Protein misfolding diseases

1
Protein misfolding diseases
12-1

• Diseases caused by mutations in chaperones
• - a-crystallin, MKKS/BBS6 chaperonin
• Neurodegenerative diseases
• prions, Huntingtons disease

2
Neurodegenerative disorders prions
12-2

• 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

3
Prion transmission characteristics
12-3
harbours hamster PrPSc
harbours murine PrPSc
contains hamster PrP (lacks mouse PrP)
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
4
Class Presentations
12-4
5
Neurodegenerative disorders Huntingtons disease
12-5

• Huntingtons 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

6
Huntingtons disease in vitro model system
12-6

• 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

after 8 hours aggregation assayed as in (A)
time course of aggregation
time course of aggregation detected by filter
trap assay
7
Huntingtons disease in vitro model system
12-7
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
• 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)
• (B) DnaK
• (C) DnaJ
• (D) Hdj-1
• (E) Hsc70/ATP
• (F) Hsc70/Hdj-1/ATP (Hsc70/Hdj-1 21). Samples
  then were analyzed by EM. (Bar 100 nm.)

Hsp70/ATP
DnaK
DnaJ
Hsc70/Hdj-1
8
Huntingtons disease in vivo yeast model system
12-8

• 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 Ttotal, Ssoluble,
  Ppellet 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)

9
Huntingtons 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
  GAL4-dependent gene expression
• flies carrying GMR-GAL UAS127Q were crossed
  with EP-element insertion strains (7000)
• screened for suppression or enhancement of
  toxicity
• found dhdJ1, an Hsp40 homologue dtpr2 is a
  TPR-containing protein with J domain

• still see aggregates (as with the in vitro
  studies)

Esfarjani and Benzer (2000) Science 287, 1837.
10
TRANSGENIC STRAIN CARRYING GMR PROMOTER-GAL4
CONSTRUCT (HIGH-LEVEL EXPRESSION IN EYE)
GMR
GMR
GMR
GMR
GMR
GAL4
CONTRUCT CROSSED INTO THE ABOVE STRAIN (UAS
ACTIVATED BY GAL4 TO INDUCE HIGH-LEVEL EXPRESSION
OF 127Q)
UAS
127Q
11
a-crystallin and disease
12-10

• a-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 a-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 ß- and ?-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 a-crystallin occurs)
• Alexanders disease
• - the neurodegenerative Alexander's disease is
  characterized by GFAP co-aggregates with
  a-crystallin GFAP is closely related to desmin

12
a-crystallin-GFAP experiment
12-11

• R120G a-crystallin mutant is found in some
  patients
• the chaperone activity of the R120G mutant
  (located in the highly conserved a-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?

GFAP wt a -crystallin
GFAP R120G a -crystallin
Association of a-crystallin (wild-type and
mutant) with GFAP at 37ºC
Perng et al. (1999) J. Biol. Chem. 274, 33235.
13
MKKS/BBS6 mutations cause disease
12-12

• 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

14
MKKS/BBS6 and other BBS alleles
12-13

• 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, equatorial domain A, apical domain I,
intermediate domain P, protrusion
P
E
E
A
A
I
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.