Transcript Lecture Slides 3

Protein Folding
*
The production of a mature protein is a multistep process in
eukaryotic cells.
*
The final folded configuration, or shape, of a protein is
determined by its amino acid sequence.
*
Most proteins require the assistance of molecular chaperones,
like Hsp70 & Hsp60, to reach their final folded form.
*
These molecular chaperones bind to exposed hydrophobic
patches on the surface of incompletely folded proteins.
*
Hsp70 and Hsp60 act sequentially on proteins to help them
achieve their correct folded state.
*
Proteins that fail to be properly folded are ultimately targeted
for destruction.
Protein production in
eukaryotic cells is a
multistep process
Protein folding
& degradation
Figure 6-97
Steps in the
creation of a
functional protein
Figure 6-82
How a protein folds into a compact conformation
Figure 3-5
Three types of noncovalent bonds help proteins fold
Figure 3-4
A current view of the
protein folding
process
Structure of a molten globule
Figure 6-85
Co-translational protein folding
Some proteins begin to fold while still being synthesized.
Figure 6-84
The actions of the Hsp70 family of molecular
chaperones facilitate protein folding
* Molecular chaperones, like those in the hsp60 and hsp70
families, help guide the folding of most newly-synthesized
proteins.
* Hsp chaperones bind to hydrophobic patches that are exposed
on incompletely folded proteins.
* Repeated cycles of ATP binding & hydrolysis are generally
required for the proper folding of a polypeptide chain.
Figure 6-86
Structure & function of the Hsp60 family of
molecular chaperones
* Hsp60 acts on fully-synthesized proteins that have not
yet achieved their final folded form.
* The presence of incompletely folded proteins can lead
to the formation of aggregates that may have
dangerous consequences for the cell.
Figure 6-87
Proteasome-mediated protein turnover
*
The ultimate turnover of incompletely folded proteins is
mediated by an abundant ATP-dependent protease, known as
the proteasome.
*
The proteasome is a highly compartmentalized protease with
sequestered active sites.
*
The proteasome acts processively to convert the entire protein
substrate into short peptides.
*
Proteins destined for degradation are marked by polyubiquitin
chains that are added via a multistep conjugation process.
*
The process of degradation can be regulated by altering the
activity of the E3 ubiquitin ligase or the availability of the
degradation signal on the target protein.
Potential fates of newly-synthesized proteins
Figure 6-88 Molecular Biology of the Cell (© Garland Science 2008)
Processive protein digestion by the proteasome
Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008)
A hexameric
protein
unfoldase
Figure 6-91b Molecular Biology of the Cell (© Garland Science 2008)
Processive protein digestion by the proteasome
Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008)
An elaborate ubiquitin-conjugating system
marks proteins for proteasomal degradation
Figure 6-92
An elaborate ubiquitin-conjugating system
marks proteins for proteasomal degradation
Ubiquitinconjugating
system
Different ubiquitylation marks specify distinct
fates for tagged proteins
Figure 6-93 Molecular Biology of the Cell (© Garland Science 2008)
Regulation of protein degradation - I
Figure 6-94b Molecular Biology of the Cell (© Garland Science 2008)
Regulation of protein degradation - II
Figure 6-94a
Compact Intermediate
(Molten Globule)
Unfolded
Fast
Native
Slow
Slow
A failure to fold
properly can lead to
protein aggregation
Aggregation
Protein Misfolding Diseases (PMDs)
*
Many inherited diseases result from mutant proteins that evade
quality control processes, fold abnormally and ultimately form
aggregates.
*
The gradual decline of protein quality controls with age can
also lead to disease by permitting normal proteins to form
aggregates that can impair cellular functions.
*
Many neurodegenerative diseases, including Huntington’s and
Alzheimer’s, are associated with the presence of misfolded
protein aggregates in neuronal tissue.
*
The aggregates in patients can be intracellular (nuclear and/or
cytoplasmic) or extracellular.
Protein aggregates & neurodegeneration associated with
Alzheimer’s disease (AD)
A disease of
“protein folding”?
Microscopically, the brains of patients
with AD exhibit plaques composed of
beta-amyloid (a) and neurofibrillary
tangles that are associated with tau (b).
On a larger scale, AD results in the
widespread loss of neurons and
general atrophy of the brain (c, left)
compared to the normal brain (c, right).
Huntington's
Huntington's
Parkinson's
Alzheimer's
Alzheimer's
Alzheimer's
a | Intranuclear inclusions (INI) and cytoplasmic inclusions (CI) in the motor cortex of Huntington's disease brain
recognized with 1C2 antibody to expanded polyglutamine. b | Lewy body (LB) and other cytoplasmic inclusions (CI) that
contain -synuclein within a neuron of the substantia nigra of Parkinson's disease brain recognized with an antibody
specific for -synuclein. c | Neuritic plaque of Alzheimer's disease in the cerebral cortex. Hirano silver stain identifies
intracellular (neuritic) and extracellular protein aggregates. d | Intranuclear inclusion in the frontal cortex of Huntington's
disease brain recognized with anti-ubiquitin antibody. e | Neurofibrillary tangles of Alzheimer's disease in the
hippocampus immunostained with antibody specific for phosphorylated tau. f | Diffuse plaque of Alzheimer's disease in
the cerebral cortex. Amyloid (A)-specific antibody recognizes extracellular deposits of A (surrounding a neuron (N) and
a capillary (C)). Figure provided by O. Pletnikova and J. C. Troncoso, John Hopkins University, Baltimore, USA.
Autophagy-mediated protein turnover
Autophagy is a membrane trafficking pathway that targets cytoplasmic proteins &
organelles to the lysosome for degradation.
Protein aggregates can be targeted by the autophagy machinery.
Figure 13-42a
Autophagy-mediated protein turnover
Mitochondrion
Peroxisome
The autophagy pathway can also
target protein aggregates, like
those associated with
Huntington’s disease.
1 mm
Figure 13-41
Misfolded protein as the infectious agent in human disease?
Stanley Prusiner (UCSF)
Nobel Prize in 1997
Protein molecules, known as prions,
are the infectious agent responsible
for particular neurodegenerative
disorders like bovine spongiform
encephalopathy ("mad cow
disease") and its human equivalent,
Creutzfeldt-Jakob disease.
Misfolded protein as the infectious agent in human disease?
PrP protein
PrPSc
Figure 6-95
Chapter 15
• Cell Communication
Signaling pathways
 Relay signal
 Amplify signal
 Modulation of response
Figure 15-1
Cell-cell communication in an unicellular yeast
Development of cell-to-cell communication was likely important for the
evolution of multicellular organisms.
Unicellular organisms can communicate and influence the proliferation or
other aspects of nearby cells.
Figure 15-2
Signaling molecules can bind to either cell-surface or
intracellular receptors
Signaling molecules are generally secreted from the signaling cell by exocytosis.
Other signaling molecules are released by diffusion through the plasma
membrane or remain tightly bound to the cell surface.
Receptors on (or in) the target cell bind the signaling molecules and initiate an
appropriate response.
Figure 15-3
Different forms of intercellular signaling
Secreted molecules can mediate paracrine, synaptic and endocrine
modes of signaling.
Figure 15-4 Molecular Biology of the Cell (© Garland Science 2008)
Different forms of intercellular signaling
Differences between long-range signaling strategies
Figure 15-5 Molecular Biology of the Cell (© Garland Science 2008)
Figure 15-6
Extracellular signals can act slowly or rapidly to change behavior of
target cells
Cell fate is determined by multiple extracellular
signals
 Any given cell in a multicellular
organism is exposed to a
multitude of signaling molecules.
 Each cell type displays a distinct
set of receptors that allows it to
respond appropriately to the
signals present.
 Many cells require specific
signals for their continued
survival; in the absence of these
cues, they undergo apoptosis
(programmed cell death).
Figure 15-8
Different cells can respond differently to the
same signal
Figure 15-9
The persistence and rapidity of onset of a response depends
upon the turnover rate of the signaling molecules
Figure 15-11
Intracellular Receptor Signaling
Certain small hydrophobic molecules diffuse across the plasma membrane
and are bound by intracellular receptor proteins.
Figure 15-3b
Nuclear/intracellular receptor ligands
Figure 15-13 Molecular Biology of the Cell (© Garland Science 2008)
Ligands are transported through aqueous
environments by specific carrier proteins
Nuclear receptor
superfamily
The intracellular receptors are
transcriptional activators that exist in an
inactive state when unoccupied.
Ligand binding triggers a
conformational change which activates
the receptor molecules.
The activated receptor-ligand complex
then binds to regulatory sequences
upstream of target genes and initiates
their transcription.
Figure 15-14
Bipartite response to steroid hormones: Part I
Typically, the response
to these ligands is
biphasic; there is a
quick primary response
and an extended
secondary one.
Figure 15-15a
Bipartite response to steroid hormones: Part II
Figure 15-15b
Cell Surface Receptors
Cell surface receptors act as signal transducers. They convert the
extracellular signal to intracellular cues (or second messengers) that
alter the behavior of the target cell.
Figure 15-3a
Three classes of cell-surface receptors
(Neurotransmitter-gated ion channels)
Figure 15-16 Molecular Biology of the Cell (© Garland Science 2008)
G-protein-linked receptors alter the activities of target proteins
via an intermediate protein complex, the trimeric G protein.
Enzyme-linked receptors activate enzymatic activities associated
with themselves or other associated proteins.
The enzymes activated by this latter type of receptor are very often
protein kinases.
Signal Transduction Pathways
*
Most signaling events involve a signal transduction
pathway where the “message” is passed on from one set
of intracellular signaling molecules to another.
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The receptor protein performs the primary transduction
event by generating a new intracellular signal in response
to the binding of an external signal.
*
The signaling pathway components perform many different
functions.
Signal transduction pathways
Physically transfer the signal from the point
of reception to the place where the
response is to occur.
Transduce/transform the signal into a
molecular form that is able to
stimulate the appropriate response.
Amplify the initial signal such that only a
few extracellular signaling molecules
are required for a large response.
Distribute the signal so as to influence
many different processes in parallel.
Allow for additional controls at any step so
that the initial signal can be modulated
according to the conditions prevailing
inside the target cell.
Figure 15-17
Signaling by Cell Surface Receptors
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Signals received by G-protein-linked & enzyme-linked receptors
are often relayed to the nucleus where they alter gene
transcription.
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Most key intracellular signaling proteins act as molecular switches
that fall into two main classes.
*
The first are those proteins regulated by protein phosphorylation.
Some of these are protein kinases themselves and are organized
into phosphorylation cascades.
*
Protein kinases fall into two main groups, those that phosphorylate
serine and threonine residues and those that phosphorylate
tyrosine.
*
The second group of switch proteins consists of GTP-binding
proteins that oscillate between active GTP-bound and inactive
GDP-bound states.
*
Some of the intracellular signaling molecules function as integration
Two types of molecular switches in
signaling pathways
Figure 15-18 Molecular Biology of the Cell (© Garland Science 2008)
Figure 15-18 Molecular Biology of the Cell (© Garland Science 2008)
GEF
GAP
Signal
Integration
Figure 15-20
Preformed signaling complex on scaffold
The formation of intracellular assembly
complexes can enhance the speed,
efficiency and specificity of the response.
Figure 15-21a
Assembly of signaling complex on an activated
receptor
Figure 15-21b Molecular Biology of the Cell (© Garland Science 2008)
Modular interaction domains in signaling molecules
Figure 15-22
The many interactions between intracellular signaling molecules are
mediated by modular binding domains.
Activation curves as a function of concentration
Figure 15-25
The importance of single-cell measurements
Figure 15-24 Molecular Biology of the Cell (© Garland Science 2008)
Signal
Protein
(Inactive)
Kinase
Protein- P
(Active)
Phosphatas
e
Responses are sharpened when a signaling pathway activates
one enzyme and simultaneously inhibits a competing activity.
The effects of feedback loops in signaling pathways
Figure 15-26
Positive feedback can be
used to generate an all-ornone response.
Neurotransmitter
Signal
Membrane
Depolarization
Respons
e
Voltage-gated
Na+ channels
During Action Potential
Signal
Kinase
(Activated)
Autophosphorylation
Kinase- P (Activated)
A mechanism for signal pathway memorythe phosphorylated kinase is active even in the absence
of the original stimulus.
Figure 15-28a,b Molecular Biology of the Cell (© Garland Science 2008)
During muscle cell
differentiation:
Signal
Transcription of MyoD
Activates its
own promoter
Activates transcription
of muscle-specific
genes
MyoD protein
Negative feedback loops are used to attenuate responses
Figure 15-28c,d Molecular Biology of the Cell (© Garland Science 2008)