Post-translational modification

Question 1

What is biological complexity linked to?

Answer 1

Gene products

Question 2

PTM

Answer 2

Chemical modifications of a protein after its translation

Question 3

How many different types of PTMs are there

Answer 3

200

Question 4

How do PTMs alter proteins

Answer 4

Alter localisation, turnover, activity state and interaction with other proteins

Question 5

Mod form

Answer 5

Modified form of the protein (2 to the power of the number of PTM sites)

Question 6

2 most abundant forms of PTM

Answer 6

Phosphorylation and glycosylation

Question 7

What AAs can be phosphorylated

Answer 7

Ser, thr, tyr

Question 8

BK channel

Answer 8

Large conductance voltage- and Ca2+-gated K+ channel

Question 9

What is the structure of the BK channel

Answer 9

Pore forming alpha subunits and modulatory beta subunits (4 alpha subunits assemble to make functional channel)

Question 10

What is BK channel involved in

Answer 10

Neuronal firing and neuropeptide release

Question 11

How is the BK channel activated

Answer 11

Alcohol activates - increases K+ channel - causes reduced neuronal excitability and neuropeptide release - leads to depression of central neurons, motor incoordination, diuresis

Question 12

What happens to BK channel when repeated exposure to alcohol

Answer 12

Phosphorylation of T107 by CAMKII kinase - switches channel alcohol response from activation to inhibition - molecular dimmer switch - mediate alcohol tolerance

Question 13

How to detect phosphorylation

Answer 13

Phosphospecific stain, Pro-Q Diamond, 32P or 33P labelling and autoradiography), western blotting with phospho-T, S, Y antibodies

Question 14

Phosphoproteomics enrichment

Answer 14

Immunoprecipitation, affinity chromatography, chemical derivitisation

Question 15

Immunoprecipitation

Answer 15

1. Immobilised pS, pT, pY antibodies 2. Immobilised antibodies against kinase consensus sites

Question 16

Affinity chromatography

Answer 16

1. Immobilised IMAC (immobilised metal ion affinity chromatography) beads 2. Immobilised TiO2 beads 3. SIMAC - peptide mix + IMAC beads

Question 17

Chemical derivitisation

Answer 17

1. Beta-elimination 2. PAC - phosphoramidate chemistry

Question 18

Why are sugars important?

Answer 18

Act as recognition molecules

Question 19

What is the name of the sugar coat that all cells have

Answer 19

Glycocalyx

Question 20

What is key for cell-to-cell communication

Answer 20

Glycan-lectin recognition

Question 21

How is there variation in glycosidic bond formation

Answer 21

Orientation of planes (alpha or beta)

Question 22

What enzyme aids glycosidic bond formation

Answer 22

Glycosyltransferase (link to hydrolysis of phosphate bond)

Question 23

What are the 2 forms of glycosylation

Answer 23

N- and O-linked

Question 24

N-linked glycosylation

Answer 24

Sugar linked to amide nitrogen in the side chain of asparagine

Question 25

O-linked glycosylation

Answer 25

Sugar linked to oxygen in the side chain of serine or threonine

Question 26

N-glycosylation consensus sequence

Answer 26

Asn-X-Ser/Thr (X = any AA except proline)

Question 27

What type of modification is N-linked glycosylation

Answer 27

Co-translational modification

Question 28

How is N-linked glycosylation initiated

Answer 28

In ER by en bloc transfer of pre-formed lipid anchored conserved glycan

Question 29

What are the two sections of the pre-formed lipid anchored conserved glycan?

Answer 29

Dolichol (lipid), core, antennae

Question 30

N-glycan biosynthesis

Answer 30

1. Synthesis of lipid linked precursor oligosaccharide 2. En bloc transfer to protein 3. Processing

Question 31

Types of N-linked glycans

Answer 31

1. High mannose N glycans 2. Hybrid N-glycans 3. Complex N-glycans

Question 32

What sugars are antennae capped with and why are they capped

Answer 32

Fuc or NeuAc to form ligands for specific recognition by lectins

Question 33

O-glycosylation consensus sequence

Answer 33

No consensus sequence but some rules e.g. nearby proline, tandem repeats of Ser/ Thr

Question 34

How is O-glycosylation initiated

Answer 34

In golgi by addition of single sugar (usually GalNAc)

Question 35

How are O-glycans classified

Answer 35

By their core structure (8 known)

Question 36

What are the most common O-glycan core structures

Answer 36

Cores 1 and 2

Question 37

What O-glycans are most common on mucins

Answer 37

Cores 1 - 4

Question 38

What are mucins

Answer 38

Cell surface and excreted glycoproteins - protect mucus membranes by keeping them hydrated, acting as lubricants and prevent microorganism invasion

Question 39

What are O-GlcNAc proteins

Answer 39

Type of glycosylation localised in cytoplasm or nucleus - they are phosphoproteins - dynamic

Question 40

What are proteoglycans

Answer 40

Special type of O-glycoprotein found in ECM and structural tissues (e.g. cartilage) which form massive molecular aggregates (act as shock absorbers)

Question 41

What is a GPI-anchor

Answer 41

Glycosylphosphatidylinosotol-anchors. Protein anchored to membrane via diacylglycerol glycolipid.