Transcript 投影片 1

Disease related to misfolding
(Protein misfolding diseases)
Yun-Ru Chen, Ph.D.
Protein folding in biological system
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Chaperone assisted
folding
In vivo protein
folding (pulse-chase,
location/trafficking,
proyl isomerase,
disulfide isomerase)
Disease related
protein misfolding
Protein aggregations
Protein degradation
Chemical chaperone
© 1995-2007 by Michael W. Davidson
and The Florida State University.
crowding
In vitro folding without
chaperones
Definition
of molecular chaperone:
A functional class of unrelated proteins that assist the
correct noncovalent assembly of other polypeptide
containing structures in vivo, but are not components of
these assembled structures when they are performing their
biological functions
Pro-domain or pro-sequence act like chaperone to guide mature protein
(ex:subtilisin) toward the native state
Chemicals act like chaperone
TF family
Hsp70 family
(ATPase)
Hsp40
Family
Hsp60 family
(ATPase)
Hsp10 family
Small chaperone
Pro
Eu
Trigger
factor
DnaK
NAC, GimC
J domain
(DnaJ)
GroEL
Hsc/Hsp70,
Saa, Sab
Hsp40
TRiC/CCT
GroES
IbpA/B,
Hsp17
Hsp25,
aA/B crystallin
GroEL-ES system in E.Coli
Chaperonin catalytic cycle
Cell, Volume 125, Issue 5,
2 June 2006, Pages 903914
Structural Features of the GroELGroES Nano-Cage Required for Rapid
Folding of Encapsulated Protein
•the structural features of the chaperonin
cage are critical for rapid folding of
encapsulated substrates.
•Modulating the volume of the GroEL central
cavity affected folding speed in accordance
with confinement theory.
•The chaperonin cage provides a physical
environment optimized to catalyze the
structural annealing of proteins with
kinetically complex folding pathways.
Cell. 2006 Jun 2;125(5):831-3.
Figure 1. Altering the Size or Charge of the GroEL Folding Cavity Can Have a
Profound Influence on Protein Folding
(Left) Wild-type GroEL/GroES with a folding substrate (green) is shown. (Middle)
Changing the cavity size by deleting or replicating the motifs at the C terminus of
each GroEL subunit affects substrate folding. (Right) Alteration of the surface
charge of the cavity in a version of GroEL (named SR1) that contains only a single
ring can also affect the folding of some substrates.
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Hsp70
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multiple cellular location:
cytosol, nucleus, ER,
Mitochondria
Soluble or tethered to
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ribosome, to membrane
Trends in Biochemical
Sciences
Volume 32, Issue 8, August
2007, Pages 372-380
Small heat shock proteins
Enzyme that catalyzes
folding
Peptidyl prolyl isomerase
(overcome kinetic barriers presented by
incorrectly oriented peptide bonds)
(Cyclophilin, FK506-binidng proteins,
parvulins)
 Protein disulfide isomerase (PDI)
(thioredoxin, Dsb family)
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Regulation of protein folding
in the ER
Protein degradation pathway
Ubiquitin-Proteosome system
 Macroautophagy
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Before they are targeted for
proteasomal degradation,
most proteins are
covalently modified with
ubiquitin (Ub). Typically,
three enzyme types are
involved in this process —
ubiquitin-activating (E1),
ubiquitin-conjugating (E2)
and ubiquitin ligase (E3)
enzymes. Proteins tagged
with chains of four or more
ubiquitins are shuttled to
the the proteasome by
various proteins such as
CDC48/p97. In the
proteasome, proteins are
reduced to peptides, which
are then released into the
cytosol and further broken
down by peptidases. See
the text for more details.
Nature. 2006 Oct 19;443(7113):780-6
Autophagy begins with the formation of
double-membrane-bounded
autophagosomes. The origin(s) of the
autophagosome membranes are unclear.
The mammalian target of rapamycin
(mTOR) is a negative regulator of
autophagosome formation, although how
mTOR regulates this process in mammals
is not clear. When mTOR is inhibited by
rapamycin, autophagy is stimulated.
Autophagosomes fuse with lysosomes to
form autophagolysosomes, a process that
is governed by a number of factors,
including dynein activity. The contents of
autophagolysosomes are finally degraded
by acidic lysosomal hydrolases.
The green dots represent the protein LC3
(also known as ATG8). This is the only
known marker that specifically localizes to
autophagosome and autophagolysosome
membranes and not to other membranes.
LC3 localizes to these structures after it
has been processed and conjugated to
phosphatidylethanolamine.
Protein misfolding problem
Protein dysfunction
Protein misfolding is no longer viewed
as an experimental artifact.
 From gene mutation to underpins
diseases
 Studies of protein folding have
revolutionized our ideas of disease
transmission
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Protein misfolding causes
diseases
I.
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Diseases associated with the
loss of active protein
Sickle-cell hemoglobin
anemia (2 point mutations out
of 574aa.)
Cystic fibrosis (囊性纖維化 )
(D508, CFTR)
P53 tumor suppression
(mutation leads to half of all
reported cancers)
Lysosomal storage diseases
II. Diseases associated
with accumulation of
aggregate
 Amyloidosis
Sickle cell hemoglobin
Hemoglobin (oxygen-transport protein): single site mutation (E6V) in beta
subunit,
Protein Structure
The structure of normal deoxy
hemoglobin is shown in Figure 1. The
four gray clusters are noncovalently
bonded heme groups. Each heme
serves as a contact site for binding one
molecule of oxygen. The two gold
spheres near the top of the molecule
are phosphate groups. The green and
blue chains are alpha chains, and the
gold and aqua chains are beta chains.
The red box highlights the general
region of two beta chains where the
sixth glutamic acid residue is located. A
genetic variant associated with this
amino acid is the most common cause
of sickle cell anemia. Figure 2 shows
the clumping together of two deoxy
hemoglobin molecules that contain this
genetic variant.
Normal red cells maintain their shape as they pass through the capillaries and
release oxygen to the peripheral tissues (upper panel). Hemoglobin polymers form
in the sickle red blood cells with oxygen release, causing them to deform. The
deformed cells block the flow of cells and interrupt the delivery of oxygen to the
tissues (lower panel).
Cystic fibrosis
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Mutations in one gene, called the cystic fibrosis
transmembrane conductance regulator (CFTR), cause
the body to make nonfunctional CFTR protein, which
leads to the disease.
CFTR as a Cl Ion channel lost its function by single
deletion at F508
most patients with CF would be to use a ligand(s) of
CFTR that acts a pharmacological chaperone to correct
the folding defect
Imaging shows the normal
CFTR fragment (top) curling
into a helix, while the mutant
fragment unravels into random
shapes, blocking it from
migrating to the cell surface,
where it would prevent
symptoms.
http://www.hopkinsmedicine.org/hmn/F99/mu_8.html
RESEARCH
Gene Therapy
Protein
Assist/Repair
Restore Salt
Transport
Mucus Treatment
Anti-Inflammatory
Anti-Infective
Transplant Drugs
Nutritional
Supplements
PRE-PHASE
CLINICAL
PHASE
PHASE
TO
1
2
3
PATIENTS
P53 mutation and Cancer
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Half of all known cancers
involve some mutation in p53,
the so-called guardian of the
cell.
P53 is a tumor suppressor
which signals for cell death if
their DNA gets damaged. If
these cells didn't die, their
damaged DNA would lead to
the strange and unusual
growths found in cancer
tumors and this growth
would continue unchecked,
until death.
When p53 breaks down and
does not fold correctly (or
even perhaps if it doesn't fold
quickly enough), then DNA
damage goes unchecked
and one can get cancer.
http://p53.free.fr/Database/p53_cancer_db.html
http://folding.stanford.edu/English/FAQ-Diseases#ntoc2
Newly synthesized proteins in the living cell must go through a folding process to attain their
functional structure. To achieve this in an efficient fashion, all organisms, including humans,
have evolved a large set of molecular chaperones that assist the folding as well as the
maintenance of the functional structure of cellular proteins. Aberrant proteins, the result of
production errors, inherited or acquired amino acid substitutions or damage, especially
oxidative modifications, can in many cases not fold correctly and will be trapped in misfolded
conformations. To rid the cell of misfolded proteins, the living cell contains a large number of
intracellular proteases, e.g. the proteasome, which together with the chaperones comprise
the cellular protein quality control systems. Many inherited disorders due to amino acid
substitutions exhibit loss-of-function pathogenesis because the aberrant protein is eliminated
by one of the protein quality control systems. Examples are cystic fibrosis and
phenylketonuria. However, not all aberrant proteins can be eliminated and the misfolded
protein may accumulate and form toxic oligomeric and/or aggregated inclusions. In this case
the loss of function may be accompanied by a gain-of-function pathogenesis, which in many
cases determines the pathological and clinical features. Examples are Parkinson and
Huntington diseases. Although a number of strategies have been tried to decrease the
amounts of accumulated and aggregated proteins, a likely future strategy seems to be the
use of chemical or pharmacological chaperones with specific effects on the misfolded protein
in question. Positive examples are enzyme enhancement in a number of lysosomal disorders.
Metabolic diseases:
lysosomal storage diseases
A genetic disease that occurs when the
catabolism of glycocojugates is impaired
and result in accumulation of the products in
lysosome.
 More than hundred kinds of single site
mutants effect correct folding of the
enzymes which leads to deficient lysosomal
enzyme activity.
Fabry: a-galactosidase
Gaucher: b-glucosidase
(Pombe: :a-glucosidase)
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Deficient Enzyme :
Lysosomal Storage Diseases
Fabry disease - causes kidney and
Globoside
heart problems, pain and a skin rash Galb3GalNAcb4
GM1
Gaucher disease - causes the spleen
GalNAcb3Gala4
Galb4GlcCer
to enlarge, anemia and bone lesions if
Galb4GlcCer
NeuAca3
untreated
Hurler syndrome - causes deformities
b-galactosidase
b-HexA,B
GM1 Ganliosidosis
of the skeleton and facial features,
Sandhoff Disease
enlargement of the spleen and liver,
GalNAcb4
joint stiffness, clouding of the cornea,
GM2
Gala4Galb4GlcCer
Galb4GlcCer
mental retardation and deafness
NeuAca3
Niemann-Pick B disease - leads to
enlargement of the spleen and liver, as
b-HexA
a-galactosidase
GM3
well as lung disease
Tay-Sachs
Fabry Disease
Pompe disease - an often fatal
Galb4GlcCer
Disease NeuAca3
storage disease in which glycogen
builds up in the liver, heart and muscle,
Galb4GalCer
neuraminidase
especially during infancy (also known
as acid maltase deficiency)
b-galactosidase
Tay-Sachs disease - a lysosomal
GlcCer
storage disease that occurs more
commonly in people of Eastern
b-glucocerebrosidase
European Ashkenazi descent and
Gaucher Disease
causes degeneration of the brain in
Ceramide
infants
Deficient Enzyme :
Glycogen storage disease
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Type II: Pompe disease, deficient a-glucosidase
Type I: von Gierke disease, deficient glucose-6phosphatase (100% mis-sense), mouse and dog model
available. (Director YT Chen, IBMS)
Structure, Folding, Protein not available
Faces of GSD1
Glucose-6-phosphatase
Glucose-6-phosphatase converts glucose-6phosphate into free glucose and is active in the
lumen of the endoplasmic reticulum, where it is
bound to the membrane
Pombe
Gaucher
Current therapy: Enzyme replacement (costly and
not the best yet)
Chemical chaperones were shown to stabilize the
enzyme against misfolding, increasing proper
trafficking from ER.
a-galactosidase
Fabry
Yu, Zhanqian, Sawkar, Anu R. & Kelly, Jeffery W.
Pharmacologic chaperoning as a strategy to
treat Gaucher disease.
FEBS Journal 274 (19), 4944-4950.
Proc Natl Acad Sci U S A.
2007 August 7; 104(32):
13192–13197.
Three classes of
glucocerebrosidase inhibitors
identified by quantitative highthroughput screening are chaperone
leads for Gaucher disease
Selectivity of inhibitors with related hydrolases.
Inhibitors were tested on GC, α-glucosidase (αGluc), α-galactosidase (α-Gal), and β-Nacetylglucosaminidase (HEX). Data represent the
results of three independent experiments
performed with three replicates per sample. (A)
Compound 1, an aminoquinoline derivative. (B)
Compound 2, a sulfonamide derivative. (C)
Compound 3, a triazine derivative. (D) Nonyl-DNJ
(compound 5), an iminosugar.
Cell-permeable chemical
chaperones (dark red triangles) act
as a template for variant lysosomal
enzymes as they fold in the ER.
Some of these lysosomal enzymes
would otherwise misfold and be
retained in the ER and ultimately be
degraded by the proteasome.
Chemical chaperones facilitate
folding in the ER lumen, which is
necessary for proper trafficking to
the lysosome where a high
concentration of substrate (red
triangles) exists to compete with the
chemical chaperones that bind to
the active site. Although the
chemical chaperones are crucial for
folding at pH 7 (for example, in the
ER), the variant lysosomal enzymes
might not need the chaperones
when they arrive in the lysosome
(where the pH is about 5) because
their sequences are stably folded at
this pH.
transthyretin