Flashcards in O-GlcNAc signalling Deck (24):
What is protein Phosphorylation ?
A post-translational protein modification
a serine/threonine/tyrosine residue is phosphorylated by a protein kinase
many kinase and phosphatase structures (500+ kinases, 100+ phosphatases - each with their own substrate sequence specificity )
What is a common mode of regulation of protein function?
Regulation of proteins by phosphorylation is one of the most common modes of regulation of protein function, and is often termed "phosphoregulation". In almost all cases of phosphoregulation, the protein switches between a phosphorylated and an unphosphorylated form, and one of these two is an active form, while the other one is an inactive form.
What is the purpose of protein phosphorylation?
- to increase energy so it is able to participate in another reaction
how can phosphorylated proteins be detected?
- mass spectometry
(32P)- ATP - very hot ! ( radioactive marker - designed to help you see the part of the body that ATP does to )
- 600 + enzymes , targetting specific sequences !
- probing with phospho - specific antibodies
- there are many specific inhibitors available
- structures/ molecular mechanisms are well understood.
What is a kinase?
- an enzyme that transfers phosphate groups from high energy donor molecules ( eg ATP) to specific substrates - phosphorylation !
protein phosphorylation is ...
a highly complex signal transduction network , and is important in regulating almost every process in the cell !
What is protein O-GlcNAcylation ?
-O-GlcNAc is form of protein glycosylation
- It occurs within the nuclear and cytoplasmic compartments of the cell.
- The GlcNAc is not elongated or modified to form more complex structures.
- It is attached and removed multiple times in the life of a polypeptide,
- O-GlcNAcylation is more similar to protein phosphorylation than it is to “classical” protein glycosylation.
-it is a post translational modification of ser/thr with a single N-acetylglucosamine (GlcNAc)
- cytoplasmic and nucleoplastmic
- reversible , inducible , dynamic , abundant and essential
- found in metazoa ( animals )
What are the differences between protein phosphorylation Vs O-GlcNAcylation ?
- 600+ proteins
- targeting specific sequences
- detection with electrophoresis / (32P)-ATP
- site mapping routine by CID MS/MS
- probing with phospho - specific antibodies
-many specific inhibitors available.
- glutamate as a phosphomimic
- phosphoproteome with mapped functions
- structurs/ molecular mechanisms well understood.
- single genes , from C.elegans to man
- enzymes act on Ser/Thr , specificity unknown
- (3H)- GlcNac- specific antibodies
- no specific inhibitors available
- No 'GlcNa-mimic'
- O-GlcNacome with few mapped functions
- structures/ molecular mechanisms not well understood.
what are OGA / OGT essential for ?
What is OGT?
an ezyme encoded by OGT gene!
- vital for embryogenesis
necessary for intracellular glycosylation and embryonic stem cell vitality
- catalyses the post translational modification that modifies transcription factors and RNA polymerase
- involved in resistance of insulin in muscle cells and adipocytes.
examples of OGT systems?
- zebrafish - the knockdown of O-Glc Nac cycling enzymes - results in embryos small than normal , with shortened bodies / smaller heads etc
- drosophila - the accefts of OGT and OGA levels affect the adult body size if decreased.
-mice- OGA negative mice are stunted in growth , and OGT negative mice ( affects their nervous system) and they are smaller and die before post natal day 10 !
Does O-GlcNacylation and Phosphorylation require one another ?
- like Yin and Yang theory !
- O-GlcNAcylation is a mechanism for global regulation of phosphorylation
( competition between GlcNAc/Phosphorylation can be seen sometimes)
What is O-GlcNAc's role in disease?
- overexpression of O-GlcNacase in brain tumours!
in the case of Alzheimers - the tau protein is hyperphosphorylated wheras in normal conditions it is hyper O-GlcNacylated.
- increased O-GlcNAc induces insulin resistance, correlation O-GlcNAcase mutations and diabetes
- O-GlcNAc cytoprotective under ischemic stress
- OGT overexpressed in certain types of cancer.
How do enzymes recognise / act on substrates?
- active site recognition ,
- substrate binds ( either broken down or built up )
- then enzyme is still able to continue to work.
is there a O-GlcNAc Sequon?
when and why did O-GlcNAc evolve?
not sure/////// ( beth ?)
How is O-GlcNAc Regulated?
O-GlcNAc modification is common to nearly all eukaryotes, including filamentous fungi, plants, animals, and animal parasites, as well as viruses that infect eukaryotes.
- a direct role for O-GlcNAc in cellular regulation.
-the disruption of normalO-GlcNAc-regulated pathways may be responsible for the pathology of some bacteria. Moreover, disruption of the gene forO-GlcNAc transferase demonstrates that O-GlcNAc modification is essential for life, even at the single cell level
Function of O-GlcNAc in developing brain?
O-GlcNAc represents a key regulatory modification in the brain, contributing to transcriptional regulation, neuronal communication and neurodegenerative disease.
Interplay O-GlcNAc/ Phosphorylation ?
- O-GlcNAc appears to be both as abundant and as dynamic as protein phosphorylation.
- phosphorylation and O-GlcNAc modification are reciprocal, occurring at the same or adjacent hydroxyl moieties.
- all O-GlcNAc-modified proteins identified also occur as phosphorylated proteins.
- the interrelationship between Ser/Thr O-GlcNAc modification and O-phosphorylation appears to be complex.
What is an example of an OGA inhibitor ?
- GlcNAcstatin - a picomolar OGA inhibitor
what is the connection between O-GlcNAc and the brain?
- if there is an increase in glucose - which would increase O-GlcNAc ,
the high levels of O-GlcNAc in the brain result in high OGA and OGT expression levels.
N,B. O-GlcNAc on proteins is linked to Alzheimers , parkinsons etc
How can we enrich for O-GlcNAc proteins?
cpOGA inactive mutant is a substrate trap
What is TAK1 ?
- a key innate immunity signalling mode.
TAK1= TGF Beta- activated protein Kinase 1
- regulates NFkB activity / cytokine production
- activated through TLR / Cytokine receptors , NaCl
- TAK1 binds regulatory subunits
- TAK1 Binding protein (TABs) regulate TAK1 activity
TAB2/3: ubiquitin - dependent/ receptor mediated activation of TAK1