12-1
Protein misfolding diseases
Diseases caused by mutations in chaperones
- α-crystallin, MKKS/BBS6 chaperonin
Neurodegenerative diseases
- prions, Huntington’s disease
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Neurodegenerative disorders: prions
 pathogenesis of many neurodegenerative disorders is due to abnormal protein
conformation
 common theme in diseases is conversion of normal cellular and/or circulating protein into
an insoluble, aggregated, beta-sheet rich form which is deposited in the brain as an amyloid
 deposits are toxic and produce neuronal dysfunction and death
 prion-related diseases occur when conversion of a normal prion protein, PrP, into an
infectious and pathogenic form, PrPSc (Prion Protein Scrapie). Prion diseases:
 Creutzfeld Jacob disease, Kuru, Gerstmann-Straussler-Scheinker disease, Fatal
familial insomnia, Scrapie (sheep), Bovine spongiform encephalopathy (BSE or ‘mad
cow’), chronic wasting disease (mule deer, elk), feline spongiform encephalopathy
 the conversion of PrP into PrPSc is a conformational one; the PrPSc form is more
resistant to proteases and is detergent-insoluble
 PrPSc forms amyloid fibrils in the brain; injection of this material into the brains of normal
mice leads to disease
 the normal function of PrP is unknown; transgenic mice lacking this protein grow normally
 Other proteins unrelated in sequence to PrP have similar properties:
 e.g., yeast Sup35, Ure2p
Prion transmission characteristics
harbours hamster PrPSc
contains hamster PrP
(lacks mouse PrP)
12-3
harbours murine PrPSc
contains hamster PrP
(lacks mouse PrP)
Note:
- testing for infectivity with
PrPSc is done by injecting
brain material from an
infected animal into the
brain of another animal
- transgenic mice devoid of
mouse PrP cannot be
infected by mouse PrPSc
Class Presentations
 TRiC stands for TCP-1 Ring Complex and is the same eukaryotic
cytosolic chaperonin as CCT (Chaperonin containing TCP-1).
 CCT was first thought to assist the folding of only actins and tubulins, but
recently, it has been found to bind ~10% of all cell proteins and is known to
assist the folding of numerous other proteins, including a viral capsid protein,
myosin, luciferase and VHL.
Feldman et al. (1999) Formation of the VHL-Elongin BC
tumor suppressor complex is mediated by the chaperonin
TRiC. Mol. Cell 4, 1051-1061.
 there exists a cellular mechanism by which misfolded/aggregated proteins
are sequestered in the cell
 such sequestration occurs near the centrosome in an organelle-like
structure commonly termed ‘aggresome’
Johnston et al. (1998) Aggresomes: a cellular response to
misfolded proteins. J. Cell Biol. 143, 1883-98.
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Neurodegenerative disorders:
Huntington’s disease
12-5
 Huntington’s disease (HD) is a very common syndrome that affects numerous
people
 It is caused by the expansion of CAG trinucleotide repeats (encoding
polyglutamine) within a large protein (350 kDa) termed huntingtin
 the function of huntingtin is unclear; evidence points to trafficking (vesicular)
 normal and disease forms
 unaffected individuals carry between 6 and 39 repeats in exon 1 of
huntingtin
 HD patients typically have between 36-180 repeats in exon 1 of huntingtin
 mutant forms of huntingtin with expanded repeats form nuclear and
cytoplasmic aggregates in human brain tissue
Huntington’s disease:
in vitro model system
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 can express protein fusion with different numbers of CAG repeats and study
 Muchowski et al. (2000) PNAS 97, 7841. produced GST-HD proteins (HD20Q and
HD53Q) then cleaved off HD from tag using protease that cleaves between GST and HD;
aggregation was then followed in the presence or absence of chaperones
 found that combination of Hsp40 and DnaK were most effective at preventing
aggregation
time course of
aggregation detected by
‘filter trap’ assay
after 8 hours;
aggregation assayed
as in (A)
time course of
aggregation
Huntington’s disease:
in vitro model system
control
Hdj-1
 GST-HD proteins were induced to aggregate
by cleavage (as before) in the presence or
absence of chaperones
 fibrils/aggregate formation was observed by
electron microscopy
DnaK
Hsp70/ATP
Suppression of HD exon 1 fibril formation by Hsp40
and Hsp70 in vitro. GST-HD fusion protein (3 µM)
was incubated with PreScission protease for 5 h as
in previous slide:
in the absence (A) or presence (B-F) of chaperones
(6 µM)
DnaJ
Hsc70/Hdj-1
(B) DnaK
(C) DnaJ
(D) Hdj-1
(E) Hsc70/ATP
(F) Hsc70/Hdj-1/ATP (Hsc70/Hdj-1 = 2:1). Samples
then were analyzed by EM. (Bar = 100 nm.)
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Huntington’s disease:
in vivo yeast model system
 Huntingtin constructs with Exon 1 and
containing 20, 39 or 53 CAG repeats as well
as a c-myc tag (which is recognized by
antibody and can be immunoprecipitated)
were expressed in S. cerevisiae
 (A) *=SDS-insoluble aggregates that do not
penetrate the gel
 (A) **=degradation product of full-length
protein
 (B) filter-trap assay; T=total, S=soluble,
P=pellet after centrifugation
 (D) immunoprecipitation of different
proteins with anti Ssa (cytosolic) and Ssb
(ribosome-bound) Hsp70 protein homologues
from yeast, as well as anti-Ydj1 (Hsp40
homologue)
 High-level expression of Hsp70/40 in
yeast with HD53Q made the aggregates
SDS-soluble! (not shown)
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Huntington’s disease:
Drosophila model system
12-9
 expressed HA-tagged 127 CAG
repeat-protein (127Q) in the eye,
causing abnormalities/polyQ deposits
 GMR has 5 tandem copies of a
response element derived from the
rhodopsin 1 gene promoter)
 GAL4 is a transcription factor
 UAS, ‘Upstream Activating
Sequence’ required for GAL4dependent gene expression
 flies carrying GMR-GAL +
UAS127Q were crossed with EPelement insertion strains (7000)
 screened for suppression or
enhancement of toxicity
 found: dhdJ1, an Hsp40
homologue; dtpr2 is a TPRcontaining protein with J domain
Esfarjani and Benzer (2000) Science 287, 1837.
 still see aggregates
(as with the in vitro studies)
GMR GMR GMR GMR GMR
UAS
GAL4
127Q
TRANSGENIC STRAIN CARRYING
GMR PROMOTER-GAL4 CONSTRUCT
(HIGH-LEVEL EXPRESSION IN EYE)
CONTRUCT CROSSED INTO THE ABOVE STRAIN
(UAS ACTIVATED BY GAL4 TO INDUCE HIGHLEVEL EXPRESSION OF 127Q)
α-crystallin and disease
 α-crystallin belongs to the class of molecular chaperones collectively termed
small heat-shock proteins
 functions include (but is not limited to) maintaining microfilament stability
(e.g., intermediate filaments and perhaps actin and tubulin)
 present in all tissue types and ubiquitous in the three domains
mutations in α-crystallin genes A and B cause some major ailments:
 cataracts
- function is as a structural protein as well as a molecular chaperone; it
makes up nearly 1/3 of the eye lens protein, while β- and γ-crystallins
make up close to the other 2/3
 desmin-related myopathy
- desmin is an intermediate filament; mutation in the chaperone result in
the accumulation of intracellular aggregates of desmin (co-aggregation
with α-crystallin occurs)
 Alexander’s disease
- the neurodegenerative Alexander's disease is characterized by GFAP
co-aggregates with α-crystallin; GFAP is closely related to desmin
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α-crystallin-GFAP experiment
 R120G α-crystallin mutant is found
in some patients
GFAP
+
wt α -crystallin
GFAP
+
R120G α -crystallin
Association of α-crystallin (wild-type
and mutant) with GFAP at 37ºC
the chaperone activity of the R120G
mutant (located in the highly conserved
α-crystallin domain) is not completely
lost compared to the wild-type
chaperone intact (as judged by
prevention-of-aggregation experiments)
 reason why the mutant chaperone
associates more strongly with GFAP
(and desmin) is unclear
 specificity of binding causing the
problem?
Perng et al. (1999) J. Biol. Chem. 274, 33235.
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12-12
MKKS/BBS6 mutations cause disease
 MKKS/BBS6 is one of 12 genes that cause Bardet-Biedl
Syndrome (i.e., a polygenic disorder)
 mapping of BBS genes relied on screening inbred populations
(e.g., Bedoin arabs, Old Order Amish, Newfoundland)
 BBS phenotypes: obesity, kidney and liver problems, retinal
degeneration, cardiomyopathy, diabetes, mental retardation,
anosmia, hearing impairment, polydactyly, etc.
 MKKS/BBS6 is related to the chaperonin CCT
 two other chaperonin-like genes were found: BBS10, BBS12
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MKKS/BBS6 and other BBS alleles
 the chaperonin is related to the eukaryotic cytosolic chaperonin CCT
 it is found only in vertebrates and ‘more evolved’ organisms
 it is highly divergent although it is clearly a Group II chaperonin
E
I
A
P
A
I
 BBS4 is involved in microtubule anchoring; it
contains multiple TPR motifs: 34 amino acid repeats of
helix-loop-helix
- TPRs are protein-protein interaction domains
 BBS proteins are required for proper cilia function;
BBS is therefore a ciliopathy
 improper function of ciliary genes results in
numerous ailments, including retinal degeneration,
polycystic kidneys, skeletal anomalies, etc.
E
E, equatorial domain
A, apical domain
I, intermediate
domain
P, protrusion