Post Translational Modifications / Ubiquitination Flashcards
1
Q
What are post translational modifications and examples
A
- Stability, function and activity to ensure gene complexity, changes occur to polypeptide chain before it becomes an active protein Examples - Protein folding - Proteolytic cleavage of proteins - Acetylation - Glycosylation - Methylation - Phosphorylation - Ubiquitin - Targeted protein degradation
2
Q
How is gene expression regulated
A
- Folding of polypeptide into correct 3D structure
- Transport to the correct location
- Post translational processing (phosphorylation and glycosylation)
- Association with other protein co-factors
3
Q
What is the purpose of protein folding
A
- Protein structure assumes its functional shape or conformation
- AA interact with each other to produce a well-defined 3D structure, folded protein, known as the native state
- Polypeptide chains spontaneously fold so that hydrophobic AA are on the inside of the structure
- Spontaneous folding leads to formation of many different structures (most inactive)
- At high concentrations unfolded proteins form insoluble aggregates through interaction of side chains
4
Q
What are chaperones
A
- Help other proteins fold appropriately
- Prevent protein-protein interactions during folding process
- Prevents formation of insoluble aggregates
- HSP: Aid protein folding and reducing formation of insoluble protein aggregates in the cell, important as messengers
5
Q
What is aberrant protein folding
A
- Mis-folding
- Numerous diseases result from incorrectly folded proteins
- Diabetes, obesity, cystic fibrosis, haemophilia A and B, blood coagulation disease, goitre, gaucher’s disease
6
Q
What is the unfolded protein response
A
- Under normal conditions BIP binds to IRE1, ATF6 and PERK
- When unfolded proteins accumulate in ER, BIP is released
- Initiates signal transduction pathways leading to induction of gene expression
- Synthesis of chaperones in increased to decrease conc of unfolded proteins
- If unfolded proteins continue to accumulate apoptosis is induced (CHOP, caspases)
- Can cause translation attenuation which halts progression of protein synthesis in attempt to resolve production of insoluble proteins
7
Q
What occurs after the unfolded protein response
A
- Activated IRE1 recruits TRAF2 to elicit JNK-P phosphorylation, activating caspases
- Activated PERK and ATF6 increase expression of nuclear genes for apoptotic effector CHOP
- Inhibits expression of Bcl2 and induces expression of genes for apoptotic promoting factors Gadd34, Trb3 and Dr5
- Release of Ca from ER leads to mitochondrial generation of ROS, contributes to cell death
8
Q
What is proteolytic cleavage of proteins
A
- Breaking peptide bonds between AA in proteins
- Creation of a stable form
- Some proteins are synthesised as inactive precursors that are activated under proper physiological conditions by proteolysis
- Inactive precursor proteins that are activated by removal of polypeptides are termed pro-proteins
9
Q
What are examples of proteolytic cleavage
A
- Pro-Caspase 3: Will cause apoptosis if it circulates the body in activate form
- Insulin: Preproinsulin (110AA) to proinsulin to insulin (51AA)
- POMC: Undergoes glycosylation, acetylation and proteolytic cleavage to produce ACTH and MSH
- Addisons Disease: Negative effect of POMC, insufficient hormones produced,
10
Q
What is glycosylation
A
- The addition of polysaccharide side chains (sugar) to proteins (glycoprotein)
- For a given protein the pattern of glycosylation differs for different species/groups, species specific
- Determines protein structure, function, stability
- Types: N and O linked glycans
- Variations in glycosylation can lead to disease
11
Q
What is the biological significance of glycosylation
A
- Oligosaccharides may be a tissue-specific marker
- Carbohydrates may alter the polarity and solubility
- Bulkiness and -ve charge of oligosaccharide chain may protect protein from attack by proteolytic enzymes
- Markers and therapy for cancerand autoimmune disease
- More efficient production of pharmaceuticals
12
Q
What are N linked glycans
A
- Covalently attached to Asn residues within a consensus sequence (Asn-Xaa-Ser/Thr)
- Enable prediction of modification sites by protein sequence analysis
- Share common penta-saccharide core recognised by lectins and N-glycanase enzymes (PNGase F)
- Aid visualisation of proteins and enrich mass for spectrometry analysis
13
Q
What are O linked glycans
A
- Comparable tools are lacking for study of proteins bearing O-linked glycans
- Mucin-type
- N-acetylgalactosamine (GalNAc) residue linked to hydroxyl group of Ser or Thr
- Complex biosynthetic origin
- Not installed at a defined consensus motif and presence cannot be accurately predicted based on protein’s primary sequence
14
Q
What is notch1 and how is it affected by glycosylation
A
- Notch 1: Consists of a number of EGF repeats, different types of glycan side chains are found attached to these EGF repeats
- Glycosylation: Receptors lacking O or N glycans are functional, lack of O fucose glycan destroys activity
- Notch Defects: Result in perinatal lethality, defects in somitogenesis, T-cell leukaemia, cancers, and developmental defects
15
Q
What is phosphorylation
A
- Most common protein modification that occurs
- Regulates biological activity of a protein
- Numerous growth factors affected by phosphorylation
- Kinases: Phosphorylate proteins
- Phosphatase’s: Remove phosphates
- Protein Kinases: Catalyse reactions of the following type, ATP + protein to phosphoprotein + ADP
16
Q
What is ubiquitin and proteasome’s
A
- Facilitates degradation of defective and superfluous proteins, present everywhere
- Addition can lead to endocytosis, vacuole / lysosome degradation, proteasome degradation, sequestration, changes in protein - protein interaction / kinetic properties
- Proteasome: Cylindrical protein, breaks peptide bonds
17
Q
What is ubiquitination
A
- Lysine dependent addition of ubiquitin
- 4 lysine’s in ubiquitin form bonds with other ubiquitin’s
- Type of bond determines fate of target protein
- A single target protein can be modified at a number of different lysine’s with either a single ubiquitin, a poly-ubiquitin chain or both
18
Q
How is ubiquitin ligase activated
A
- Phosphorylation
- Ligand binding inducing conformational changes
- Accessory protein binding inducing conformational change
19
Q
How does a molecules susceptibility to ubiquitination change
A
- Phosphorylation by a protein kinase
- Unmasking by protein dissociation
- Creation of a new terminus by proteolytic cleavage
20
Q
How does ubiquitin regulate cellular processes
A
- Regulates by selective degradation of master regulatory proteins
- Affects signal transduction and cell cycle
- Addition of ubiquitin is regulated by accessory factors that monitor activity of E3
- E3 regulates EGFR tyrosine kinase signal transduction
- E3 regulates transcription by ubiquitinating activator rendering it inactive
21
Q
What is the EFGR tyrosine kinase signal transduction pathway and Cbl proteins
A
- Regulates growth, survival, proliferation, and differentiation
- Receptor: EGF binding site (extracellular) and tyrosine kinase residues / cytosolic tail (cytosolic)
- Cbl: Specific subset of E3 proteins, interact with EGFR tyrosine kinase and regulate amount of EGFR protein that is activated
22
Q
How is the EFGR pathway regulated
A
- Nedd: Facilitates degradation of Cbl (E3) and down regulates the binding of Cbl with EGFR, thus inhibiting EGFR degradation
- Spry: Facilitates sequestering of Cbl (E3), temporarily inhibiting the EGFR pathway, upon appropriate trigger, Cbl is released and EGFR / Spry degraded
23
Q
Describe the steps involved in the EFGR pathway
A
- EGF receptor and ligand bind and aggregation occurs
- Auto phosphorylation of tyrosine and conformational change leads to downstream events
- Binding of cytosolic proteins with SH2 domains (PLCγ and Sos / GRB2)
- Activated PLCγ stimulates IP3-DAG pathway
- Up-regulation of IP3 (ER) increases cytosolic Ca
- Activated GRB2-Sos pathway stimulates Ras pathway
- Promotes gene expression
- Ras pathway is then inactivated by a GAP
24
Q
What is the difference between degradative and non-degradative
A
Degradative:
- Ubiquitin addition leads to degradation of target protein
Non-Degradative:
- Ubiquitin addition changes susceptibility of protein or cause methylation / phosphorylation
- Changes regulatory role
- Can lead to changes in function, localisation or activity of protein (TLR pathway)
- Can affect susceptibility to other modifications (histone modification)
25
Q
What is the TLR pathway (non-degradative)
A
- Invading pathogens recognised by PRRs (TLR)
- Sense pathogens and communicate via NF-kB transcriptional activator (alters gene expression in nucleus)
- Activation leads to ubiquitination of TRAF
- Increases susceptibility of NF-kB/IkB to phosphorylate
- Causes release of NF-kB activator and enters nucleus
26
Q
What is histone modification (non-degradative
A
- Addition of ubiquitin to H3 / H2B proteins
- Affects susceptibility to methylation
- Rad6 adds ubiquitin to lysine 123 on histone H2B
- Causes methylation of H3K4 and H3K79
- Result is telomere induced gene silencing or activation of genes in euchromatin
27
Q
What is the N end rule
A
- Determines susceptibility of a protein to ubiquitin mediated degradation
- Degradation initiated with binding of a ubiquitin to the N-end
28
Q
What is involved in ubiquitin conjugation
A
- E1: Ubiquitin activating enzyme, through high energy thioester linkage to a Cys side chain on E1
- E2: Ubiquitin conjugating enzyme, specific E2’s function in specific pathways, added to N terminus
- E3: Ubiquitin ligase enzyme, does not handle ubiquitin, facilitates transfer of ubiquitin from E2 to the target molecule, requires activation
