Affecting Form and Function Fayig El-Migdadi, MD, PhD

Why are proteins modified? Secreted and Membrane-Associated Proteins Acylation Methylation Glycosylation Membrane proteins Proteolytic processing Phosphorylation Acetylation Sulfation Small Molecule Binding Regulated degradation Selenoproteins

Proteins that are membrane bound or are destined for excretion are synthesized by ribosomes associated with the membranes of the endoplasmic reticulum (ER). The ER associated with ribosomes is termed rough ER (RER). This class of proteins all contain an N-terminus termed a signal sequence or signal peptide.

The signal peptide is usually 13-36 predominantly hydrophobic residues. The signal peptide is recognized by a multi-protein complex termed the signal recognition particle (SRP). This signal peptide is removed following passage through the endoplasmic reticulum membrane. The removal of the signal peptide is catalyzed by signal peptidase.

Proteins that contain a signal peptide are called preproteins to distinguish them from proproteins. However, some proteins that are destined for secretion are also further proteolyzed following secretion and, therefore contain pro sequences. This class of proteins is termed preproproteins.

Many proteins are modified at their N-termini following synthesis. In most cases the initiator methionine is hydrolyzed and an acetyl group is added to the new N-terminal amino acid. AcetylCoA is the acetyl donor for these reactions. Some proteins have the 14 carbon myristoyl group added to their N-termini. The donor for this modification is myristoyl-CoA. This latter modification allows association of the modified protein with membranes. The catalytic subunit of cyclicAMPdependent protein kinase (PKA) is myristoylated.

Post-translational methylation of proteins occurs on nitrogens and oxygens. The activated methyl donor is S-adenosylmethionine (SAM). The most common methylations are on the -amine of lysine residues. Methylation of lysine residues in histones in DNA is an important regulator of chromatin structure and consequently of transcriptional activity. Lysine methylation was originally thought to be a permanent covalent mark, providing longterm signaling, including the histone-dependent mechanism for transcriptional memory.

Major form of protein modification Sugars are added in the ER and Golgi Most proteins formed in the ER are glycoproteins Many different forms and functions

A precursor oligosaccharide is formed on a dolichol lipid. This is transferred to the growing protein.

by glucosidase

Stabilise proteins against proteolysis

Limit approach of macromolecules to protein surface

Modulation of immune response

Selectins (weakly) bind to oligosaccharides Helps to concentrate lymphocytes in lymphoid organs Attracts white blood cells & platelets to inflammation sites

Provide sorting signals

M6P for lysosomal hydrolase GPI anchors (see later)

Contributes to differentiation events in organism development

Removing N-acetylglucoasmine transferase I in mice causes embryo death
Neural tube development and left-right body plan asymmetry impaired

Made of core protein and polysaccharide chains

Extremely diverse

Form hydrated gel

Resists compressive forces Regulate traffic (perlecan in kidney)

Can regulate secreted protein activity

e.g. chemokines in inflammatory response

GPI-anchored proteins are delivered to the apical plasma membrane Trypanosomes can shed these proteins to avoid immune attack

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

Why is this common for secreted enzymes?

Some peptides (e.g. enkephalins) too short by themselves Prevent premature activation of hydrolytic enzymes

Most common posttranslational modification to proteins in eukaryotes Enzymes and regulators are turned on and off Energy from ATP

Broadcasts signals from cell surface

Cell proliferation Differentiation

Move chromosomes during mitosis Move organelles along molecular tracks Move enzymes along DNA during DNA synthesis

ATP binding - conformation 1 to conformation 2 ATP hydrolyzed to ADP Pi conformation 2 to conformation 3. Release of ADP and Pi - back to conformation 1. Irreversible one direction only

Acetylation enhances transcription Deacetylation represses transcription

Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen and in some secreted proteins (eg gastrin). The universal sulfate donor is 3'phosphoadenosyl-5'-phosphosulphate (PAPS).

Retinal Heme group

.A
.B

Degradation mechanisms:
Ubiquitin ligase Degradation signal

Multiubiquitin chain marks protein for degradation in proteosome

Sulphydryls Amines

Disulphide bond Cysteinylation Methylation Acetylation Farnesylation Biotinylation Stearoylation Pyroglutamic acid Carboxylation Phosphorylation

Oxidation Glutathionylation Formylation Lipoic acid Myristoylation Palmitoylation Geranylgeranylation Deamidation Sulphation

Acids & amides Hydroxyl groups

Carbohydrates

Pentoses Hexosamines N-acetylhexosamines

Deoxyhexoses Hexoses Sialic acid

Post-translational modifications necessary for protein function

Correct protein folding Organism developmen Cellular Signaling Motor Proteins Regulating degradation others

Selenium is a trace element and is found as a component of several prokaryotic and eukaryotic enzymes that are involved in redox reactions. The selenium in these selenoproteins is incorporated as a unique amino acid, selenocysteine, during translation. A particularly important eukaryotic selenoenzyme is glutathione peroxidase. This enzyme is required during the oxidation of glutathione by hydrogen peroxide (H2O2) and organic hydroperoxides.

Details (small ubiquitin-related modifier)