BIOL 588

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BIOL
588 LECTURERS NOTES FOR SEPT 21, 2006
Macromolecular quality control
Ken Hastings
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Macromolecular Quality Control
Mechanisms Cellular macromolecules, especially
proteins, have a specific folded 3-D shape that
is essential for their function. The expression
of incomplete proteins or of mutant or damaged
proteins that are not properly folded would be
bad. Nonfunctional molecules would dilute active
components, incomplete proteins could act as
dominant-negative interfering mutants, and
misfolded proteins could aggregate and interfere
with intracellular movement.
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Cells have evolved mechanism for avoiding
aberrant protein production and or for detecting
and destroying or neutralizing aberrant proteins.
This quality control is expensive. Even under
ideal conditions a significant fraction (1/3 or
more) of newly-synthesized protein molecules do
not assemble correctly and are destroyed.
There are two main classes of macromolecular
quality control mechanism 1) those that work at
the RNA level to prevent the synthesis of
abnormal proteins. 2) those that work at the
protein level to detect, and destroy, aberrant
proteins.
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Preventing the synthesis of abnormal proteins
Dealing with incompletely processed, broken or
mutant mRNAs. Export of mRNA from
nucleus. Nuclear pore complex recognizes and
exports only mRNPs containing
complete, fully processed mRNAs Cap-binding
complex (5') and poly(A)-binding protein (3')
must be present in mRNP, and snRNPs (involved
in RNA splicing) must be absent.
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-40
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Dealing with incompletely processed, broken or
mutant mRNAs (contd) Translation Initiation of
mRNA translation requires interactions between
5-cap-binding proteins and poly(A)-binding
proteins, so broken mRNA 5 or 3 halves are not
translated
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-75
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Dealing with incompletely processed, broken or
mutant mRNAs (contd) Nonsense-mediated mRNA
decay mRNAs with misplaced in-frame stop
(nonsense) codons (due to mutation or failed
intron removal) are degraded by NMD. During
pre-mRNA splicing a set of specific proteins, the
exon-exon junction complex (EEJC), associates
with mRNA at sites of intron removal. During
translation ribosomes displace the EEJCs. But
premature stop codons cause the ribosomes to fall
off, leaving some EEJCs on the mRNA. The
non-displaced EEJC recruits decapping and poly(A)
ribonucleases and exonuclease to degrade the
mRNA.
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 7-106
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Protein folding and chaperones The folding of
many proteins is facilitated by chaperones.
Chaperones are proteins that help guide protein
folding along productive pathways, by permitting
partially misfolded proteins to return to the
proper folding pathway.
What do chaperones recognize? Exposed
hydrophobic patches
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-82
Many chaperones are upregulated under conditions
where misfolded proteins accumulate, e.g.
heat-shock, and were originally identified as
heat shock (or other stress) - induced proteins.
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Chaperones are ATPases that bind to, and release
from, proteins Two major classes hsp60-like and
hsp70-like. hsp70-like act early in the life of
proteins, during synthesis, by binding to
hydrophobic patches in nascent peptide, clamping
down through ATP hydrolysis, and shielding the
hydrophobic patch from dangerous interactions.
Hsp70 later dissociates (while rebinding ATP)
presumably when the rest of the newly
synthesized protein is ready to interact with the
hydrophobic patch.
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-83
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hsp60-like proteins form a large macromolecular
machine. Misfolded proteins are fed into a
chamber where conditions are appropriate for
refolding on a productive pathway.
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-84
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Irretrievably misfolded proteins are targeted for
degradation by multiubiquitination which directs
them to the proteasome. ubiquitin a 76 amino
acid protein that can be C-terminally coupled
by ubiqutinin ligases to the epsilon-amino
group of lysine on target proteins recognized
by a wide variety of ubiquitin ligase E3
subunits.
E2 ca 30 isoforms E3 gt100 isoforms of varying
specificty
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-87B, C
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The proteasome a macromolecular machine for
processive degradation of multiubiquitinated
target proteins within an inner chamber.
Cap selectively binds multi-ubiquitinated
proteins, unfolds them (ATPase), and feeds them
into central cylinder.
Cylindrical stack of inward-facing
protease subunits.
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-86
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protein quality control failure Ideally,
misfolded/aberrant proteins that cannot be
properly refolded with chaperone assistance are
degraded by the multiubiquitination/proteasome
mechanism. However, this system is imperfect in
some situations, leading to the accumulation of
aggregates of insoluble proteins.
Bad outcome ! But could take a long time
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-85
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Many neurological diseases are associated with
abnormal misfolded protein aggregates. Alzheime
rs disease Parkinsons disease some genetic
forms caused by mutant E3 ubiquitin ligase
(parkin) Huntingtons disease CAG triplet
repeat, spinocerebellar ataxia polyQ
protein diseases Creutzfeldt-Jacob disease
(prion disease) many more
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A wide variety of proteins can form a similar
protease-resistant structure, the cross-beta
filament. This is based on peptide chain
backbone, rather than amino acid side chains.
This type of insoluble protein is called amyloid,
and stains characteristically with certain dyes
- Congo red, or thioflavin S/T.
Amyloid filament helical stack of flat
beta-sheet subunits
Abnormal conformation (colored elements beta-sheet
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Normal conformation (colored elements alpha-helix)
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-89 CD
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Prion diseases Creutzfeldt-Jacob
(humans) scrapie (sheep) bovine spongiform
encephalopathy (BSE mad cow disease) An
infectious conformation of an otherwise normal
cellular protein of unknown function, the prion
protein PrP. Abnormally folded PrP (PrP)
catalyzes the conversion of normally folded to
abnormally folded protein, presumably by subunit
interactions in an oligomeric complex.
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 6-89A
Prp
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N.B. experimental overexpression of chaperones
can protect against neurodegenerative disease
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response to unfolded proteins in the endoplasmic
reticulum 1. export of misfolded proteins back
into cytoplasm, for ubiquitination/ proteosomal
degradation.
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 12-55
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response to unfolded proteins in the endoplasmic
reticulum (contd) 2. ER unfolded protein
response Shown (next slide) for yeast - a
similar pathway operates in mammals, and Also a
parallel pathway based on proteolytic cleavage of
ATF6, an ER transmembrane protein whose
cytoplasmic domain is a transcription factor for
ER chaperone genes) Unfolded ER protein
activates a transmembrane protein kinase/
ribonuclease that specifically cleaves the
(nonfunctional) mRNA for a transcription factor
involved in ER chaperone gene expression. The
cleaved mRNA is respliced (nonspliceosomally) to
give functional mRNA. Result Upregulated
expression of ER chaperones
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ER unfolded protein response
Alberts et al Molecular Biology of the Cell
Garland Press 4th ed Fig 12-56
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Given the long lives of neurons, especially in
humans, basic cellular homeostatic mechanisms
such as macromolecular quality control, may be
operating near their limits.