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

ADPPhosphorylasekinase

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

AGGTCAnnnnAGGTCAInverted Repeat

TGACCCnnnnnnAGGTCAPalindrome

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