Intracellular proteolysis Flashcards
What is proteolysis?
Proteolysis is the reversal of protein synthesis through hydrolysis
What are proteases and how are they classified?
Proteases are the enzymes that catalyse this reaction
One way these are classified is based on their catalytic mechanism
Serine proteases contain a serine residue at their active site
Cysteine proteases contain a cysteine
Aspartyl proteases an aspartic acid
Metalloproteases form complexes with specific metal ions in order to be active
Another key distinction between proteases is whether they recognise specific amino acid sequences in their substrate-
If so, the cleavage serves to activate the protein
If proteases are non-specific these are generally involved in proteins degradation e.g. during digestion
What is the difference between endo- and exopeptidases?
Endopeptidases cleave proteins somewhere in the middle of the protein
Exopeptidases remove one or two amino acids from the end of a polypeptide chain
If they work on the amino end they are ‘Aminopeptidases’
If they work on the other end of the peptide chain they are ‘Carboxypeptidases’
What names are used to describe a protein in its inactive state?
Zymogen, proprotein and proenzyme
How are proteins activated by proteolysis? What are the examples?
-One example is enzymes active in the gastrointestinal tract
Chymotrypsinogen is initially a protein of 245 amino acids which must be cleaved by another protein called trypsin between AA 15 and 16 to generate pi-Chymotrypsin which is active
pi-Chymotrypsin cleaves itself between 146 and 149
After those two cuts, we are left with 3 peptide chains connected by disulphide bonds to produce the final active Chymotrypsin enzyme
Trypsin must itself be activated by another enzyme, enteropeptidase which yields the active trypsin enzyme
-Another example is that of proopiomelanocortin in the Golgi which gives rise to multiple peptide hormones
-Insulin must also be processed by proteolysis before it becomes active
The initial protein that is synthesised at the ER is called preproinsulin which carries the signal sequence used to direct it to the RER
Its removal produces proinsulin, that also requires a number of disulphide bonds
This must be cleaved again to release an internal fragment called the C peptide which then produces the final mature insulin
-The clotting cascade involves a whole series of different enzymes that are all serine proteases
At each step of the cascade, one serine protease cleaves another
How are proteases involved in certain disorders?
Relating to the clotting cascade, deficiencies of Factor VIII or IX are the cause of X-linked haemophilia (only males are affected by this?)
HIV-1 Protease (retropepsin) is a prominent aspartyl protease (thanks to its discovery new drugs and therapies were developed)
How is protein degradation controlled?
Non-specific protein degradation is compartmentalised because otherwise, they could cause damage elsewhere
Proteases with low specificity are found in the gastrointestinal tract, but inside cells are compartmentalized inside lysosomes or inside a proteosome
The proteases found in lysosomes are also designed in a way to minimise potential damage, by operating only at a low pH
What is the ubiquitin-proteasome pathway?
A two-step process whereby a protein that needs to be degraded is first covalently modified with a chain of ubiquitin molecules and then targeted to the proteasome where the degradation takes place
In both cases, the products are amino acids and sometimes small peptides
Ubiquitin is a very small protein and highly conserved with 76 amino acids
What is the three-step pathway for protein ubiquitylation? What happens in this process?
E1- Ubiquitin-activating enzyme
1.In the first step, ubiquitin is attached to E1, which reacts with the carboxyl-terminal via the thiol groups on the cysteine residue on the E1
This is a reaction that requires ATP
It generates what’s called a thioester bond (O=C-S-E1)
E2- Ubiquitin-conjugating enzyme
2.Ubiquitin is transferred from E1 to E2 (O=C-S-E2)
E3- Ubiquitin ligase
3. It’s only through E3 that ubiquitin is now transferred onto the lysine residue of the target protein
If a protein is to be degraded by ubiquitylation, then a chain must be formed where additional ubiquitin molecules are to either lysine 48 (k48) or sometimes lysine 11 (k11)
A minimum of 4 ubiquitin molecules must be attached in order for the protein substrate to be marked for proteolysis
What is the N-end rule?
What determines protein half-life is the first amino acid at the beginning of the N-terminus
The very first AA is actually always methionine by that is quickly removed
It is because these AAs are recognised by E3 ubiquitin ligases
So some AA are very stabilising because they are not very recognised by E3 ubiquitin ligase
If it isn’t recognised the proteins isn’t ubiquitylated and hence will not be degraded
