Proteases 3 Flashcards
What is the purpose of protein degradation?
regulation of levels of specific proteins, tidying up damaged/worn out proteins, liberate energy (amino acid consituents can be used for fuel) and free up amino acids better needed in alternative proteins
Through which route are most ExtraCellular matrix and membrane proteins degraded?
The lysosomal pathway
What is the lysosomal degradative pathway?
Proteins enter the lysosome through either endocytosis, micro/macro autophagy or chaperone mediated autophagy
Once inside all material taken in is digested, proteins through non-specific endo and exo peptidases
And other material through various acid hydrolases
The resulting dipeptides and amino acids are able to pass through the membrane and into the cytosol
How does chaperone mediated autophagy occur?
Proteins which have a consensus peptide sequence get recognized by the binding of a chaperone or cochaperone complex
This complex is recognized by lysosome associated membrane protein type-2A
How are most cytosolic, ER and nuclear proteins digested?
The ubiquitin-proteasome pathway
What is the ubiquitin-proteasome pathway?
The protein target has multiple copies of ubiquitin added in an ATP dependant reaction
These marked proteins are the degraded by the 26S proteasome via ATP dependant reactions
The remnants of the proteins are then either degraded by end/exopeptidases in the nucleus or cytosol or it is displayed as an antigen
How does ubiquitination occur?
A selective process where the highly conserved small (76 amino acids) Uquitin is attached to ubiquitin activating enzyme E1 in an ATP dependant reaction
it is the transferred to ubiquitin activating enzyme E2
E3 recognizes specific degron sequences on target proteins and catalyses the transfer of ubiquitin between the E2 protein and a lysine residue on the target protein
What are examples of degrons?
Usually large, or charged amino acids
Internal degradation signals
Phosphorylation
What is the proteasome?
A complex made of a 20S core flanked by two 19S caps which is located in the cytosol
The 20S core is made of 28 units divided into 4 rings the top and bottom rings are alpha rings and the middle rings are beta rings which hold the catalytic chamber
How does the proteasome cleave protein?
The 19S caps cleave off the ubiquitin chain using ATPases to unfold the protein and translocate it to the inside the 20S core which has the proteolytic cores
The two alpha rings act as gates which will hold and release the target protein in response to the 19S regulatory particle
The beta rings all act as threonine proteases, there are three catalytic units
Beta1= cleavage after acidic residues
Beta2= cleavage after basic amino acids
Beta 5= cleavage after large hydrophobic amino acids
The target protein is held in place until it is completely degraded to short peptides not free amino acids
These short peptides must be degraded further or these form cytotoxic aggregates
What are the differences between the lysosomal proteases and the 26S proteasome?
The lysosomal proteases have a low optimal pH (4-6) while the 26S works at neutral pH
The lysosomal proteases have cysteine, serine and aspartic catalytic classes while 26S has only threonine
The 26S is much larger, with a greater number of subunits and requires energy in the form of ATP in comparison to the energy independent lysosome
The protein needs to be unfolded before it can enter the 26S but whole proteins can enter the lysosome
How do proteins differ in their half-lives?
Collagen and other structural proteins have long half lives while regulatory proteins such as ornithine decarboxylase have very short half lives
How does the lysosome maintain its low pH in order for its enzymes to be at their optimum pH?
A v-type proton ATPase is used to pump H+ into the cell through a soluble V1 component (A and B units) and a rotor part (c subunits)
What is the difference between micro and macro autophagy?
In microphagy the lysosome directly engulfs the cytoplasm through invagination, protusion and/or separation of the lysosomal membrane
In macrophagy damaged organelles or longlived proteins are sequestered in a double-membrane vacuole (autophagosome) formed from the elongation of small membrane structures (autophagosome precursors), the autophagosome fuses with the lysosomal membrane so the contents are degraded
