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This class:
Regulation of protein activities
(1) What is a protein activity?
(2) How to change the rate of a specific cellular
activity?
(3) Rapid vs slower change
(4) Varying amount vs specific activity of a protein
(5) Coordinating simultaneous changes in related
proteins
(6) How to achieve fine/differential regulation
What is meant by a protein activity?
Overall cellular activity vs specific activity
Specific activity of a protein = amount of event
performed per unit time per molecule of that protein
Overall activity of a protein = amount of event per unit
time per cell (or unit tissue mass)
Regulation of protein function
How to change the rate of a protein’s overall cellular
activity?
(1) Change specific activity of that protein
(2) Change amount of that protein
Important additional considerations:
(1) What rate of change is required?
(2) Do activities of any other proteins need to
be changed simultaneously?
Post-translational regulation of protein
function
• Affects existing proteins (does not ∆ amt,
but ∆ specific activity)
• Can be rapid
• Can be short- or long-lived
• Multiple proteins may be affected
• Multiple modifications are possible within a
protein
Post-translational regulation
1. Reversible phosphorylation
the first example (historically): mobilization of
glucose from glycogen
Sugar stored in skeletal muscle and liver
Polymer of glucose
The enzyme glycogen phosphorylase releases individual
subunits of glucose from the polymer
Glycogen phosphorylase
How to control glycogen phosphorylase so it catalyzes
this reaction only when necessary?
Glycogen phosphorylase - P
ADP
Phosphorylase
kinase
ATP
Glycogen phosphorylase - P
Phosphoprotein
phosphatase
Protein phosphorylation: a ubiquitous strategy
ATP cleaved to ADP; the P released covalently attached to
a protein
Phosphorylation is often of just a single
amino acid residue :
Serine
Tyrosine
Threonine
Reversible protein phosphorylation: a widespread
regulatory strategy
Post-translational regulation
2. Other chemical modifications of individual amino
acids
- egs. reversible acetylation, hydroxylation
- Use of mass spectrometry to identify prosthetic
groups:
Post-translational regulation
3. Cleavage of an internal domain
Post-translational regulation
3. Cleavage of internal domain
Pro-caspase-3
activated to
caspase-3 to
initiate apoptosis
Post-translational regulation
4. Movement between subcellular compartments
Post-translational regulation
4. Movement between subcellular compartments
Post-translational regulation
5. Reversible association-dissociation
Heat shock factor-1 (HSF-1)
Post-translational regulation
6. Modification of immediate environment
- eg. oxidation of cardiolipin causes cytochrome c
release
• Post-translational modifications change specific
activity of proteins
• Only change the absolute amount of proteins
secondarily (because transcription factors may also
be reversibly phosphorylated)
Regulation by altering absolute amount of a protein
Regulation by altering absolute amount of a protein
(1) change synthesis rate
(2) change degradation rate
Steps on the road to protein synthesis
http://vcell.ndsu.nodak.edu/animations/transcription/
movie.htm
Assembly of the basal transcriptional complex
on DNA
Various factors interact with transcriptional
complex to alter gene transcription rate
Affecting transcription rate
Some terminology:
Regulatory elements on DNA (cis-acting):
Positive = enhancers
Negative = silencers
Regulatory elements not on DNA (protein factors;
trans-acting)
Positive = activators
Negative = repressors
Some definitions
• Transcription factor: interacts with basal
transcriptional complex and DNA
• Co-transcriptional activator: interacts with
transcription factors to activate or repress (eg.
PGC-1)
Hormones can activate gene transcription
Hormones regulate transcription of broad suites of genes
due to presence of response elements
Hormones regulate transcription of broad suites of genes
due to presence of response elements
Example: thyroid hormone (thyroxine)
Stimulates metabolism and metabolic rate (many genes)
Thyroxine Response Elements (TREs)
Direct Repeat
AGGTCAnnnnAGGTCA
Inverted Repeat
TGACCCnnnnnnAGGTCA
Palindrome
AGGTCATGACCT
Half-Site Promiscuity modulates effect
Achieves finer control of transcriptional activation
Perfect Palindrome (= `the ideal` TRE)
AGGTCATGACCT
Promiscuity = substitution of “non-essential” bases
CGGTCATGACCA
AGGTCATGACCC
* The greater the divergence of RE from the ideal, the less
strongly it enhances gene transcription
Linking hormone response elements (HREs)
to modulate effect
HRE
HRE
HRE HRE
Hormone receptors that act as transcription factors
tend to share a modular design
Regulation by altering absolute amount of a protein
Other ways?
Regulation by altering absolute amount of a protein
Other ways?
Regulated degradation
Wide variability in cellular protein half-lives
N-terminal
amino acid
Protein halflife
Ala (A)
4.4 hour
Cys (C)
1.2 hour
Asp (D)
1.1 hour
Glu (E)
1 hour
Phe (F)
1.1 hour
Gly (G)
30 hour
His (H)
3.5 hour
Ile (I)
20 hour
Lys (K)
1.3 hour
Leu (L)
5.5 hour
Met (M)
30 hour
Asn (N)
1.4 hour
Pro (P)
>20 hour
Gln (Q)
0.8 hour
Arg (R)
1 hour
Ser (S)
1.9 hour
Thr (T)
7.2 hour
Val (V)
100 hour
Trp (W)
2.8 hour
Tyr (Y)
2.8 hour
Half-lives of cellular proteins
vary widely, depending on:
• identity of N-terminal amino
acid (table)
• damage
• specific chemical modifications
(eg. ubiquitinylation)
Regulated protein degradation via ubiquitinylation and
proteosomal digestion
A ubiquitous (pun intended) regulatory strategy
Next week:
More of chapter 2Receptors and signal transduction
1. Relatively rapid adjustments in activity