modification by Ebselen Reduces the Stability and Cellular Levels of 14-3-3 Proteins.

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.

DOPA Homeostasis by Dopamine: A Control-Theoretic View.

Dopamine (DA) is an important signal mediator in the brain as well as in the periphery. The term "dopamine homeostasis" occasionally found in the literature refers to the fact that abnormal DA levels can be associated with a variety of neuropsychiatric disorders. An analysis of the negative feedback inhibition of tyrosine hydroxylase (TH) by DA indicates, with support from the experimental data, that the TH-DA negative feedback loop has developed to exhibit 3,4-dihydroxyphenylalanine (DOPA) homeostasis by using DA as a derepression regulator. DA levels generally decline when DOPA is removed, for example, by increased oxidative stress. Robust DOPA regulation by DA further implies that maximum vesicular DA levels are established, which appear necessary for a reliable translation of neural activity into a corresponding chemical transmitter signal. An uncontrolled continuous rise (windup) in DA occurs when Levodopa treatment exceeds a critical dose. Increased oxidative stress leads to the successive breakdown of DOPA homeostasis and to a corresponding reduction in DA levels. To keep DOPA regulation robust, the vesicular DA loading requires close to zero-order kinetics combined with a sufficiently high compensatory flux provided by TH. The protection of DOPA and DA due to a channeling complex is discussed.

Serine 19 phosphorylation and 14-3-3 binding regulate phosphorylation and dephosphorylation of tyrosine hydroxylase on serine 31 and serine 40.

Multisite phosphorylation and structural flexibility allow for complex regulation of proteins through cellular signaling. Tyrosine hydroxylase (TH), a key enzyme of catecholamine synthesis, is regulated by multiple neuronal signaling pathways through phosphorylation at serine 19 (Ser19), serine 31 (Ser31), and serine 40 (Ser40) located in the flexible, far N-terminal region of the regulatory domain. Phosphorylated Ser19 (pSer19) provides a binding site for 14-3-3 proteins, a family of multi-target binding adaptor proteins. We hypothesized that pSer19 and 14-3-3 binding can regulate access to the Ser31 and Ser40 sites and modulate the dynamics of their phosphorylation state. To avoid complications from upstream signal interactions and have good control of TH-phosphorylation and 14-3-3 binding stoichiometry, we used purified recombinant human TH and 14-3-3 dimer types. We found that pSer19 strongly stimulated Ser31 phosphorylation (4.6-fold), but inhibited pSer31 dephosphorylation (3.4-fold). Binding of 14-3-3ζ counteracted the stimulatory effect of pSer19 on phosphorylation at Ser31, but amplified the effect on its dephosphorylation. In contrast, phosphorylation at Ser19 had moderate effect on pSer40 dephosphorylation, but 14-3-3ζ binding inhibited dephosphorylation, an effect that was consistent across different homo- and heterodimeric 14-3-3s. Additional phosphorylation of Ser31 or Ser40 had little impact on the binding affinity of pSer19 TH to 14-3-3s. Mathematical modeling was performed to elucidate possible physiological implications of these observations. We propose a role of Ser19 and 14-3-3 proteins as modulators of TH phosphorylation in response to neuronal co-signaling events. These mechanisms add to our understanding of the multifaceted roles of phosphorylation and adaptor proteins in cellular signaling.

Tyrosinemia Type 1 and symptoms of ADHD: Biochemical mechanisms and implications for treatment and prognosis.

Hereditary tyrosinemia Type 1 (HT-1) is a rare metabolic disease where the enzyme catalyzing the final step of tyrosine breakdown is defect, leading to accumulation of toxic metabolites. Nitisinone inhibits the degradation of tyrosine and thereby the production of harmful metabolites, however, the concentration of tyrosine also increases. We investigated the relationship between plasma tyrosine concentrations and cognitive functions and how tyrosine levels affected enzyme activities of human tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2). Eight Norwegian children between 6 and 18 years with HT-1 were assessed using questionnaires measuring Attention Deficit Hyperactivity Disorder (ADHD)-symptoms and executive functioning. Recent and past levels of tyrosine were measured and the enzyme activities of TH and TPH2 were studied at conditions replicating normal and pathological tyrosine concentrations. We observed a significant positive correlation between mean tyrosine levels and inattention symptoms. While TH exhibited prominent substrate inhibition kinetics, TPH2 activity also decreased at elevated tyrosine levels. Inhibition of both enzymes may impair syntheses of dopamine, noradrenaline, and serotonin in brain tissue. Inattention in treated HT-1 patients may be related to decreased production of these monoamines. Our results support recommendations of strict guidelines on plasma tyrosine levels in HT-1. ADHD-related deficits, particularly inattention, should be monitored in HT-1 patients to determine whether intervention is necessary.

Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states.

Mathematical Modelling of Nitric Oxide/Cyclic GMP/Cyclic AMP Signalling in Platelets.

Platelet activation contributes to normal haemostasis but also to pathologic conditions like stroke and cardiac infarction. Signalling by cGMP and cAMP inhibit platelet activation and are therefore attractive targets for thrombosis prevention. However, extensive cross-talk between the cGMP and cAMP signalling pathways in multiple tissues complicates the selective targeting of their activities. We have used mathematical modelling based on experimental data from the literature to quantify the steady state behaviour of nitric oxide (NO)/cGMP/cAMP signalling in platelets. The analysis provides an assessment of NO-induced cGMP synthesis and PKG activation as well as cGMP-mediated cAMP and PKA activation though modulation of phosphodiesterase (PDE2 and 3) activities. Both one- and two-compartment models of platelet cyclic nucleotide signalling are presented. The models provide new insight for understanding how NO signalling to cGMP and indirectly cAMP, can inhibit platelet shape-change, the initial step of platelet activation. Only the two-compartment models could account for the experimental observation that NO-mediated PKA activation can occur when the bulk platelet cAMP level is unchanged. The models revealed also a potential for hierarchical interplay between the different platelet phosphodiesterases. Specifically, the models predict, unexpectedly, a strong effect of pharmacological inhibitors of cGMP-specific PDE5 on the cGMP/cAMP cross-talk. This may explain the successful use of weak PDE5-inhibitors, such as dipyridamole, in anti-platelet therapy. In conclusion, increased NO signalling or PDE5 inhibition are attractive ways of increasing cGMP-cAMP cross-talk selectively in platelets.

Regulation of tyrosine hydroxylase is preserved across different homo- and heterodimeric 14-3-3 proteins.

Tyrosine hydroxylase (TH) is regulated by members of the 14-3-3 protein family. However, knowledge about the variation between 14-3-3 proteins in their regulation of TH is still limited. We examined the binding, effects on activation and dephosphorylation kinetics of tyrosine hydroxylase (TH) by abundant midbrain 14-3-3 proteins (ß, η, ζ, γ and ε) of different dimer composition. All 14-3-3 homodimers and their respective 14-3-3ε-heterodimers bound with similar high affinity (K d values of 1.4-3.8 nM) to serine19 phosphorylated human TH (TH-pS19). We similarly observed a consistent activation of bovine (3.3- to 4.4-fold) and human TH-pS19 (1.3-1.6 fold) across all the different 14-3-3 dimer species, with homodimeric 14-3-3γ being the strongest activator. Both hetero- and homodimers of 14-3-3 strongly inhibited dephosphorylation of TH-pS19, and we speculate if this is an important homeostatic mechanism of 14-3-3 target-protein regulation in vivo. We conclude that TH is a robust interaction partner of different 14-3-3 dimer types with moderate variability between the 14-3-3 dimers on their regulation of TH.

Phosphorylation dependence and stoichiometry of the complex formed by tyrosine hydroxylase and 14-3-3γ.

Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15-0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [(32)P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18-0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in the TH:14-3-3γ complex. This adds to our understanding of the fine-tuned physiological regulation of TH, including hierarchical phosphorylation at multiple sites.

Cell Death Inducing Microbial Protein Phosphatase Inhibitors--Mechanisms of Action.

Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity.

Epistatic and gene wide effects in YWHA and aromatic amino hydroxylase genes across ADHD and other common neuropsychiatric disorders: Association with YWHAE.

Monoamines critically modulate neurophysiological functions affected in several neuropsychiatric disorders. We therefore examined genes encoding key enzymes of catecholamine and serotonin biosynthesis (tyrosine and tryptophan hydroxylases-TH and TPH1/2) as well as their regulatory 14-3-3 proteins (encoded by YWHA-genes). Previous studies have focused mainly on the individual genes, but no analysis spanning this regulatory network has been reported. We explored interactions between these genes in Norwegian patients with adult attention deficit hyperactivity disorder (aADHD), followed by gene-complex association tests in four major neuropsychiatric conditions; childhood ADHD (cADHD), bipolar disorder, schizophrenia, and major depressive disorder. For interaction analyses, we evaluated 55 SNPs across these genes in a sample of 583 aADHD patients and 637 controls. For the gene-complex tests, we utilized the data from large-scale studies of The Psychiatric Genomics Consortium (PGC). The four major neuropsychiatric disorders were examined for association with each of the genes individually as well as in three complexes as follows: (1) TPH1 and YWHA-genes; (2) TH, TPH2 and YWHA-genes; and (3) all genes together. The results show suggestive epistasis between YWHAE and two other 14-3-3-genes - YWHAZ, YWHAQ - in aADHD (nominal P-value of 0.0005 and 0.0008, respectively). In PGC data, association between YWHAE and schizophrenia was noted (P = 1.00E-05), whereas the combination of TPH1 and YWHA-genes revealed signs of association in cADHD, schizophrenia, and bipolar disorder. In conclusion, polymorphisms in the YWHA-genes and their targets may exert a cumulative effect in ADHD and related neuropsychiatric conditions, warranting the need for further investigation of these gene-complexes. © 2015 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc.

Functional studies of tyrosine hydroxylase missense variants reveal distinct patterns of molecular defects in Dopa-responsive dystonia.

Congenital tyrosine hydroxylase deficiency (THD) is found in autosomal-recessive Dopa-responsive dystonia and related neurological syndromes. The clinical manifestations of THD are variable, ranging from early-onset lethal disease to mild Parkinson disease-like symptoms appearing in adolescence. Until 2014, approximately 70 THD patients with a total of 40 different disease-related missense mutations, five nonsense mutations, and three mutations in the promoter region of the tyrosine hydroxylase (TH) gene have been reported. We collected clinical and biochemical data in the literature for all variants, and also generated mutant forms of TH variants previously not studied (N = 23). We compared the in vitro solubility, thermal stability, and kinetic properties of the TH variants to determine the cause(s) of their impaired enzyme activity, and found great heterogeneity in all these properties among the mutated forms. Some TH variants had specific kinetic anomalies and phenylalanine hydroxylase, and Dopa oxidase activities were measured for variants that showed signs of altered substrate binding. p.Arg233His, p.Gly247Ser, and p.Phe375Leu had shifted substrate specificity from tyrosine to phenylalanine and Dopa, whereas p.Cys359Phe had an impaired activity toward these substrates. The new data about pathogenic mechanisms presented are expected to contribute to develop individualized therapy for THD patients.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression.

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

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.

The effects of phenylalanine and tyrosine levels on dopamine production in rat PC12 cells. Implications for treatment of phenylketonuria, tyrosinemia type 1 and comorbid neurodevelopmental disorders.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in phenylalanine accumulation and impaired tyrosine production. In Tyrosinemia type 1 (TYRSN1) mutations affect fumarylacetoacetate hydrolase, leading to accumulation of toxic intermediates of tyrosine catabolism. Treatment of TYRSN1 with nitisinone results in extreme tissue levels of tyrosine. Although PKU and TYRSN1 have opposite effects on tyrosine levels, both conditions have been associated with neuro-psychiatric symptoms typically present in ADHD, possibly indicating an impaired dopamine (DA) synthesis. However, concrete in vivo data on the possible molecular basis for disrupted DA production under disease mimicking conditions have been lacking. In pursuit to uncover associated molecular mechanisms, we exposed an established, DA producing cell line (PC12) to different concentrations of phenylalanine and tyrosine in culture media. We measured the effects on viability, proteomic composition, tyrosine, DA and tyrosine hydroxylase (TH) levels and TH phosphorylation. TH catalyzes the rate-limiting step in DA synthesis. High extracellular levels of phenylalanine depleted cells of intracellular tyrosine and DA. Compared to physiological levels (75 µM), either low (35 µM) or high concentrations of tyrosine (275 or 835 µM) decreased cellular DA, TH protein, and its phosphorylation levels. Using deep proteomic analysis, we identified multiple proteins, biological processes and pathways that were altered, including enzymes and transporters involved in amino acid metabolism. Using this information and published data, we developed a mathematical model to predict how extracellular levels of aromatic amino acids can affect the cellular synthesis of DA via different mechanisms. Together, these data provide new information about the normal regulation of neurotransmitter synthesis and how this may be altered in neurometabolic disorders, such as PKU and TYRSN1, with implications for the treatment of cognitive symptoms resulting from comorbid neurodevelopmental disorders.

ADHD symptoms in neurometabolic diseases: Underlying mechanisms and clinical implications.

Neurometabolic diseases (NMDs) are typically caused by genetic abnormalities affecting enzyme functions, which in turn interfere with normal development and activity of the nervous system. Although the individual disorders are rare, NMDs are collectively relatively common and often lead to lifelong difficulties and high societal costs. Neuropsychiatric manifestations, including ADHD symptoms, are prominent in many NMDs, also when the primary biochemical defect originates in cells and tissues outside the nervous system. ADHD symptoms have been described in phenylketonuria, tyrosinemias, alkaptonuria, succinic semialdehyde dehydrogenase deficiency, X-linked ichthyosis, maple syrup urine disease, and several mitochondrial disorders, but are probably present in many other NMDs and may pose diagnostic and therapeutic challenges. Here we review current literature linking NMDs with ADHD symptoms. We cite emerging evidence that many NMDs converge on common neurochemical mechanisms that interfere with monoamine neurotransmitter synthesis, transport, metabolism, or receptor functions, mechanisms that are also considered central in ADHD pathophysiology and treatment. Finally, we discuss the therapeutic implications of these findings and propose a path forward to increase our understanding of these relationships.

Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.

Nostocyclopeptide-M1: a potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and OATP1B1.

We have isolated a novel cyanobacterial cyclic peptide (nostocyclopeptide M1; Ncp-M1) that blocks the hepatotoxic action of microcystin (MC) and nodularin (Nod). We show here that Ncp-M1 is nontoxic to primary hepatocytes in long-term culture. Ncp-M1 does not affect any known intracellular targets or pathways involved in MC action, like protein phosphatases, CaM-KII, or ROS-dependent cell death effectors. In support of this conclusion Ncp-M1 had no protective effect when microinjected into cells. Rather, the antitoxin effect was solely due to blocked hepatocyte uptake of MC and Nod. The hepatic uptake of MC and Nod is mainly via the closely related organic anion transporters OATP1B1 and OATP1B3, which also mediate hepatic transport of endogenous metabolites and hormones as well as drugs. OATP1B3 is also expressed in some aggressive cancers, where it confers apoptosis resistance. We show that Ncp-M1 inhibits transport through OATP1B3 and OATP1B1 expressed in HEK293 cells. The Ncp-M1 molecule has several nonproteinogenic amino acids and an imino bond, which hamper its synthesis. Moreover, a cyclic all L-amino acid heptapeptide analogue of Ncp-M1 also inhibits the OATP1B1/1B3 transporters, and with higher OATP1B3 preference than Ncp-M1 itself. The nontoxic Ncp-M1 and its synthetic cyclic peptide analogues thus provide new tools to probe the role of OATB1B1/1B3 mediated drug and metabolite transport in liver and cancer cells. They can also serve as scaffolds to design new, exopeptidase resistant OATP1B3-specific modulators.

Dipyridamole synergizes with nitric oxide to prolong inhibition of thrombin-induced platelet shape change.

We and others have previously demonstrated that nitric oxide (NO)-induced inhibition of platelet shape change is important in regulating platelet adhesion and aggregation, and therapeutic intervention of this pathway is clinically relevant for secondary prevention of stroke with dipyridamole. In the present study, we investigated whether dipyridamole affected the shape change of aspirinated platelets. Platelet shape change was inhibited using both authentic NO and sodium nitroprusside, as monitored by light scattering and mean platelet volume measurements. Dipyridamole synergized with NO, even at supra-therapeutic levels, to inhibit thrombin-induced shape change and further potentiated cAMP dependent protein kinase (PKA) mediated phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser157, even without altered levels of platelet cAMP. The effect of dipyridamole on NO-inhibited shape change depended on cGMP synthesis as evaluated by inhibition of soluble guanylyl cyclase. Measured increases in cGMP levels by dipyridamole and NO was assessed by mathematical modeling and found to be consistent with inhibition of phosphodiesterase 5 (PDE5). The model could explain the unexpected efficiency of dipyridamole in inhibiting PDE5 at the measured cGMP levels, by the majority of cGMP being bound to cGMP-dependent protein kinase (PKG). Still, selective activators of PKG failed to extend NO-mediated inhibition of the thrombin-induced platelet shape change, suggesting that PKG was not responsible for the inhibitory effect of NO and dipyridamole on shape change. The effects of dipyridamole were independent of the prostanoid and ADP pathways. Thus, the effect of dipyridamole on NO-mediated inhibition of platelet shape change may be an important and additional beneficial therapeutic effect of dipyridamole, which we suggest, is acting though localized amplification of the NO/cGMP/Phosphodiesterase3/cAMP/PKA-pathway. Probably, the efficiency of dipyridamole could be amplified clinically with NO donors.

Personalized Medicine to Improve Treatment of Dopa-Responsive Dystonia-A Focus on Tyrosine Hydroxylase Deficiency.

Dopa-responsive dystonia (DRD) is a rare movement disorder associated with defective dopamine synthesis. This impairment may be due to the fact of a deficiency in GTP cyclohydrolase I (GTPCHI, GCH1 gene), sepiapterin reductase (SR), tyrosine hydroxylase (TH), or 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) enzyme functions. Mutations in GCH1 are most frequent, whereas fewer cases have been reported for individual SR-, PTP synthase-, and TH deficiencies. Although termed DRD, a subset of patients responds poorly to L-DOPA. As this is regularly observed in severe cases of TH deficiency (THD), there is an urgent demand for more adequate or personalized treatment options. TH is a key enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis, and THD patients often present with complex and variable phenotypes, which results in frequent misdiagnosis and lack of appropriate treatment. In this expert opinion review, we focus on THD pathophysiology and ongoing efforts to develop novel therapeutics for this rare disorder. We also describe how different modeling approaches can be used to improve genotype to phenotype predictions and to develop in silico testing of treatment strategies. We further discuss the current status of mathematical modeling of catecholamine synthesis and how such models can be used together with biochemical data to improve treatment of DRD patients.

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 Ser(19)-phosphorylated (THp-(1-43)) states by surface plasmon resonance. THp-(1-43) showed high affinity for 14-3-3 proteins (K(d) approximately 0.5 microM for 14-3-3gamma and -zeta and 7 microM for 14-3-3eta). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S(0.5) = 25-58 microM (TH-(1-43)) and S(0.5) = 135-475 microM (THp-(1-43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3gamma showed a preferential binding to membranes, compared with 14-3-3zeta, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3gamma for negatively charged membranes (S(0.5) = 1-9 microM) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser(19)-phosphorylated TH, 14-3-3gamma, 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.