Question

) What are the functions of protein degradation? (2 pts)

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    ii) What are amino acid or other sequence aspects that appear to correlate with the half-lives of proteins? (2 pts)

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    iii) Then discuss the following:

                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. (3 pts)

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                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. (4 pts)

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    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? (1 pt)

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5. There are some differences in the way unsaturated fatty acids are catabolized versus standard beta oxidation. What would be the products of the complete oxidation of the cis fatty acid 15:1^D9 ? Show your work by including full reactions, names of enzymes and cofactors required for the steps necessary for the catabolism of the given fatty acid. However, if you are just repeating rounds of fatty acid oxidation, you may just state that in a manner similar to what you see in your text on page 677. Be sure to make this specific to the complete catabolism of cis 15:1^D9.

Answer 1

1. Function of protein degradation

Proteins degradation helps in removal of unfolded or improperly folded protein during unfolded protein response in Endoplasmic reticulum. If degradation does not take place such abnormal protein would accumulate in ER and hamper its normal functioning.

It also helps indirectly in regulation of gene expression and cell cycle progression as various transcription factors and cell cycle regulation proteins like cyclins undergo controlled degradation. In cell cycle, cyclin B is essential for entry into mitosis. Cyclin B is a regulatory subunit of Cdc2 ,this protein complex activates Cdc 2 Kinase. Kinase then breaks down nuclear envelope and causes chromatin condensation and mitosis start. Cyclin B is degraded towards the end of mitosis so that cells can proceed towards interphase. If cyclin B was always present cells would not move past mitosis step.

Protein degradation is a major function of lysosomes. Proteins of microbes ingested by macrophages undergo degradation in phagolysosomes. Short peptide released from these peptides are complexed with MHC molecules and presented to T cells. This process is crucial for immune response.

It also helps in recycling of amino acids.

It also required for activation of zymogen like trpsinogen. by cleavage .

Cleavage of polyproteins.

2. The N terminal amino acid composition of proteins is correlated to their half lives. A ccording to N-end rule, the certain stabilizing and destabilizing amino acid present in the N terminal dictates the half live of proteins in vivo.

Proteins with Stabilizing amino acids like alanine ,glycine, serine, methionine, threonine, valine at N terminus have a half life of more than 20 h.

Destabilizing amino acids like isoleucine, glutamic acid have approximately 30 min half life

Highly destabilizing amino acids like leucine, phenylalanine, asparagine and lysine have approximately 3 minute half life.

PEST sequences rich in proline, glutamic acid, serine, threonine, are also correlated to proteins half lives . More the number of PEST sequences shorter is the half life.

3 (a) Lysosomes contains proteases like cathepsins, peptidases and collagenase for protein breakdown. These are active only in the acidic pH of lysosomes. lysosomal protein degradation can be selective or non-selective. Non- selective process is autophagy where autophagosme vesicles are formed. These vesicles engulf portions cytosol and cellular organelles and later fuse to lysosome for degradation. Membrane of autophagosome is derived from endoplasmic reticulum.

Selective process is when lysosomes in response to cellular starvation ,uptake cellular proteins having KFERQ sequence, for degradation. Hsp 70 chaperone is required for unfolding protein during their transport across the lysosomal membrane.

b) ubiquitin , a 76 amino acid long polypeptide is covalently attached to protein for degradation via proteosomal pathway. It is attached to amino group of lysine residue. Later more ubiquitin molecules are attached to make a polyubiquinated protein. This process of ubiquitinylation and degradation requires energy from ATP.

Ubiquitin is attachhed in a step wise manner. First E1 (ubiquitin activating complex) activates ubiquitn by attaching a adenylate group to it , adenylated ubiquitin then binds to cysteine residue to E1. Second E1 transfer ubiquitin to E2 ( Ubiquitin- conjugating enzyme). Finally E3 ( ubiquitin ligase) attaches the molecule to target protein.

Ubiquitnylated protein is recognized by 19S regulatory subunit of proteosome in an ATP- dependent way. Protein then enters the core of 20S subunit to come in contact with the active site. As the 20S channel is narrow protein must be slightly unfolded to enter inside.

Cyclin B, a cell cycle regulator for entry into mitosis is degraded by ubiquitinylation . Cdc2 with which cyclin B binds, activates the ubiquitinated degradation of cyclin B.