Cellular Protien turnover.docx

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Department
Biomedical Sciences
Course
BMS2021
Professor
Prof.Christina Mitchell
Semester
Fall

Description
Cellular Protien turnover, the proteasome and autophagy. All proteins have a limited lifetime within a cell. They need to be removed as they can be denatured, damaged by oxidation or mutated and are not in correct tertiary/quaternary structure. There is no enzyme catalysed repair mechanism for proteins. Constitutive enzymes are present in constant concentrations in the cell and have life spans that can range from several days to months. It concentration does not change with changes in metabolic state or environment and are usually enzymes of central metabolic pathways. Some enzymes, such as inducible/repressible enzymes have very short lifespans within a cell, from minutes to hours and are destroyed by proteases. These enzymes are ones that influence the rate of synthesis by specific substances (metabolites, hormones, growth factors). Their short life span is due to either particular amino acid sequences that makes them susceptible to attack by proteolytic enzymes or ubiquitination, which makes them targets for specific proteases. Ubiquitination a process by that marks a specific protein for destruction. This may be due to the presence of a PEST region, and amino terminal amino acid residue or being an abnormal protein. The process of ubiquitination is as follows. The protein ubiquitin is found free floating within the cytosol of all cells. When a protein is destined for degradation it is marked by a small single ubiquitin protein. This is primed for action by an E1 ubiquitin activating enzyme, which requires ATP. This is transferred to E2 which acts as a transporter of the ubiquitin to E3, this is a ligase enzyme that binds to the E2 transporter, with a bound and activated ubiquitin protein. The E3 acts as a platform where the targeted protein can interact with the activated ubiquitin. When both the protein and the e2 is bound by the e3, the activated ubiquitin is added to the protein. This process repeats many times to form a poly ubiquitin chain made up of multiple ubiquitins. This is then transported to the proteasome. This is a cylindrical complex that removes the ubiquitin chain and digests the protein, breaking it down into amino acids that can be reused. The ubiquitin can also be reused. Discuss the functions and importance of cellular protein turnover Discuss the signals for protein degradation Amino-terminal amino acid residues. This is a protein destined for degradation. It is first phosphorylated by protein kinase, unmasked by protein dislocation and marked for destruction via creation of a destabilising N-terminus. PEST region. These are internal regions of the protein rich in pro, glu, der and thr. These create domains that are recognised by specific proteases. The protein is marked for degradation should it be denatured, damaged by oxidation, mutated. Explain the molecular mechanisms of cellular protein turnover. Intracellular trafficking of Protiens. This is the importance of getting proteins to the correct place within cells. Discuss the importance of protein sorting and trafficking in the accurate formation and function of organelles and other cellular components. Living cells create an maintain order, not only by making the right molecules but also by getting the proteins into the correct place inside and outside the cell. A simplified road map of protein trafficking: There are three main ways that membrane bound organelles import proteins. Though nuclear pores: these are done by gated, selective, active transport and also diffusion of small molecules. Transport across membranes. Done via transmembrane traslocators, specific, protein usually unfolds. Transported by vesicles. This is membrane enclosed, from one compartment to another. The sorting signal can be built in to a protein by either having a signal sequence or a signal patch. Explain the complementary roles of signal sequences and trafficking machinery. Discuss the central roles of ER and Golgi in protein trafficking. During vesicular transport the ‘sidedness’ of membranes is preserved and soluble molecules (cargo) are transferred from lumen to lumen. I.e. enables budding and fusion of transport vesicles to target compartment. The function of membrane bound ribosomes is to enable proteins to be translocated to lumen of ER. Leader is a signal sequence that directs secreted protein to ER membrane. The Signal recognition particle (SRP) directs the ribosomes to the ER membrane. Binding of SRP to signal peptide causes a pause in translation. SRP-bound ribosome attaches to the SRP receptor on the ER membrane, SRP and SRP receptor is displaced and recycled. Translocation continues and translation begins, within the ER lumen. Once the signal peptide is cleaved off, this creates a mature and soluble protein within the ER lumen. Protein glycosylation occurs in the rough ER . This is a major synthetic function of the ER, 50% of all eukaryotic proteins are glycosylated. To most proteins an asparagine-linked precursor oligosaccharide is added in the rough ER. Precursor oligosaccharide is initially made on the cytosolic side of the ER. Dolichol then flips across the membrane to place the maturing precursor inside the ER lumen. Transport of proteins out of the ER. Chaperone proteins bind to unfolded or misfolded proteins preventing their movement out of the ER. Transport vesicles form containing the newly synthesised proteins within the cytosol, but are surrounded by various proteins, embedded onto the vesicle surface membrane. These vesicels, due to the outer proteins are able to fuse to one another and function as transport container, bringing material from ER to Golgi. The Golgi is the ‘post office’ of the cell as the function of the Golgi apparatus packages and tags for proper destination. Vesicles from the ER move to the cis side of the golgi first. As it moves though the proteins are sorted and tagged for identification for sending to the proper destination. The trans golgi network package the proteins into vesicles that modulate their release from the cell. There are 2 methods of release, the constitutive method involves the vesicle merging with the cell membrane and releasing contents into the extracellular space, without any need for regularity molecules. The regulatory secretory pathway relies on the binding of a signal such as a hormone or neurotransmitter to enable secretory vesicles to fuse with the membrane and release proteins into the extracellular space. Protein Trafficking to Lysosomes. One step in the protein sorting step, that takes place within the trans Golgi network is signal mediated diversion to lysosomes. A lysosome is a vesicle that contains hydrolytic enzymes that are active under acidic conditions. This acidic condition is maintained at an acidic pH by H+ATPase that pumps H+ into the lumen. By hydrolytic enzymes, nucleases, proteases, glycosidases, lipases, phosphatases, sulphatises and phospholipases are contained within the lysosome. To recognise lysosomal proteins, phosphate is added to mannose of glycoproteins destined for the lysosome when within the golgi apparatus. The recognition of mannose-6-phosphate occurs by the binding of the lysosomal protein bound with the n-linked oligosaccharide, however unlike other proteins, it has a mannose terminal residue. This binds, along with UDP-GlcNAc to UDP-GlcNAc phosphotrasnferase, where this UDP-GlcNAc and phosphate is attached to the lysosomal mannonse terminal residue. The UDP-GlcNAc is cleaved by another enzyme, leaving the phosphate bound to the mannose. This lysosomal hydrolase binds to M6P receptors in the golgi and released within a transport vesicle, where it makes its way to lysosome. Receptor dependent transport occurs enables
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