Protein phosphorylation and O-GlcNAc signals Flashcards
How can phosphorylation be used in cell detection.
Phosphorylated proteins detectable by MS / electrophoresis and [32P]-ATP
Probing with phospho-specific antibodies
Many specific inhibitors available; there are 100+ phosphatases, each with their own substrate sequence specificity due to specific peptide recognition groves in their structure
As a result, structures/molecular mechanisms well understood.
How is protein phosphorylation important to the cell.
With its very complex signal tranduction pathways, phosphorylation regulates EVERY CELL PROCESS!
What is Protein O-GlcNAcylation? What role can it have in the cell?
It is the post modification of SERINE or THR with a GlcNAc (a derivative of glucose/glycoprotein).
This process is Reversible, everywhere, essential through Metazoa.
O-GlcNAcylation is involved in numerous cellular processes such as transcription, cell cycle progression, and signal transduction
Name some differences between Phosphorylation and O-GlcNAcylation.
Phosphorylation: is very detectable with lots of mapped functions and understood structures due to specific antibodies, inhibitors and target sequences.
A LOT LESS IS KNOWN about O-GlcNAcylation: no specific inhibitors, mimics, known function, structures, antibodies, unknown action of enzymes too.
No precise O-GlcNAcylation sequence motif (sequon) has so far been defined
What is OGA and OGT? What are they good for?
OGA: O-GlcNAcase
OTA: O-GlcNAc transferase.
O-GlcNAcylation is achieved by the action of the two enzymes
Needed in EMBRYOGENESIS
without these enzyme, embryos had smaller heads, eyes,bodies; with disfigured notocords. In mice, it is fatal.
How is O-GlcNAcylation connected to phosphorylation?
It has been suggested that O-GlcNAc is needed to REGULATE phosphorylation.
Indeed, has been linked in metabolism; OGT, an enzyme responsible for modification of proteins with N-acetylglucosamine, may act as a nutrient sensor that links hexosamine biosynthesis pathway for signaling and regulation of factors involved in glucose and lipid metabolism.
If O-GlcNAc malfunction what effects can it have in the body?
1) brain tumours and cancer (overexpression of enzymes)
2) Alzheimers (when Tau protein is hyperphosphorylated instead of O-GlcNAcylated)
3) Insulin resistance and DIABETES (Increased levels)
How do OTA and OGA recognize the correct protein substrates? What is their role.
O-GlcNAc transferase (OGT) is the glycosyltransferase that acts to install O-GlcNAc onto proteins and O-GlcNAcase (OGA) is the glycoside hydrolase that acts to remove O-GlcNAc from proteins.
OGT possesses an unusual N terminus, consisting of up to 13.5 tetratricopeptide repeats (TPRs) that are thought to play a role in recognition of intact protein substrates. Its sequence is conserved.
OGA interacts with glycopeptide substrates, and achieves O-GlcNAc removal from O-GlcNAc sites in a sequence-independent manner from HEK293 Lysates
Name an inhibitor of OGA
GlcNAcstatin competitivly inhibits
What role does O-GlcNAc have in the brain?
O-GlcNAc is very abundant within brain and is present on hundreds of proteins including several, such as tau and the amyloid precursor protein.
If absent it can be linked to Alzheimer’s, Parkinson’s, Pick’s disease, BSE.
For example in the brains of AD patients O-GlcNAc levels have been found to be decreased and aggregates of tau appear to lack O-GlcNAc entirely.
What does TAK1 do
What is it inhibited by
Transforming growth factor β-activated kinase 1 (TAK1) is a key signaling component of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways that regulates NFkB activity / cytokine production
Activated through TLR/cytokine receptors and NaCl
Activation of TAK1 is tightly regulated through its binding protein (TAB) and modifications such as ubiquitin.
Describe the relationship with TAB1 and TAK1 and PHOSPHORYLATION and O-GlcNAc.
TAK1 is regulated by TAB1 and is required for FULL ACTIVITY.
PHOSPHORYLATION of TAB1 stops TAK1
Proteomics shows TAB1 is an O-GlcNAc protein and acts as a substrate for OGT, binding at residue SER395
TAB1 O-GlcNAc modulates IkBa phosphorylation; stops inactivation.
TAB1 O-GlcNAc also modulates cytokine production.
