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 (β, 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 (Kd 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.publishedVersio

Мотивационная политика предприятия как основа новой философии управления

В современной концепции управления ключевое место отводится человеку. Именно люди, характеризующиеся не только специальным образованием и профессиональными навыками, но и яркой индивидуальностью, способны генерировать новые идеи и решать непростые задачи в высококонкурентной и динамичной бизнес-среде. Такова закономерность в развитии экономических отношений и совершенствовании менеджмента организаций. Акценты в управлении компанией смещаются к идеологическим (культурным, духовным) ценностям предприятия, носителем которых является его персонал, конкретные сотрудники, способные в этих условиях обеспечивать эффективную работу в высококонкурентной среде

Functional and structural analysis of five mutations identified in methylmalonic aciduria cbIB type

ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349-1G>C, and c.290G>A affect the splicing process. Wild-type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a K M for ATP and K D for cob(I)alamin similar to wild-type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild-type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations. Hum Mutat 31:1033–1042, 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78057/1/21307_ftp.pd

Research in new therapeutical targets

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22/2/2011Vitamin B12, or cobalamin, is an essential nutrient that humans must take in their diet so that it serves as the cofactor of two enzymes: methionine synthase and methylmalonyl-CoA mutase, which need methylcobalamin and adenosylcobalamin as active cofactors, respectively. The study of the inborn intracellular defects of cobalamins has given rise to eight complementation groups, cblA-G and mut. In particular, defects in the synthesis of adenosylcobalamin (groups cblA, B and D) or in the apoenzyme methylmalonyl-CoA mutase give rise to isolated methylmalonic aciduria (MMA). Defects in the common pathway of synthesis of methylcobalamin and adenosylcobalamin causes methylcobalamin aciduria combined with homocistinuria (MMAHC; complementation groups cblC, D and F). The objective of this work has been to study the molecular basis of MMAHC and isolated MMA in order to identify molecular targets for the development of new therapeutical approaches to improve the outcome of these severe disorders. We have analyzed 48 patients of MMAHC cblC type, identifying the mutational spectrum of the Spanish population for the first time. We have identified eleven nucleotidic changes, including: one small duplication (c.271dupA), two missense (p.M1L and p.R189S), seven nonsense (p.W30X, p.R73X, p.R111X, p.R132X, p.R153X, p.R161X, p.Y205X) and one change that affects the splicing process (IVS1nt+2T>G). Two changes, p.W30X and IVS1nt+2T>G, are novel mutations and the rest have been previously described. The change c.271dupA accounts for 85% of studied alleles, and causes a frameshift and truncation of the protein. All but one of the analyzed patients bear the duplication in at least one allele, so we established a high resolution melting protocol for rapid and cost-effective screening of the mutation in MMACH patients. The specific gene expression analysis of 17 fibroblasts cell lines has indicated that transcripts bearing c.271dupA undergo NMD degradation, while p.R132X and p.R153X bearing mRNA surprisingly escape the NMD system. The transcriptional analysis of eight MMACHC patients and seven controls treated with or without hydroxycobalamin (OHcbl) has not shown any particular group of genes differentially expressed that could account for the B12 responsiveness found in our group of cblC patients. To gain insights into the physiopathology of the disease, we have analyzed the differential gene expression of 84 apoptosis-related genes. We have found that MMAHC cblC samples exhibit mostly up-regulated extrinsic pathway apoptotic genes while MMA cblB mostly to over-express the intrinsic pathway genes. The differences could be attributed to homocysteine which is the differential metabolite in cblB and cblC affected patients. In this study six MMA allelic variants were analyzed by expression studies in the appropriated expression system. c.733G>A mutation, analyzed by ex vivo splicing expression system using minigenes, affects the splicing process of MMAA gene and causes the skipping of exon 4, although some molecules do not undergo the skipping and show the misssense mutation p.G245S. The p.V153D and p.R629G mutations in MUT gene, drastically affect the enzyme’s activity. Since p.V153D responds in vitro to OHcbl, it belongs to the less severe mut- group and since p.R629G does not respond it is classified as the more severe mut0. Finally mutations p.I96T, p.H183L and p.R191W in MMAB gene have been studied using stability/activity assays in prokaryotic expression systems combined with molecular modeling, and residual activity in a cellular disease model in eukaryotic expression systems. Taken together these results suggest that p.I96T, p.H183L and p.R191W are misfolding mutations that destabilize ATR protein, retaining however some biological activity. Based on these findings we have tested gene expression modulators specific for lipid metabolism genes, such as statins and bezafibrate, which did not render conclusive results, probably due to the use of a not totally adequate cellular disease model. Pharmacological chaperone stabilization of ATR protein was carried out by screening a commercial library of compounds and selecting six hits by physical-chemical analysis (compound I-VI). Compound V and VI have not inhibited wild-type purified ATR; moreover compound V (currently patent pending) has increased the activity in different degrees in wild-type, p.I96T and p.H183L ATR. Compound V has also increased the activity to the control range in a cblB cell disease model. In summary, the knowledge gained from the comprehensive study of the genetic and functional basis of the disease has enabled us to identify new therapeutical targets and to find a pharmacological chaperone that could be a potential treatment for MMA cblB type patients

Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-beta interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised

Phosphorylation at serine 31 targets tyrosine hydroxylase tovesicles 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

Phenylalanine hydroxylase variants interact with the co‐chaperone DNAJC12

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co‐chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild‐type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu1/1 mouse model, homozygous for the V106A‐Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin‐tagged PAH. This form represented a major fraction of PAH in the Enu1/1 but was also present in liver of wild‐type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin‐dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients

Stabilization of Human Tyrosine Hydroxylase in Maltodextrin Nanoparticles for Delivery to Neuronal Cells and Tissue

Enzyme replacement therapy (ERT) is a therapeutic approach envisioned decades ago for the correction of genetic disorders, but ERT has been less successful for the correction of disorders with neurological manifestations. In this work, we have tested the functionality of nanoparticles (NP) composed of maltodextrin with a lipid core to bind and stabilize tyrosine hydroxylase (TH). This is a complex and unstable brain enzyme that catalyzes the rate-limiting step in the synthesis of dopamine and other catecholamine neurotransmitters. We have characterized these TH-loaded NPs to evaluate their potential for ERT in diseases associated with TH dysfunction. Our results show that TH can be loaded into the lipid core maltodextrin NPs with high efficiency, and both stability and activity are maintained through loading and are preserved during storage. Binding to NPs also favored the uptake of TH to neuronal cells, both in cell culture and in the brain. The internalized NP-bound TH was active as we measured an increase in intracellular L-Dopa synthesis following NP uptake. Our approach seems promising for the use of catalytically active NPs in ERT to treat neurodegenerative and neuropsychiatric disorders characterized by dopamine deficiency, notably Parkinson’s disease