Protein degradation(molecular biology)

description of protein degradation pathways in lysosomes and ubiquitin mediated pathway and n-end rule in different organisms

1. PROTEIN DEGRADATION & N-end rule D.INDRAJA

2. The levels of  proteins  within cells are determined not only by rates of synthesis, but also by rates of degradation. The half-lives of proteins within cells vary widely, from minutes to several days, and differential rates of protein degradation are an important aspect of cell regulation. Many rapidly degraded proteins function as regulatory molecules, such as  transcription  factors. Other proteins are rapidly degraded in response to specific signals, providing another mechanism for the regulation of intracellular enzyme activity . I n addition, faulty or damaged proteins are recognized and rapidly degraded within cells, thereby eliminating the consequences of mistakes made during protein synthesis. In   eukaryotic cells , two major pathways: lysosomal   proteolysis —mediate protein degradation . ubiquitin - proteasome  pathway

3. WHY? Cells also degrade other types of proteins while they are still functional(remodeling involves proteolysis of one set of structural and metabolic proteins and its replacement by another specialized for a different purpose.) Removal of damaged or harmful proteins(proteins can be misfolded or denature they may fail to assemble properly in to complexes,or they can be altered by some abnormal post translational modifications. Such aberrant proteins are potentially toxic and need to be eliminated) The eukaryotic cell has a remarkable ability to distinguish normal from abnormal proteins and selectively degrade the letter When the capacity is compromised, disease often results

4. The first type of protein degradation identified in the cell is lysosome and autophagosome mediated degradation Lysosomal proteolysis The  lysosome  is a membrane-bound intracellular compartment full of nonspecific proteases that will cleave into individual amino acids any protein they come into contact with.   Proton pumps  fill the lysosome with H+ from the cytosol , making it acidic (pH 4.8) — the proteases function optimally at this pH and not at all at cytosolic pH (7.2), thus minimizing the risk to the cell in the event of lysosome rupture.   The lysosome is formed by budding off from a compartment of the late Golgi – it represents an alternate endpoint for some proteins in the secretary pathway that neither stay in the ER or Golgi nor undergo exocytosis to the cell surface

5. Proteins destined for lysosomal degradation can reach the lysosome by a variety of means. receptor-mediated endocytosis  endocytic vesicles from the cell surface can fuse with the lysosome ; this is a mechanism for degradation of cell surface receptors and thus the down regulation of incoming signals Phagocytosis  the cell engulfs foreign bodies – say, invading bacteria, or apoptotic bodies from other cells – and delivers them to the lysosome .  The membrane of the lysosome itself can invaginate , creating exosome -like vesicles full of cytosolic proteins to be degraded receptor-mediated endocytosis Phagocytosis

6. 3 . “canonical”, starvation-induced  autophagy ,  In this double membrane forms around material (such as unneeded organelles) in the cytosol and delivers them to the lysosome .   The degradation of proteins in the lysosomes is  catabolic  – it releases energy – so this response to nutrient starvation recovers some of the energy originally put into synthesizing proteins and other cellular components.   But autophagy isn’t induced only by starvation – unfolded protein stress in the endoplasmic reticulum can cause chunks of ER to be degraded by autophagy

7. The proteins degraded by lysosomal proteases under the starvationconditions contain  amino acid  sequences similar to the broad consensus sequence Lys- Phe - Glu - Arg - Gln , which presumably targets them to lysosomes . A member of the Hsp70 family of molecular chaperones is also required for the lysosomal degradation of these proteins, presumably acting to unfold the  polypeptide  chains during their transport across the lysosomal membrane. The proteins susceptible to degradation by this pathway are thought to be normally long-lived but dispensable proteins. Under starvation conditions, these proteins are sacrificed to provide amino acids and energy, allowing some basic metabolic processes to continue.

8. ubiquitin - proteasome  pathway The major pathway of selective protein degradation in  eukaryotic cells  uses  ubiquitin  as a marker that targets cytosolic and nuclear  proteins  for rapid  proteolysis   Ubiquitin is a 76-amino-acid  polypeptide  that is highly conserved in all eukaryotes ( yeasts , animals, and plants). Proteins are marked for degradation by linkage occurs between the carboxyl group of the C-terminal glycine residue of ubiquitin and the E aminogroup of a lysine residue within the substrate forming a type of amide bond often referred to as an isopeptide bond.

9. POLYUBIQUITINATION Additional ubiquitins are then added to form a multi ubiquitin chain. Such polyubiquinated proteins are recognized and degraded by a large, multisubunit protease complex, called the  proteasome . Ubiquitin is released in the process, so it can be reused in another cycle. It is noteworthy that both the attachment of ubiquitin and the degradation of marked proteins require energy in the form of ATP.

10. Ubiquitination is a multistep process. First, ubiquitin is activated by being attached to the ubiquitin -activating enzyme, E1 . The ubiquitin is then transferred to a second enzyme, called ubiquitin -conjugating enzyme (E2). The final transfer of ubiquitin to the target protein is then mediated by a third enzyme, called ubiquitin ligase or E3 , which is responsible for the selective recognition of appropriate  substrate  proteins.

11. In some cases, the ubiquitin is first transferred from E2 to E3 and then to the target protein . In other cases, the ubiquitin may be transferred directly from E2 to the target protein in a complex with E3. Proteins in the other major class of E3 have a single subunit with a segment called the HECT domain. This domain act as a direct carriers of the activated ubiquitin For the HECT E3s the activated ubiquitin is transferred from the E2to a conserved cysteine side chain in the HECT domain 

12. Structure of E3 The E3 protein contains a structural motif called a RING FINGER that constitutes either a motif with in a larger polypeptide or one of several subunits within a complex Most cells contain a single E1, but have many E2s and multiple families of E3  enzymes . (E1<E2<E3)

13. It has different substrate binding subunits can be used with the same framework subunit producing E3s with similar architecture but different substrate specificities The variable subunits not only provide sites for binding different substrates but also produce gaps of different sizes between the site and the E2. This presumably helps the E3 adapt to substrates of different dimensions

14. Recognition of degron by E3 A  PEST sequence  is a  peptide sequence  that is rich in  proline  ( P ),  glutamic acid  ( E ),  serine  ( S ), and  threonine  ( T ). This sequence is associated with  proteins  that have a short intracellular  half-life ; therefore, it is hypothesized that the PEST sequence acts as a  signal peptide  for  protein degradation . This protein degradation may be mediated via the  proteasome The degrons may be degraded by specific signals such as phosphorylation , methylation , glycosylation exposure of hydrophobic molecules outside of the protein Phosphorylation is the most common degron modification that triggers substrate binding to E3

15. Hidden degrons Some times the degrons may be hidden due to complex formation so that they live in the cell at a longer time than the ususal Eg : phosphorylation dependent degron found in a yeast inhibitor of the onset of DNA replication. Atleast six of nine potential sites in this inhibitor must be phosphorylated by a cyclin dependent kinase before the inhibitor is recognized by an E3 ligase and ubiquinated

16. proteasome The  proteasome  is a cylindrical protein complex found in the cytosol which cleaves up proteins tagged with  ubiquitin . The proteasome itself weighs in at 26 S , and is composed of a 19S gate and a 20S core.  The 19S gate recognizes and binds ubiquitinated proteins, powered by ATP – unlike the lysosome , the proteasome is an energy-losing operation The 19S subunits act as a gates for entry and exit site of proteins. which has lid and a base Lid  deubiquination Baseunfolding

17. Once recognized, these proteins must be de- ubiquitinated and unfold in order to pass through the narrow channel of the 19S and enter the 20S core, a cylindrical complex which does the actual chopping up of proteins.   Unlike the lysosome , where proteases shear proteins up into individual amino acids, the proteasome just chops proteins into small peptides, usually of 7 – 9 amino acids each.

18. Active site of proteolysis Central part which is a processive proteolysis part has a two α and two β types they are seven subunits in both α and β subunits This central cylinder has a certain pH and temp that is required to degrade a protein They are three types of active sites in the β subunits, each with a different specificity, but all employ an N-terminal threonine . The hydroxyl group of the threonine residue is converted in to a nucleophile that attacks the carbonyl groups of peptide bonds to form acyl -enzyme intermediates

19. Path of protein degradation In the path of protein degradtaion the ATP is required for the entry of protein only

20. ubiquitin - proteasome  pathway

21. N-END RULE The  N -end rule  is a rule that governs the rate of  protein degradation  through recognition of the N-terminal residue of proteins. The rule states that the  N -terminal amino acid  of a protein determines its half-life (time after which half of the total amount of a given polypeptide is degraded). The rule applies to both eukaryotic and prokaryotic organisms, but with different strength, rules, and outcome.  In eukaryotic cells, these N-terminal residues are recognized and targeted by  ubiquitin ligases , mediating  ubiquitination  thereby marking the protein for degradation Discovery The rule was initially discovered by  Alexander Varshavsky  and co-workers in 1986.

22. Rules in different organisms Aminoacids present Half-life Met, Gly , Ala, Ser, Thr , Val, Pro  > 20 hrs (stabilizing) Ile, Glu    approx. 30 min (stabilizing) Tyr, Gln approx. 10 min (destabilizing Leu , Phe , Asp, Lys approx. 3 min (destabilizing) Arg approx. 2 min (destabilizing) For s.cervisiae For Mammals

23. Bacteria In  Escherichia coli , positively-charged and some aliphatic and aromatic residues on the N-terminus, such as arginine , lysine, leucine , phenylalanine, tyrosine, and tryptophan , have short half-lives of around 2-minutes and are rapidly degraded.   Other amino acids on the other hand may have half-lives of more than 10 hours when added to the N-terminal of the same protein However, a complicating issue is that the first residue of bacterial proteins is normally expressed with an N-terminal  formylmethionine  (f-Met). Once the f-Met is removed, the second residue becomes the N-terminal residue and are subject to the N-end rule.