Post-translation modification II.pptx

Post-translational modification II Ubiquitination and glycosylation Protein degradation For a given protein, the total amount of molecules of such protein is highly regulated. To regulate the amount of protein, cells can use two major strategies: – Control how much protein is being made Gene transcr...

Post-translational modification II Ubiquitination and glycosylation Protein degradation For a given protein, the total amount of molecules of such protein is highly regulated. To regulate the amount of protein, cells can use two major strategies: – Control how much protein is being made Gene transcription regulation, mRNA transport, microRNA, translation regulation ect.. – Control how much protein is being degraded Ubiquitination dependent proteosome degradation Lysosomes Protein degradation Protein life spans vary a lot. – Some proteins like cyclins are involved in cell cycle and only last few minutes while other can last the life span of the cell. – Most proteins are degraded at a rate of 1-3% per hour Approximately 30% of newly synthesized protein is marked for removal and degraded due to lack of proper folding Other reason for degradation includes: – Toxic protein – Damaged protein that is not functioning properly Protein degradation Protein degradation not only removes misfolded, toxic or damaged proteins but it is also used to regulate a vast network of pathways. Controlled degradation can remove almost instantly an entire protein from cells. – This is very important to make transitions fast Examples: cell cycle transitions Metabolic changes Presence of DNA damage Receptors bind ligands And so on…… Protein degradation Two major systems for protein degradation: – Lysosomes: an organelle with an acidic ph (4.5) and filled with plenty of hydrolytic enzymes – Ubiquitin and Proteosome dependent degradation. Proteosome Large complex of more than 50 subunits There are aprox 30,000 proteosome per animal cell Made of a central core and two caps at each end Proteins to be degraded would come at the top cap, will be degraded in the core and will come as small pieces from at the bottom cap The core region contains subunits with active sites that cut proteins after hydrophobic, positive or negative aminoacids Proteosome inhibitors are now part of many chemotherapy recipes as it has been proven to be effective in several types of cancer. How cells know which protein should be degraded? A protein post-translation modification called ubiquitination tags proteins for degradation. Polyubiquitinated proteins are recognized by the proteosome and degraded. To be recognized by the proteosome protein must have at polyubiquitin chain of at least 4 ubiquitin proteins Ubiquitin A 76 aminoacid protein Highly conserved from yeast to human Several lysines perform critical roles: – They mediate linkage to target proteins – And they mediate binding to other ubiquitin molecules to form polyubiquitin chains Ubiquitin is covalently attached to specific lysines in the target protein. Ubiquitination Ubiquitination requires a series of steps all marked by specific enzymes First: Ubiquitin Activating Enzyme (E1) binds and activates an ubiquitin molecule using one molecule of ATP Second: Ubiquitin is then transferred from EI to an Ubiquitin Conjugating Enzyme (E2) Third: Ubiquitin ligase enzyme (E3) binds both the E2 and the target protein and catalyzes the transfer of ubiquitin from the E2 to the target Two types of E3: – RING type: catalyzes the transfer of ubiquitin from E2 directly into the target. – HECT type: Catalyzes first the transfer from E2 to E3 and then from E3 to target E3 specificity There are 2 types of E1 in cells, ~60 types of E2 and ~600-800 types of E3. Therefore the specificity of the system depends on E3 enzyme that recognizes both targets and E2 enzymes HECT ligases They all contain the HECT domain which is able to bind both the E2 and the target protein. This domain has a catalytic site that contains a critical cysteine that will bind ubiquitin from E2 and attached to a lysine in the target protein. HECT ligases There are many types of HECT ligases in cells involved in a great diversity of pathways Each one provides specificity for each target and pathway. RING ligases They all posses the RING domain and form part of a complex with multiple subunits: – A protein containing a RING finger domain – A scaffold protein – An adaptor protein – And a protein containing an F-box domain that will bind the substrate. There are two main types of such complexes: SCF and APC/C which play critical roles in cells, especially during cell cycle Example: APC/C Anaphase Promoting Complex/Cyclosome It Regulates critical aspects of the transition from metaphase to anaphase where it catalyzes the degradation of securing that triggers chromosome separation It gets more complicated Recently, it has been discovered that proteins can be also just monoubiquitinated. In other cases polyubiquitination occurs using different types of lysines to produce the ubiquitin chain. In these cases, the target protein is not degraded but addition of a single ubiquitin or a different type of polyubiquitin chain, either activates/inactivate the target or it is used to proper localize that protein. SUMOLATION Much like ubiquitin, SUMO is a small protein that can be attached to other proteins and modify their properties It also requires E1, E2 and E3 proteins and it can also form chains. Opposite to ubiquitin, it is never used to tag proteins for degradation. Types of protein modifications Phosphorylation Acetylation Methylation Glycosylation Ubiquitination Sumolation …and several others Glycosylation Proteins are also frequently modified by adding sugars to their structure. These sugars can be very simple or form very complicated structures Glycosylation is especially important in membrane proteins and proteins trafficking between the ER and the Golgi apparatus Glycosylation Proteins are also frequently modified by adding sugars to their structure. These sugars can be very simple or form very complicated structures Glycosylation is especially important in membrane proteins and proteins trafficking between the ER and the Golgi apparatus