1. Post-translational Modifications
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The Hard Truth

2. Major Modes of Activity Regulation
Control of activity of pre-existing enzymes using post-translation modification
Control amount (presence or absence) of enzyme using transcriptional regulation – transcription and translation
Activity control is rapid but synthesis control is slow

3. Post-Translational Protein Modification
Proteolytic cleavage
Fragmenting protein
Addition of chemical groups (>300 types)
Phosphorylation: activation and inactivation of enzymes
Acetylation: protein stability, used in histones
Methylation: regulation of gene expression
Glycosylation: cell–cell recognition, signaling
GPI anchor: membrane tethering
Hydroxyproline: protein stability, ligand interactions
Sulfation: protein–protein and ligand interactions
Disulfide-bond formation: protein stability
Deamidation: protein–protein and ligand interactions
Ubiquitination: destruction signal
Nitration of tyrosine: inflammation
Protein function may be altered by posttranslational modifications as well. Posttranslational modifications are defined as any changes to the covalent bonds of a protein after it has been fully translated. These changes can be broken into two broad categories: proteolytic cleavage (i.e., fragmenting the protein) and the addition of chemical groups to one or more amino acids on the protein.

4. Why are proteins modified?
Regulation of activity
modification may turn activity on
modification may turn activity off
modification may generate a different function
Protein-protein interaction
modification site may be a binding interface
Subcellular localization
modification site may be a targeting signal
modification may be a membrane anchor
Aging
modification may identify the protein for degradation
modification may target a protein to be scavenged

5. Phosphorylation
Most common posttranslational modification to proteins in eukaryotes
Enzymes and regulators are turned ‘on’ and ‘off’
Energy from ATP
Phosphate added to Serine, Threonine or Tyrosine
Kinases usually S/T specific or Y
Phosphatases usually take STY

6. The On-Off Switch: Phosphorylation
Protein switched off
Protein switched on by P

7. Glycosylation Most common form of protein modification
~50% of all mammalian proteins are glycosylated
Sugars are added in the ER and Golgi
Most proteins formed in the ER are glycoproteins
Many different forms and functions
CHO structures are very heterogeneous
In mammals, 9 different sugars are used
2 types: N-linked and O-linked
CHO means carbohydrate. N-linked means the sugar is attached to Asparagine (Asn, N). O-linked means that it is attached to the hydroxyl OH of serine or threonine.
Allow there are 9 different sugars used they can be joined together in many more ways that amino acids can. Thus a carbohydrate made of just 10 sugars can form thousands of possible structures.

8. Initial glycosylation in the ER
A precursor oligosaccharide is formed on a dolichol lipid
This is transferred to the growing protein

9. Processing in the Golgi

10. Membrane proteins: GPI anchors
GPI-anchored proteins are delivered to the plasma membran4

11. Myristylation and Farnesylation
Attaches cytosolic proteins to the plasma membrane
Protein usually involved in signal transduction

12. Proteolytic processing
Why is this common for secreted enzymes?
To store in an inactive state without affecting host cell
To allow rapid activation of hydrolytic enzymes outside

13. Eucaryote DNA is Packaged as Chromatin
Human cell has 2m of DNA
Nucleus is mm in diameter
Two opposing requirements:
Compaction
Access for Transcription, Replication & Repair
Euchromatin –
Partially decondensed
Transcribed genes
Heterochromatin –
Hypercondensed in interphase
Transcriptionally inert
Formation of chromosomal structures such as Centromeres and Telomeres

14. Chromosome Structure Nucleosome 2(H2A/H2B) + (H3/H4)2
fundamental unit of chromatin
147bp DNA wound 1.75 turns around histone core (octamer)
2(H2A/H2B) + (H3/H4)2
11 nm fiber (“beads on a string”)

15. Chromosome –Open and Closed

16. Post-Translational modification of Histones
Histones consist of a
Globular core domain
Unstructured N- and C-terminal tails
Post-translational modifications:
Acetylation – Lys
Methylation (mono-, di- and tri-) – Lys and Arg
Phosphorylation – Ser and Thr
Ubiquitination (mono- and poly-) – Lys
Sumoylation (Lys)
ADP-ribosylation
glycosylation
biotinylation
carbonylation

17. Histone modifications

18. Acetylation and Histones
Histones provide a Structure
Histones regulate gene expression
Short term
Long term i.e. epigenetic regulation
Acetylation enhances transcription
Deacetylation represses transcription
The histone acetylation switch. Targeted HAT and HDAC activities negotiate the acetylation status of chromatin. Acetylation establishes a structure that permits ATP-dependent chromatin remodeling factors to open promoters. Deacetylation, frequently followed by histone methylation, may form a solid base for highly repressive structures, such as heterochromatin. Acetylated histone tails are shown as yellow circles. Methylations are indicated as gray rectangles. HAT, histone acetyltransferase; HDAC, histone deacetylase; HMT, histone methyltransferase; HP1, heterochromatin protein 1.
Acetylation of the lysine residues at the N terminus of histone proteins removes positive charges, thereby reducing the affinity between histones and DNA.  This makes RNA polymerase and transcription factors easier to access the promoter region.  Therefore, in most cases, histone acetylation enhances transcription while histone deacetylation represses transcription.
Figure Covalent modification of core histone tails. (A) Known modifications of the four histone core proteins are indicated: Me = methyl group, Ac = acetyl group, P = phosphate, u = ubiquitin. Note that some positions (e.g., lysine 9 of H3) can be modified in more than one way. Most of these modifications add a relatively small molecule onto the histone tails; the exception is ubiquitin, a 76 amino acid protein also used in other cellular processes (see Figure 6-87). The function of ubiquitin in chromatin is not well understood: histone H2B can be modified by a single ubiquitin molecule; H2A can be modified by the addition of several ubiquitins. (B) A histone code hypothesis. Histone tails can be marked by different combinations of modifications. According to this hypothesis, each marking conveys a specific meaning to the stretch of chromatin on which it occurs. Only a few of the meanings of the modifications are known. In Chapter 7, we discuss the way a doubly-acetylated H4 tail is "read" by a protein required for gene expression. In another well-studied case, an H3 tail methylated at lysine 9 is recognized by a set of proteins that create an especially compact form of chromatin, which silences gene expression. The acetylation of lysine 14 of histone H3 and lysines 8 and 16 of histone H4 usually associated with gene expression is performed by the type A histone acetylases (HATs) in the nucleus. In contrast, the acetylation of lysines 5 and 12 of histone H4 and a lysine of histone H3 takes place in the cytosol, after the histones have been synthesized but before they have been incorporated into nucleosomes; these modifications are catalyzed by type B HATs. These modified histones are deposited onto DNA after DNA replication (see Figure 5-41), and their acetyl groups are taken off shortly afterwards by histone deacetylases (HDACs). Thus, the acetylation at these positions signals newly replicated chromatin. Modification of a particular position in a histone tail can take on different meanings depending on other features of the local chromatin structure. For example, the phosphorylation of position 10 of histone H3 is associated not only with the condensation of chromosomes that takes place in mitosis and meiosis but also with the expression of certain genes. Some histone tail modifications are interdependent. For example methylation of H3 position 9 blocks the phosphorylation of H3 position 10, and vice versa.

19. Histone Methylation Histone lysine demethylases
Histone lysine methyl transferases
Methylate specific Lys (K) residues depending on histone type,
Activation or repression of transcription
depends on specific residue and number of methyls
Histone lysine demethylases
Demethylates mono- or di-methylated lysine
Repression or activation; depends on cofactors
Histone arginine methyl transferases
Methylate Arg (R) residues
Mainly linked to transcription activation
Histone arginine demethylases
Converts arginine to citrulline
Results in transcriptional repression
Varying number of methyl groups:
Lys – mono- di- or tri-methylated
Arg – mono- or di-methylated

20. Histone Acetylation Histone acetyl transferases Histone de-acetylases
Add acetyl groups to histone tails
Reduces interaction of histones with DNA
Facilitates transcription
Associated with inducible expression
Histone de-acetylases
Remove acetyl groups from histone tails
Increases interaction of DNA and histones
Represses transcription (usually)
May involve same Lys residues as for methylation

21. Histone Phosphorylation
Phosphorylation by aurora AIR2–Ipl1 kinase
Required for chromosome condensation
Phosphorylation by ATM or DNA-PK
response to DNA damage
signal transduction leading to gene activation
Prevents nearby histone methylation
Alters recruitment of binding proteins

22. Histone Ubiquinitation
E3 Ubiquitin ligases
Mono- or poly ubiquitination recruits proteasome
Alters chromatin structure
Regulates H3 methylation
Ubiquitination opens nucleosome so accessible to methyl transferases
Regulates transcription
Ubiquitination of H2A interferes with transcription
Ubiquitin hydrolases
De-ubiquitination regulates mono- vs tri-methylation of H3-K4

23. Histone variants (Translational regulation)

24. Histone code hypothesis
Model 1 – structural role for modifications
Based on charge density of histone tails
Modulates protein-DNA interactions
Model 2 – modifications as recognition sites
Recruit effector molecules
Bromodomains – bind K-acetyl
Chromodomains, Tudor domains, WD40 repeats – bind K-methyl and are residue specific

25. Reading the Histone code
H3K4 HKMT
(activation)
HAT & de-Ub
Heterochromatin
Gene silencing
DNA repair