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Protein folding and misfolding Hyperphenylalaninemia, phenylketonuria Familial hypercholesterolemia Protein expression Protein expression is the multistep process involving regulation at the level of transcription, mRNA turnover, protein translation, and posttranslational modifications leading to the formation of a stable product. Protein folding Scheme of the free-energy surface that proteins explore as move towards the native state. The accumulation of conformations that need to traverse energy barriers to reach a favourable downhill path. When molecules fold in the same compartment, the free-energy surface of folding may overlap with intermolecular aggregation. This state is prevented by molecular chaperones. NATURE | VOL 475 | 21 JULY 2011 Protein folding Partially folded states are problematic - they tend to aggregate in concentrationdependent manner. Aggregation primarily results in amorphous structures. Alternatively, fibrillar aggregates called amyloid may form. Formation of these aggregates in vivo is strongly restricted by the chaperone machinery. Molecular chaperone interact with, stabilize or help proteins to acquire its functionally active conformation, without being present in its final structure. Different classes of structurally unrelated chaperones exist in cells. Protein folding • The nascent proteins are synthesized vectorially and the N-terminal sequences will be available for folding before the C-terminal fragments. • Translation is coupled to co-translational folding. The rate of translation is non-uniform along mRNAs and is shaped by the asymmetric tRNA abundance for the different codons. Codons that pair to lowly abundant tRNAs are translated slower that codons read by highly abundant tRNA. • Slow-translating segments are not randomly distributed along the coding mRNA sequences; they are predominantly located downstream of the domain boundaries of multidomain proteins. Hyperfenylalaninemie (HPA), fenylketonurie (PKU) Popis stavu: HPA je způsobená deficitem jaterního enzymu fenylalaninhydroxylázy. Dědičnost: autosomálně recesivní Incidence: 1:13000 (v ČR 1 : 6500) Symptomy: Pozvolná mentální retardace začínající po porodu, ale obvykle není zjevná před šestým měsícem života. Rozsah retardace je závislý na stupni enzymového deficitu (typu mutace). Léčba: Standardní péče je nízkobílkovinná dieta s omezením fenylalaninu. Dieta je doporučována po celý život a její dodržování je považováno za nejdůležitější faktor normálního vývoje mozku. Průběh onemocnění bez léčby: většinou těžká mentální retardace, změny na bílé hmotě při hladině fenylalaninu nad 1500 μmol/l, mírnější poškození mozku při hladině fenylalaninu 600 – 1500 μmol/l. Mohou se přidat křeče a ekzém. Průběh onemocnění s léčbou: není mentální retardace, mohou být specifické problémy v učení. Při přerušení diety dochází k poklesu IQ, poruchám chování a soustředění. Ženy fenylketonuričky mají 95% šanci narození poškozeného dítěte (mikrocefalie a postižení mozku plodu, vrozené srdeční vady), jestliže nedržují přísnou dietu během těhotenství. Enzym a lokalizace: Fenylalaninhydroxyláza, játra MS/MS profil: Zvýšený fenylalanin, abnormální poměr fenylalanin/tyrosin Phenylalanin hydroxylase gene (PAH) The basic structure of the human PAH gene; chromosome 12 (12q23.2), 13 exons encoding a polypeptide of 452 amino acids. Structural domains of PAH. The catalytic domain of PAH contains a motif of 26 amino acids which are responsible for ferric iron, cofactor (tetrahydrobiopterin, BH4), and substrate binding. Phenylalanine hydroxylase (PAH) • Homotetrameric enzyme • Each subunit is composed of three functional domains: the N-terminal regulatory domain; the catalytic domain; and the C-terminal oligomerization domain. • In most HPA cases, PAH mutations are associated with decreased stability, increased susceptibility toward aggregation and degradation, folding defficiency of PAH mutant proteins. Phenylalanine hydroxylase (PAH) PAH converts phenylalanine into tyrosine and requires the cofactor tetrahydrobiopterin (BH4), molecular oxygen, and iron to do so. Loss of PAH activity results in increased concentrations of phenylalanine. Phenylalanine hydroxylase (PAH) Lancet 2010; 376: 1417–27 During the hydroxylation of phenylalanine by PAH (O2, Fe+3), tetrahydrobiopterin (BH4) is oxidised to 4a-hydroxyBH4 intermediate, which is subsequently regenerated back to BH4 by the enzymes carbinolamine-4adehydratase (PCD) and by the NADH-dependent dihydropteridine reductase (DHPR). BH4 is synthesised from guanosine triphosphate (GTP) by three additional enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Mutations in genes coding for PCD, DHPR, GTPCH, PTPS, and SR result in BH4 deficiency. Tetrahydrobiopterin (BH4) metabolism HPA, PKU is due to • mutations in the PAH gene (98% of cases) • mutations in genes coding for enzymes involved in BH4 biosynthesis or regeneration (2% of cases) Hyperphenylalaninemia (HPA), history Until the 1960s, most children born with HPA became profoundly mentally disabled. The foundations for the early detection and modern management of HPA were laid by three key findings: • In the 1930s, Asbjorn Folling identified raised levels of phenylalanine in the blood as the underlying cause of the neuropsychological deficits. • In the 1950s, Horst Bickel introduced a low-phenylalanine diet to treat HPA. • In the 1960s, Robert Guthrie introduced a diagnostic test suitable for mass screening for HPA (the Guthrie test). Nowadays, many countries around the world include a test for HPA in neonatal screening programmes - the Guthrie test or more modern systems based on tandem mass spectrometry. Guthrie test The Guthrie test – the semiquantitative assay designed to detect elevated blood levels of phenylalanine, using the ability of phenylalanine to facilitate bacterial growth in culture medium with inhibitor. • A small disk of filter paper is punched out and placed on agar gel plate containing Bacillus subtilis and B-2-thienylalanine. The agar gel is able to support bacterial growth but B-2-thienylalanine inhibits bacterial growth. • In presence of extra phenylalanine leached from filter paper, inhibition is overcome and the bacteria grow. Within a day the bacterial growth surrounding the paper disk is visible to the eye. The amount of growth, measured as the diameter of the colony, is proportional to the amount of phenylalanine in the serum. • The result is read by comparing the diameter of each sample disk's colony to the colonies of a series of reference disks with standard phenylalanine content included on each plate. Hyperphenylalaninemia (HPA), phenotypes Little or no PAH enzyme activity results in classic phenylketonuria phenotype. Other mutations only partly inhibit enzyme activity, giving rise to mild phenylketonuria or mild hyperphenylalaninaemia. • The normal range of blood phenylalanine concentrations is 50–110 μmol/L. • Individuals with blood phenylalanine concentrations of 120–600 μmol/L before starting treatment are classified as having mild hyperphenylalaninaemia. • Individuals with blood phenylalanine concentrations of 600–1200 μmol/L are classified as mild phenylketonuria. • Individuals with blood phenylalanine concentrations above 1200 μmol/L denote classic phenylketonuria. Hyperphenylalaninemia (HPA), molecular pathology of neurotoxicity • Although PAH deficiency occurs at the hepatic level, the clinical effects of HPA are on brain function. • The competition between Phe and other large neutral amino acids (LNAA: Tyr, Trp, .....) for transport across the blood brain barier level (the amino acid transporter LAT1). Phe has the highest affinity for LAT1. Thus, high plasma Phe concentrations impairs uptake of the other LNAA into the brain → a disturbance in brain uptake of the other LNAA. • The competition for LAT1 has the effect of blocking transport tyrosine and tryptophane (precursors of dopamine and serotonine). Hyperphenylalaninemia (HPA), tetrahydrobiopterin (BH4) therapy • The observation that levels of Phe can be reduced significantly by administration of exogenous BH4 in a subset of patients with HPA. • The response to BH4 therapy, dependent upon PAH gene mutation(s). About one third of HPA patients (mostly those with mild phenotypes) have been estimated to be potential candidates for BH4 treatment. • Sapropterin dihydrochloride (Kuvan, Biomarin Pharma) is an orally active synthetic form of BH4. Clinical trials have shown that Kuvan is a safe and effective therapy in selected patients with mild PKU and mild HPA. Sapropterin dihydrochloride Hyperphenylalaninemia (HPA), tetrahydrobiopterin (BH4) therapy Efficacy of sapropterin dihydrochloride in the management of phenylketonuria. (A) Response rates (%) according to blood phenylalanine levels after sapropterin treatment (10 mg/kg/day) over a period of 8 days. (B) Randomized comparison of the effect of sapropterin dihydrochloride and placebo on blood phenylalanine levels in responders to sapropterin therapy (10 mg/kg/day) over a period of 6 weeks. Molecular Genetics and Metabolism 96 (2009) 158–163 Hyperphenylalaninemia (HPA) • In most HPA cases, PAH mutations can lead to protein misfolding, aggregation, and early degradation and thus to a loss of functional PAH proteins • BH4 – molecular chaperone – the restoration of enzyme function which might be transmitted by correction of protein misfolding. • About 75% of PAH mutations, characterized by high residual activity, have been found to be associated with BH4 responsiveness. predicted 3D structure of phenylalanine hydroxylase Hyperphenylalaninemia (HPA), tetrahydrobiopterin (BH4) therapy Schematic view of the molecular pathophysiology of PAH deficiency. PAH mutations can lead to protein misfolding, aggregation, and early degradation and thus to a loss of functional PAH. All processes are mutually dependent and contribute to loss of function. J Inherit Metab Dis (2010) 33:649–658 Hyperphenylalaninemia (HPA), tetrahydrobiopterin (BH4) therapy Molecular mode of action of BH4. At the protein level, BH4 prevents misfolding, aggregation, and degradation, and thus induces an increase in the effective PAH concentration resulting in rescue of its function. J Inherit Metab Dis (2010) 33:649–658 PAH mutations in all three domains can lead to aggregation or impaired tetramer assembly Oligomerization profiles of wild-type and variant PAH were determined by sizeexclusion chromatography. Arrows mark the elution volumes of soluble aggregates, tetramers, dimers, and monomers. (A) Profiles of variants arising from mutations located in the regulatory domain. I65S showed increased amounts of dimers. (B) Profiles of variants arising from mutations located in the catalytic domain. S310Y and R408W eluted as high-molecular-weight aggregates without any detectable tetramers. (C) Profiles of variants arising from mutations mapping to the dimerization motif of the oligomerization domain (O). Y417H showed significant amounts of monomers and increased amounts of dimers. S.W. Gersting, The American Journal of Human Genetics 83, 5–17, July 2008 Familial hypercholesterolemia (FH) • Mutations in the LDLR (low-density lipoprotein receptor) or the APOB (apolipoprotein B) genes • Autosomal dominant inheritance • Incidence: 1:500 Familial hypercholesterolemia • LDLR is synthesized by ribosomes bound to the endoplasmic reticulum (ER), partially glycosylated (molecular mass 120 kDa). • LDLR is transported to the Golgi apparatus, glycosylated (molecular mass 160 kDa). • LDLR is transported to the cell surface, where it mediates the uptake of lipoprotein particles, mainly low-density lipoproteins (LDLs), by receptor-mediated endocytosis. • The internalized LDL particle is subsequently released in the endosome, and the receptor returns to the cell surface in a process called receptor recycling. Cell. Mol. Life Sci. Vol. 61, 2004 Familial hypercholesterolemia, protein folding • Folding of LDLR occurs in a vectorial manner, domain by domain. • The newly synthesized LDLR chains fold rapidly into compact structures containing non-native disulfide bonds linking distant regions of the protein. • With time, non-native disulfides are reshuffled, allowing extension of the molecule. In the native conformation, disulfide bonds only exist between cysteine residues within individual repeats. Despite the extensive formation of non-native disulfide bonds during folding, LDLR rarely aggregates - nonnative disulfide bond formation are part of the normal LDLR folding pathway. • Efficient folding of LDLR may be caused by assistance of chaperones and folding enzymes in ER. Cell. Mol. Life Sci. 61 (2004) Familial hypercholesterolemia, protein folding FEBS Journal 274 (2007) 1881–1893: Transport defective mutations (class 2) causing partial or complete retention of LDLR in the endoplasmic reticulum (G544V, G528D and W556R, the betapropeller), to study the ability of chemical chaperones to assist folding and to facilitate the transport of the mutant LDLR out of the ER. A molecular model of the LDLR beta-propeller. Left: the six blades in the betapropeller. Right: side viev. The amino acids G528, G544 and W556 are shown in red. Familial hypercholesterolemia, protein folding The wild-type LDLR appeared primarily with molecular mass of 160 kDa on the western blot, representing the mature form of the LDLR, whereas the mutant LDLRs appeared with molecular mass of 120 kDa, representing the ER-localized form of the receptor. Western blot analysis of the rescue of LDLR mutants. Stably transfected CHO cells expressing G544V-mutant, G528D-mutant or W556R-mutant LDLR were incubated with different chemical chaperones. Cell lysates were prepared, and equal amounts of proteins were subjected to SDS ⁄ PAGE and western blot analysis using an antibody to LDLR. A cell lysate from CHO cells stably transfected with wild-type (WT) LDLR is included on the left. FEBS Journal 274 (2007) 1881–1893 Familial hypercholesterolemia, protein folding Confocal laser microscopy on CHO cells stably transfected with wild-type LDLR or G544Vmutant LDLR. The upper panels show cells expressing wild type LDLR, the middle panels show cells expressing G544-mutant LDLR, and the lower panels show cells expressing G544Vmutant LDLR treated with 5 mM 4-PBA for 24 h. FEBS Journal 274 (2007) 1881–1893 The appearance of a 160 kDa mature form of LDLR indicates that the receptor has escaped from the ER and has been subjected to oligosaccharide modifications in the Golgi apparatus. To determine whether the mature form of G544V-mutant LDLR appeared at the cell surface, confocal laser microscopy on intact cells was performed. The wild-type LDLR was present on the plasma membrane, whereas the G544V-mutant LDLR was almost undetectable. When the cells were grown in the presence of 4-PBA, the G544V-mutant LDLR could be observed. Protein folding • ER contains stringent quality-control systems, which ensure that only correctly folded proteins are transported to their final destinations. • The ER quality control system involves molecular chaperones that transiently associate with newly synthesized proteins and promote their folding. • Misfolded proteins are retained and subsequently degraded by the ER-associated degradation. • Protein misfolding is the cause of several genetic diseases. • Chemical chaperones are small molecules that bind to a protein, stabilize the folded state, and thereby reduce protein misfolding. It has been proposed that chemical chaperones promote folding of mutant proteins, allowing them to escape from ER retention and subsequent degradation. • Accumulation of misfolded proteins in the ER has been shown to cause ER stress and activation of a protective response known as the unfolded protein response (UPR).