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[Vierstra, 2003 TIPS]
[Vierstra, 2003 TIPS]
Ubiquitin/26S proteasome pathway
Simplified
Ubiquitination
Proteolysis
+ ATP
Ub
E1
E2
E3
Target
Ub
Ub Ub
Ub
Target
+ ATP
26S proteasome
Loss of 26S proteasome function
WT
rpn10-1
Smalle et al., 2003
Loss of proteasome function in the rpn10-1 mutant, leads to growth
inhibition and the accumulation of polyubiquitinated target proteins.
Diversity in Ubiquitination Machinery is
largely provided by the many E3s
Single E1
Few E2’s
Many E3’s
E3 structure/function
Ub
E2
Target protein
E3 (Ubiquitin ligase)
Target binding
E2 binding
Ub
Target protein
E2
Target binding
E2 binding
E3 (Ubiquitin ligase)
E3 structure/function
Ub
Ub
Ub
Ub
UbUb
Ub
Ub
Target protein
26S proteasome
Ub
UbUb
Ub
Target protein
E3
Target binding
E2 binding
Number of E3s per genome
Saccharomyces cereviseae
68
Caenorhabditis elegans
657
Drosophila melanogaster
189
Homo sapiens
527
Arabidopsis thaliana
1156
Advantages of proteolysis control in signal transduction
1) Fast response to a change in signal intensity:
direct control of protein activities in contrast to transcriptional
regulation that involves transcription, transcript processing and
translation steps before protein abundance is increased.
2) Proteolysis control can rapidly increase as well as
decrease a proteins activity (only an increase is possible
with transcriptional regulation).
3) Accurate reflection of signal intensity in response
output: secundary modifications such as
phosporylation/dephosphorylation can also directly change a
proteins activity. However since such controls tend to be leaky,
i.e. are the result of modification/demodification equilibria, their
outcome depends on the initial abundance of the target protein.
Why more E3s in plants?
* More E3s means more proteolysis control of signaling.
* Energetically wasteful?
* Animals can side-step adverse environmental conditions.
•The sessile plant must endure.
•Plants need to be more sensitive to environmental changes.
* Proteolysis control of signaling allows for quick responses
to changes in signal intensities (changes in environmental
conditions).
* Proteolysis control also allows for an accurate responsestrength to signal-intensity ratio.
* Allows for a constant state of readiness.
* Plants are less energy-limited.
From Kepinski and Leyser, 2003
Proteolysis control of signaling
Signal transduction leads to destabilization of a
repressor of the response or stabilization of a
response activator.
This is accomplished via secundary modification
(phosphorylation or dephosphorylation) of the target
protein that leads to or prevents its detection by a
Ubiquitin ligase (E3). Alternatively, signaling directly
controls E3 affinity for the target protein.
Controlling the activity of a protein via its degradation
rate allows for faster and more accurate responses to
changing concentrations/intensities of the signal
(changing environment).
The Ub/26SP pathway and signaling
Describe two mechanisms that can be used to transform a
signal into a response via the regulated degradation of a
repressor of this response. Show how increased signal intensity
leads
to
an
increased
response
output.
The Ub/26SP pathway and signaling
Describe two mechanisms that can be used to transform a
signal into a response via the regulated degradation of an
activator of this response. Show how increased signal intensity
leads to an increased response output.
Control of gene expression via conditional proteolysis
EXAMPLE 1:
Signal (variable)
DNA
RNA
Constitutive expression
Response
repressor
*
Response
repressor
E3
Response
(variable)
ors
e s r
n pee
se p s
ro R
Control of gene expression via conditional proteolysis
EXAMPLE 2:
Signal (variable)
DNA
RNA
Constitutive expression
Response
activator
*
Response
activator
E3
ao r s
i t v
n pe c
e o Rs
Response
(variable)
ABA response
(Vierstra, 2009)
Control of gene expression via conditional proteolysis
EXAMPLE 3:
Signal (variable)
DNA
RNA
Constitutive expression
Response
activator
Response
(variable)
E3
ao r s
i t v
n pe c
e o Rs
Control of gene expression via conditional proteolysis
EXAMPLE 3: Photomorphogenesis
Light (variable)
DNA
RNA
HY5
Constitutive expression
COP1 a o
i tr sv
n pe c
e o Rs
Light responses
(variable)
COP1 acts as an E3 to target
HY5 for degradation
Coil
RING
E2*
WD-40 repeats
Ub
bZIP
COP1
COP1
HY5
HY5
Ub
E2
Ub
Ub
Ub
Ub
E1
Degradation
Degradation
via
the 26S
26Sby
Proteasome
proteasome
Ub
(Osterlund et al.,2000)
Photomorphogenesis
Light intensity
LIGHT
COP1
HY5
LIGHT RESPONSES
HY5
(Osterlund et al., 2000)
Control of gene expression via conditional proteolysis
EXAMPLE 4:
Signal (variable)
DNA
RNA
Constitutive expression
Response
repressor
Response
(variable)
E3
ors
e s r
n pee
se p s
ro R
Control of gene expression via conditional proteolysis
EXAMPLE 4: Auxin response pathway
Auxin (variable)
DNA
RNA
Constitutive expression
AUX/IAA
factors
TIR1
Auxin Response
(variable)
ors
e s r
n pee
se p s
ro R
Auxin response
(Vierstra, 2009)
Jasmonate response
(Vierstra, 2009)
Summary: important to remember
1) How does a target protein become polyubiquitinated through the
sequential action of E1, E2 and E3 enzymes?
2) 26S Proteasome: structure/function. How does the proteasome detect
and then degrade target proteins?
3) Where in the cell does the Ubiquitin/26S Proteasome pathway act?
4) ATP requiring steps in the pathway? Energy is needed to establish
specific proteolysis (as opposite to non-specific).
5) Predict the effects of loss of function of different components of the
pathway (proteasome --- pleiotropic; E3 --- highly specific phenotype).
6) Why proteolysis control of signal transduction (what are the
advantages)?
7) Possible mechanisms of conditional protein degradation to control
signal/response ratios (see examples 1-4).
2011
PLS 623
Spring Semester 2011
Exam: Targeted protein Degradation
March 25, 2011
Name: _____________________________
E-mail:
1) Describe in detail how a target protein is degraded via the Ubiquitin/26S
Proteasome pathway. (20 pts)
2) W hy does protein degradation via the Ubiquitin/26S Proteasome pathway require
ATP? (10 pts)
3) W hy are gene families that encode E3s (ubiquitin ligases) more complex (larger)
than families that encode E2s or E1s? (10 pts)
4) Describe two mechanisms that can be used to transform a signal into a response
via the regulated degradation of a repressor of this response. Show how
increased signal intensity leads to an increased response output. (10 pts)
2009
PLS 623
Spring Semester 2009
Exam: Targeted protein Degradation
March 27, 2008
Name: _____________________________
E-mail:
1) Describe in detail how a target protein is degraded via the Ubiquitin/26S
Proteasome pathway. (20 pts)
2) W hy does protein degradation via the Ubiquitin/26S Proteasome pathway require
ATP? (5 pts)
3) I n which cellular compartments is the Ubiquitin/26S Proteasome pathway active?
(5 pts)
4) Describe two mechanisms that can be used to transform a signal into a response
via the regulated degradation of an activator of this response. Show how
increased signal intensity leads to an increased response output. (20 pts)
2008
PLS 623
Spring Semester 2008
Exam: Targeted protein Degradation
March 21, 2008
Name_____________________________
1) Describe in detail how a target protein is degraded via the Ubiquitin/26S
Proteasome pathway. (20 pts)
2) W hy are gene families that encode E3s (ubiquitin ligases) more complex (larger)
than families that encode E2s or E1s? (10 pts)
3) W hat are the advantages of controlling the abundance of a regulatory protein via
targeted proteolysis as compared to transcriptional regulation. (10 pts)
4) Describe two mechanisms that can be used to transform a signal into a response
via the regulated degradation of a repressor of this response. Show how increased
signal intensity leads to an increased response output. (10 pts)