L16: Post-translation Modifications

Question 1

Post translational modification (PTM)

Answer 1

Covalent processing events -> change properties of protein by proteolytic cleavage or by addition of modifying group to one or more AA

Change size, charge, structure & conformation of proteins

Involve AA residues

Question 2

Consequence of PTM

Answer 2

Protein folding/conformation (modification may be important for 3D structure)

Regulation of activity (modification may turn activity on or off)

Protein-protein interaction

Subcellular localisation (modification site may be targeting signal and may be a membrane anchor)

Protein degradation (modification may identify protein for degradation)

Question 3

Covalent modification of..

Answer 3

N terminus (formulylation, acetylation, pyroglutamate formation)

C terminus (GPI-anchoring, amidation, polyglycylation)

AA residues (side chains)

Peptide bonds

Question 4

Major types of PTMs

Answer 4

Proteolysis

Phosphorylation

Lipidation

Glycosylation

Question 5

Proteolytic cleavage

Answer 5

Most common form of PTM

Partial proteolysis of proteins: common maturation step

E.g. insulin. Schematic processing of preproinsulin to proinsulin by signal peptidase in ER

Proinsulin to insulin by proteases in Golgi network

Question 6

Advantages over synthesis and binding of 2 separate polypeptides

Answer 6

Ensure production of equal amts of A & B chains without coordination of 2 translational activities

Proinsulin folds into 3D structure in which cysteine residues placed for correct disulfide bond formation

Question 7

Phosphorylation

Answer 7

Most common mechanism of regulating protein function & used for transmitting signals throughout cell

Critical roles in regulation of many cellular processes including cell cycle, growth, apoptosis & signal transduction pathways

Question 8

Protein kinases

Answer 8

Substrates include lipids, carbohydrates, nucleotides and proteins

Serine, threonine, tyrosine

Question 9

Phosphoprotein phosphatases

Answer 9

2 major families: PP1 & PP2A

Question 10

How does phosphate group affect protein function?

Answer 10

Cause conformational changes in phosphorylated protein

• > regulate catalytic activity of protein m
• > recruit neighbouring proteins that recognise and bind to phosphomotifs

Question 11

Lipidation

Answer 11

Many proteins undergo covalent alterations before they become functional. Conversion of inactive apo forms of proteins by covalent installation

Question 12

Lipid-anchoring motifs

Answer 12

One or more lipid anchors that help to target the modified proteins to particular membranes

Purpose: anchors proteins to membranes, facilitates protein protein interaction

Types: Palmitoyl group on internal Cys (or Ser), N-Myristoyl group on amino-terminal Gly, Farnesyl (or geranylgeranyl) group on carboxyl-terminal Cys

Exterior: GPI anchor on carboxy terminus

Question 13

Lipidation-anchoring motifs example

Answer 13

N-myristoylation or amide-linked myristol anchor

Always myristic acid (14 carbon fatty acid)

Always N-terminal

Always a Gly residue that links

Question 14

Glycosylation

Answer 14

Glycoproteins consist of proteins covalently linked to carbohydrate

Question 15

Glycosylation classified into 2 groups

Answer 15

O-linked oligosaccharides (O-glycans): O-glycosidic bond; no discernable AA sequence motif

N-linked oligosaccarides (N-glycans): N-glycosidic bond; consensus site or motif on protein is Asn-X-Ser/Thr

Question 16

N-linked saccharides

Answer 16

High mannose type- contains all mannose (Man) outside core in varying amts

Hybrid type- contains various sugars such as galactose (Gal) and amino sugars such as N-acetyl glucosamine (GlcNAc)

Complex type- is similar to hybrid type. Contains sialic (Sia) acids to varying degrees

Common pentasaccharide core and are synthesised from common precursor oligosaccharide

Question 17

Synthesis of N-linked saccharides

Answer 17

In ER

Complex carbohydrate chain with 23 or more separate enzyme steps involved in assembly, trimming and maturation of branched carbohydrate structures

Carbohydrate core synthesised -> attached to growing protein

Question 18

N linked glycoproteins

Answer 18

Functions:

Proper of folding of newly synthesised proteins: antibiotic tunicamycin block N-glycosylation -> non-functional proteins

Question 19

Proteolytic cleavage example: Insulin

Answer 19

Schematic processing of proinsulin to proinsulin by signal peptidase in ER

Prepoinsulin (insulin synthesised as one long polypeptide) -> cleavage -> proinsulin -> cleavage insulin with c peptide as by product & disulfide bonds form between A & B chain (cysteine forms bond)

Measure c peptide as diabetes test

Question 20

Phosphorylation example: Glycogen Phosphorylase

Answer 20

Glycogen phosphorylase : an enzyme that breaks down glucose 1 phosphorylate

Glycogen + Pi -> glycogen (n-1) + glucose 1-phosphate

Non-phosphorylated : exist as dimer. Change in conformation for activation. Peptide blocks access to active site -> moves aside -> substrate (phosphate residue) can enter active site

Question 21

Lipidation example: pyruvate dehydrogenase

Answer 21

Pyruvate -> cleavage -> CO2 -> attach TPP

Disulfide forms on lipoate with redox reaction (acetyl groups transfer to thiol group sitting on lipoate)

Enzyme catalysed reaction: coenzyme A attached to thiol group -> acetyl CoA

Reduce lipoate -> thiol groups reoxidise by dehydrogenase

Question 22

Lipidation example

Answer 22

Hormone bind to receptor

Hydrophobic lipid group bind to anchor alpha -> GTP binds to alpha -> subunits dissociate from receptor

If alpha wasnt anchored -> drift into cytoplasm

GTP can move from through membrane -> interact with adenyl cyclase (cause synthesis cAMP)

Overtime, GTP breaks down to GDP -> reassociation of complex -> inactivation of G protein and lose hormone