Post-translational Modifications Amoeba 670 Trillion letters 23,000 Bufo bufo 6 Trillion letters Homo sapiens 3 Billion letters Homo-Centric View The Hard Truth

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

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.

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

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

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

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.

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

Processing in the Golgi

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

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

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

Eucaryote DNA is Packaged as Chromatin Human cell has 2m of DNA Nucleus is 0.006 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

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”)

Chromosome –Open and Closed

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

Histone modifications

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 4-35. 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.

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

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

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

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

Histone variants (Translational regulation)

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

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