Question

a. What are the steps/characteristics of lysosome centered protein degradation? Discuss any specificities and give an example of at least one protein which is cleared via the lysosomal degradation.

  

b. What are the steps in the proteasome centered protein degradation via ubiquitination? Include the labeling with ubiquitin and the steps in proteasome and post proteasome processing. Discuss any specificities and give an example of at least one protein which is degraded via ubiquitination and the proteasome.

  

iv) Protein degradation may also be a way of regulating enzyme activities. What is an example of regulation of a key enzyme of a pathway where a product or effector regulates enzyme activity by increasing or decreasing the rate of degradation of the enzyme protein?

Answer 1

a. Lysosomal degradation of proteins: In eukaryotic cells, one of the major pathways of protein degradation is uptake of proteins by lysosomes. This pathway is mainly targeted towards extracellular proteins, membrane proteins taken up by endocytosis and is quite selective for cytosolic proteins and organelles. Lysosomes are membrane enclosed organelles with a multitude of hydrolytic enzymes like proteases (around 40 different types), which break down waste products, fats, carbohydrates and are recycled to the cytoplasm, such that they can be used as building blocks for macromolecules. Lysosomes are found in most mammalian cells, except RBCs. In order to be degraded by the lysosome, the target protein needs to be first taken up by the lysosome, which is achieved by any one of the following process: endocytosis, microautophagy, macroautophagy or chaperone-mediated autophagy. In endocytosis, the cell takes in materials to be degraded from the external environment and subsequently fuses with the plasma membrane. Endocytosis is either by phagocytosis (cell eating), pinocytosis (cell drinking) and receptor mediated endocytosis. During phagocytosis, particles are engulfed and ingested by a phagosome, which fuse with the lysosome, forming a phagolysosome, inside which the material is eventually digested. During pinocytosis, which is a non-specific process, narrow channels are formed through the plasma membrane which pinch off and fuse with the lysosome, degrading the extracellular fluid taken up. In receptor mediated endocytosis, macromolecules are taken up by receptors that are located in coated pits which pinch off forming coated vesicles and eventually fuse with the lysosome. Damaged organelles or unused proteins are engulfed by autophagosomes or by lysosomes during autophagy, by invagination and protrusion of the lysosomal membrane. The autophagosomes fuse with the lysosomes forming autophagolysosomes.Once inside the lysosomes, proteins are degraded by endo- and exo-peptidases and non-protein molecules are degraded by acid hydrolases. The most common cystein protease inside the lysosome is cathepsins. These aggressive enzymes are contained within the lysosomes, such that the other cellular proteins are not degraded. Usually these lysosomal hydrolases work best at an acidic environments (pH 4~6), which is maintained by the action of V-type proton ATPase. Example: Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates adaptive responses to oxygen deprivation. HIF-1 interacts with cyclin dependent kinase Cdk2. Cdk2 activity promotes lysosomal degradation of HIF-1? at the G1/S phase transition.

b. Proteasomal degradation by ubiquitination: Ubiquitin is a 76 residue protein that is conserved in all eukaryotes, which marks cytosolic and nuclear proteins for rapid proteolysis. Ubiquitin is attached to the amino group of the lysine side chain in the target protein. Additional ubiquitins are then added such that polyubiquitinylated proteins are made which are easily recognized by a multi-subunit protease complex, called proteasome. The marking of proteins by ubiquitin and the subsequent degradation is an energy utilising process involving ATP. The resulting peptides post degradation are further targeted by endo and exo-peptidases in the cytosol and nucleus, or else used for antigen presentation during immune response. The steps involved are : 1) Ubiquitin is attached to E1 or ubiquitin activating enzyme, a process requiring ATP. 2) Ubiquitin is transferred from E1 to ubiquitin activating enzyme E2. 3) E3 finally catalyzes transfer of ubiquitin from E2 to the lysine side chain of the target protein. This process is very specific as various classes of E3 proteins recognize specific target proteins containing a particular degradation signal or degron. Once a protein is tagged with a single Ub molecule, further molecules add up, such that proteins tagged with atleast 4 Ub moleucles are targeted for destruction by the 26S proteasome.

In contrast to the lysosomal degradation pathway, which targets proteins of molecular weights 20-40 kDa in an acidic environment with its cysteine/serine/aspartate protease degrading proteins in a non-processive manner, proteasomal degradation can target proteins of larger sizes (~ upto 2.5 million Da) using Threonine proteases in a neutral environment, strictly in an ATP dependent, processive manner. Example: Microtubule-associated protein Tau is a common target for 26S proteasomal degradation.

iv) Enzyme turnover is regulated by increasing or decreasing the rate of degradation or synthesis of the targte enzyme. Many a times pathway regulation is achieved by products or effectors which have an effect on the protein turonover. For example, the enzyme liver arginase, a key enzyme of the urea cycle , which mediates catabolism of L-arginine to L-ornithine/urea or L-citrulline/NO, involves changes in protein synthesis and degradation. After a protein-rich meal, liver arginase levels will rise and subsequently the arginine synthesis decreases. But during starvation, arginase levels also rise.However in this case, arginase enzyme degradation decreases.

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