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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 liverPolymer of glucoseThe 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
ATP
ADP
Phosphorylasekinase
Glycogen phosphorylase - P
Phosphoproteinphosphatase
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 :
SerineTyrosineThreonine
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 = enhancersNegative = silencers
Regulatory elements not on DNA (protein factors; trans-acting)
Positive = activatorsNegative = 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
Example: thyroid hormone (thyroxine)
Stimulates metabolism and metabolic rate (many genes)
Hormones regulate transcription of broad suites of genes due to presence of response elements
Thyroxine Response Elements (TREs)
Direct Repeat
AGGTCAnnnnAGGTCA
Inverted Repeat
TGACCCnnnnnnAGGTCA
Palindrome
AGGTCATGACCT
Half-Site Promiscuity modulates effect
Achieves finer control of transcriptional activationPerfect 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
Other ways?
Regulation by altering absolute amount of a protein
Regulated degradation
Other ways?
Regulation by altering absolute amount of a protein
Wide variability in cellular protein half-lives
N-terminal amino acid
Protein half-life
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 2- Receptors and signal transduction
1. Relatively rapid adjustments in activity