Lecture 16 PTMs Flashcards
What is the definition of a PTM?
Covalent additions that regulate protein function, determining their activity state, cellular location and dynamic interactions with other proteins.
How much do PTMs increase the complexity of the proteome by?
A 10 fold increase.
What are the general characteristics of PTMs?
More than 200 types, present on at least 80% of eukaryotic proteins, found in all eukaryotic species, located at specific sequence motifs, often transient/reversible, usually present at sub-stoichiometric levels (less than 50% of the protein is modified), often act together.
What is the most common phosphorylation?
HPO3 is added to Ser, Thr or Tyr. One of the most important is abundant PTMs. More than 30% of all proteins are phosphorylated. Many proteins multiple phosphorylated. E.g., gives a protein charge to facilitate an interaction.
How does insulin depend on phosphorylation?
When insulin binds a conformational change occurs to get auto phosphorylation of the insulin receptor. The negative charge allows binding of the IRS1. IRS is phosphorylated on a tyrosine residue to interact with PI3K. PI3K produces phosphate inositol triphosphate. This activated PIP3 dependent kinase one which phosphorylates protein kinase b and c. Further downstream phosphorylation and overall leads to increase glucose transport and glycogen synthesis.
What does this pathway cause?
Insulin resistance leading to diabetes.
What is ubiquitin?
A 76 aa protein. Targets proteins for degradation by the 26S proteosome. Found in all eukaryotes. Forms an isopeptide bond between the C terminal gly of ub and NH2 of lys on substrate.
What is fanconis anaemia?
FA occurs as a result of mutations in a cluster of protein involved in DNA repair by homologous recombination. Affects primarily bone marrow in stem cells. Lots of cell division without the necessary DNA repair mechanisms. Leads to bone marrow failure and subsequent anemia.
What are they key steps in the fanconi anemia pathway for DNA repair?
Phosphorylation – FANCD2 & FANCI are phosphorylated in response to DNA damage.
Monoubiquitination – FA core complex (FANCA, FANCB, etc.) monoubiquitinates FANCD2 & FANCI.
Deubiquitination Regulation – USP1/UAF1 prevents excess monoubiquitination in normal conditions but decreases in response to DNA damage.
Damage Site Localization – Monoubiquitinated FANCD2 & FANCI move to damaged DNA with other FA proteins (BRCA2, PALB2, etc.).
Repair Process – The FA repair complex fixes the damage with other repair proteins.
Disassembly – FANCD2 & FANCI are deubiquitinated post-repair, leading to complex dissociation.
What is glycosylation?
Most common PTM. Around 50% of all mammalian proteins are glycosylated. CHO structures are very heterogenous. In mammals 9 different sugars are commonly used. 2 major types N-linked and O-linked. (nitrogen or oxygen depending on amino acid). Very complex.
How is glycosylation regulated?
There is no code. Expression of enzymes in the ER/golgi are higher and lower in different tissues. Modifications such as sulphation and acetylation can block further glycosylation. Glycosidases can modify glycosylation in the ER/golgi.
Where is N-linked glycosylation always linked to?
Always hooked to Asn. Asn-X-Ser/Thr/Cys (X is not Pro). Can be completely removed by PNGase F.
What is O-linked glycosylation linked to?
Hooked to ser or thr. No known sequon. No easy removal method.
What do complex carbohydrate groups impact?
Protein folding, protein extension (to keep two functional domains held apart), add a huge negative charge, cell signaling and binding.
What PTMs do HIV undergo?
HIV env consists of gp120 soluble portion bound non-covalently to TM gp41. Role is to bind to CD4 and chemokine receptors during HIV-1 entry. Co-translationally translocated into ER as gp160. Has around 30 potential sites for N-linked glycosylation in ER. If non-glycosylated won’t bind CD4. Some glycosylations are dispensible for proper folding; others are needed. Forms 10 disulfide bonds in ER (9 are in gp120 portion). Trimerization of HIV-1 env in ER. Protein folding/trimerization equired BiP, calnexin and celreticulum and PD1. In golgi protease mediated cleavage of gp160 to gp120 and gp41.
What is lipidation?
Myristic acid (C14:O)-cytosolic proteins. Attaches to NH2-terminal Gly. Consensus sequence Met-Gly X-X-X-Ser/Thr. HIV-1 Gag and HIV-1 Nef are examples of myristoylated proteins.
What is another lipidation?
Palmitic acid C16:O-intracellular proteins attached to Cys.
What is prenylation?
Addition of prenyl groups to S in internal cysteine. A) Farsenylation-C15 fatty acid to C terminus by thioester linkage occurs at CAAX sequences, 2 aliphatic residues and a C-terminal residue. After attachment, last 3 residues are removed and new C terminal methylated creates a highly hydrophobic C terminus.
Geranylgeranylation- Similar to above but addition of C20 to C terminal Cys.
What is S-palmitoylation?
Formed by the reversible addition of the 16-carbon lipid palmitate to the thiol group of Cys residues. When this occurs on an N-terminal residue there is a chemical rearrangement resulting in the attachment of the palmitate through a stable amide linkage known as N-palmitoylation.
What is the role of N-myristoylation in apoptosis?
Activation of the extrinsic pathway begins with binding of a death ligand e.g., Fas ligand (FasL) to its corresponding death receptor Fas. Binding of adaptor proteins leads to formation of death induced signalling complex DISC and activation of caspase-8. Caspase-8 cleaves the pro-apoptotic protein BID, which is then PT myristoylated by NMT at an N terminally exposed glycine of C terminal fragment which is essential for ctBid’s translocation to the mitochondria and progression of apoptosis by the release of cytochrome c. Beta-actin, gelsolin and p21-activated kinase 2 are all cleaved by caspase-3 to yield caspase-truncated products which are post-translationally myristoylated. These translocate to their new respective membrane locales to affect apoptosis.
What does glycophosphatidyl inositol do GPI?
Attached to the C terminus. Composed of oligosaccharides and inositol phospholipids. Provides a mechanism for anchoring cell surface proteins to the membrane as a flexible leash allowing the entire protein to be in the EC space. Typically associated with membrane microdomains enriched with sphingolipids and cholesterol.
What is the GPI anchor’s role in prion diseases?
When exposed to PrPsc, GPI-anchored PrPc is converted to aggregates of PrPsc. These interfere with the normal signaling events involving PrPc leading to neuron death. However transgenic mice expressing PrPc lacking a GPI anchor still form PrPsc aggregates upon infection but this may be unable to disrupt signal transduction due to the lack of a GPI anchor.
