Transcript Protein folding

 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).