Flashcards in Lecture 22 Deck (31)
Where can polypeptide chain cleavage occur along a polypeptide?
1) N-terminal end, most often including Methionine (Met) as well
2) C-terminal end
3) Internal sequence
What are function of polypeptide chain cleavage?
1) Maturation - generation of active products from inactive precursors
2) Inactivation or partial inactivation - regulation of activity
Who was the first person to sequence a protein and what protein was it?
Fred Sanger; insulin
Describe the process of insulin maturation
1) pre-prohormone undergoes signal sequence cleavage (signal peptidase) in ER to form proinsulin
2) In ER, C peptide holds A and B peptides adjacent to each other to allow disulfide bridges to form between A and B peptides
3) C peptide is cleaved off to form insulin (A and B peptides only)
What type of cleavages occur to activate insulin?
1) Tryspin-like (cleaves lysine and arginine - basic amino acids)
2) Carboxypeptidases (chew off amino acids from C-terminal end)
What is familial hyperproinsulinemia?
It is an autosomal dominant disorder characterized by an excess of proinsulin in the blood. There is defective proinsulin cleavage resulting in equal amounts of insulin and partially-cleaved proinsulin in blood. No diabetes will ensue however in a heterozygote affected by the disorder
What are uses of insulin C-peptide?
1) Because the C-peptide is relatively stable in plasma, one can measure insulin production, which is elevated in insulinoma. This can also be used to distinguish type 1 from type 2 DM
2) Monitor endogenous insulin production in patients receiving injected insulin
3) Insulin overdose. In factitious hypoglycermia (self-inflicted insulin overdose) can use forensic tests
4) *It is thought to have physiological properties preventing some DM complications (kidney & CNS)
Proteolytic cleavage is ________ specific
Tissue specific; You can get different combinations of products based on which tissue the proteolytic cleavage occurs
What is a polyprotein?
Viruses create a polypeptide that is destined to be cleaved into multiple proteins
What are two types of proenzymes?
Zymogens & Activation cascades
What do the synthesis of prothrombin and factors VII, IX & X require?
Formation of gamma carboxyglutamate residues via vitamin K. They are inhibited by coumarins
What activates cell death by apoptosis?
Proteolysis cascade. During apoptosis chromatin condenses, DNA is fragmented, cells shrink, and cells are engulfed by macrophages
What does the poliovirus protease do to induce polio?
Poliovirus protease 2A cleaves a polyprotein for activation and also cleaves some cellular proteins, including translation initiation factor eIF4G. Intact eIF4G is required for initiation on capped cellular mRNAs. Cleaved eIF4G still works, but only for poliovirus IRES-mediated initiation. Therefore, there is reduced cap-dependent scanning
What does the poliovirus RNA do?
1) Initiates by internal ribosome entry mechanism (IRES)
2) unaffected by eIF4G cleavage
What are advantages of precursor proteins?
1) Protection of cell/organism (digestive proteases)
2) Allows folding & modification (insulin)
3) Multiple gene products (proopiomelanocortin, poliovirus)
4) Control (apoptosis, blood clotting, poliovirus/eIF4G)
How much of the total body protein turns over or is reused per day?
About 2% (250g or 1/2lb for a 75kg person)
Which proteins last less than an hour in the body?
1) Ornithine decarboxylase
2) Phosphokinase C
Which proteins last days in the body?
Typical housekeeping proteins
Which proteins last months in the body?
Hemoglobin & Histones
Which proteins last a lifetime in the body?
When is protein turnover rate elevated?
During starvation and in hypercatabolic states, especially in skeletal muscle (Due to cortisol induction of ubiquitin-mediated proteolysis)
What is the mechanism of extracellular protein turnover?
Dietary protein digestion (proteases & amino- and carboxy- peptidases)
What are the four mechanisms of intracellular protein turnover?
1) Proteasome - ubiquitin system
2) Lysosomal digestion
3) Calpains (calcium dependent proteins)
4) Caspases (apoptosis, activation cascade, & De-regulated in cancer and viral infection)
What is the proteasome-ubiquitin system?
1) It is a proteolysis system that is ATP-dependent
2) Proteins are degraded to 3-24 amino acid peptides
3) It turns over most cytosolic proteins
4) A PEST sequence (pro, glu, ser, thr - rich) & N-terminal amino acids are determinants (PEST sequence leads to a higher rate of turnover - destabilization element)
What is ubiquitin?
Ubiquitin is a 76 amino acid protein that targets proteins to the proteasome. Usually a chain of ubiquitins is attached to lysine residues. Ubiquination is a signal for digestion of that protein
What is a proteasome?
A proteasome is a large complex structure, forming a hollow cylinder. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds
What does Ubiquitination require?
ATP and 3 enzymes (E1-E3)
What are the functions of E1, E2, and E3 in ubiquitination?
1) Ubiquitin is activates by E1 (Ubiquitin activating enzyme)
2) Ubiquitin is conjugated by E2 (Ubiquitin conjugating enzymes)
3) Ubiquitin is ligated by E3 (Ubiquitin ligases)
What is Bortezomib (Velcade)?
Proteasome inhibitor anti-cancer drug (for example - relapsed multiple myeloma)
What is the defect in Angelman syndrome?
An ubiquitin ligase (E3) is defective because of a defective E6AP/UBE3A gene