1. 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

2. What is meant by a protein activity?

3. 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)

4. 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

5. Important additional considerations: (1) What rate of change is required? (2) Do activities of any other proteins need to be changed simultaneously?

6. 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

7. Post-translational regulation 1.Reversible phosphorylation the first example (historically): mobilization of glucose from glycogen

8. Sugar stored in skeletal muscle and liver Polymer of glucose The enzyme glycogen phosphorylase releases individual subunits of glucose from the polymer

9. Glycogen phosphorylase

10. How to control glycogen phosphorylase so it catalyzes this reaction only when necessary?

11. Glycogen phosphorylase - P ATP ADP Phosphorylase kinase

12. Glycogen phosphorylase - P Phosphoprotein phosphatase

13. Protein phosphorylation: a ubiquitous strategy ATP cleaved to ADP; the P released covalently attached to a protein

14. Phosphorylation is often of just a single amino acid residue : Serine Tyrosine Threonine

15. Reversible protein phosphorylation: a widespread regulatory strategy

16. Post-translational regulation 2. Other chemical modifications of individual amino acids -egs. reversible acetylation, hydroxylation - Use of mass spectrometry to identify prosthetic groups:

17. Post-translational regulation 3.Cleavage of an internal domain

18. Post-translational regulation 3.Cleavage of internal domain Pro-caspase-3 activated to caspase-3 to initiate apoptosis

19. Post-translational regulation 4. Movement between subcellular compartments

20. Post-translational regulation 4. Movement between subcellular compartments

21. Post-translational regulation 5.Reversible association-dissociation

22. Heat shock factor-1 (HSF-1)

23. Post-translational regulation 6.Modification of immediate environment - eg. oxidation of cardiolipin causes cytochrome c release

24. Post-translational modifications change specific activity of proteins Only change the absolute amount of proteins secondarily (because transcription factors may also be reversibly phosphorylated)

25. Regulation by altering absolute amount of a protein

26. (1) change synthesis rate (2) change degradation rate

27. Steps on the road to protein synthesis

28. http://vcell.ndsu.nodak.edu/animations/transcription/ movie.htm

29. Assembly of the basal transcriptional complex on DNA

30. Various factors interact with transcriptional complex to alter gene transcription rate

32. 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

33. 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)

34. Hormones can activate gene transcription

35. Hormones regulate transcription of broad suites of genes due to presence of response elements

36. 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

37. Thyroxine Response Elements (TREs) Direct Repeat AGGTCAnnnnAGGTCA Inverted Repeat TGACCCnnnnnnAGGTCA Palindrome AGGTCATGACCT

38. 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

39. Linking hormone response elements (HREs) to modulate effect HRE

40. Hormone receptors that act as transcription factors tend to share a modular design

41. Other ways? Regulation by altering absolute amount of a protein

42. Regulated degradation Other ways? Regulation by altering absolute amount of a protein

43. Wide variability in cellular protein half-lives

44. 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)

45. Regulated protein degradation via ubiquitinylation and proteosomal digestion

46. A ubiquitous (pun intended) regulatory strategy

47. Next week: More of chapter 2- Receptors and signal transduction

56. 1. Relatively rapid adjustments in activity