Protein Degradation Flashcards
Protein turnover
-Proteins turn over continually
-Adult humans turn over ~2% of their protein per day
-Cells continuously synthesize proteins from and degrade proteins to amino acids
—Recycling
-A protein’s level is determined by synthesis and degradation rate
—To study one rate, the other process must be inhibited
-Half-lives range from 10 min for Cyclin D1 to 4 months for hemoglobin
Why are cells wasting so much energy turning proteins over?
Continual turnover serves to:
-Respond to environmental changes
—Ramp up/down metabolic processes
-Control cell cycle
-Eliminate damaged proteins, arising from chemical modifications in cell’s reactive environment
What are lysosomes?
-Membrane bound organelles involved in protein degradation
-Maintain an internal pH of 5
-Contain ~50 hydrolytic enzymes, including proteins called cathepsins
-Lysosomal degradation is largely non-selective
What is a characteristic of lysosomal enzymes?
-They have acidic pH optima
-They are non-functional at cytosolic pH
-Occurs because if they get out of lysosome, you don’t want them to harm anything
How is intercellular material degraded in the lysosome?
-Material is degraded in lysosome by fusing with other vacuoles
-Intercellular “garbage” can be packaged into vacuoles through a process called “autophagy”
How is extracellular material degraded in the lysosome?
Material is degraded in lysosome when taken up by endocytosis
What is chloroquine
-Antimalarial drug that inhibits the lysosome
-A weak base that freely penetrates the lysosome where it becomes pronated, this charged form accumulates and raises lysosomal pH
-Inhibits the lysosome
What maintains low lysosomal pH
-Vacuolar ATPAses
Ubiquitin-based degradation basic characteristics
-Selective
-ATP-dependent
-Occurs in nucleus and cytosol
-Ubiquitin is ubiquitous and abundant 76 amino acid protein
-Highly conserved protein
How are proteins selected for degradation in ubiquitin-based degradation?
-Proteins are marked by covalently linking ubiquitin to them
How is ubiquitin expressed? What are DUBs?
-Expressed as a polyprotein containing several ubiquitin units (no gene that encodes a single ubiquitin)
-These polyproteins are processed by proteases, called deubiquitinases (DUBs) to yield single ubiquitin
-DUBs also remove ubiquitin from targets in the cell
How does ubiquitin attach and where?
-Attaches to substrates/targets by its carboxyl-terminal Gly residue to the ε-amino group of Lys residues on target proteins
-Bond to protein is similar to an amino bond
Steps to attachment/ligation of ubiquitin to target proteins
-C-terminus of ubiquitin is activated with ATP by ubiquitin activating enzyme (E1)
—aka AMPylation
-Terminal carboxyl group of ubiquitin is conjugated via a thioester bond to a Cys on E1
—Yeast and humans only have one E1
-Ubiquitin is then transferred to Cys on a ubiquitin conjugating enzyme (E2), forming a second thioester bond
—11 in yeast; 20+ in mammals
-A ubiquitin-protein ligase (E3) transfers the activated Ub from the E2 to a lysine group on the target protein, forming an isopeptide bond
—~644 E3 ligases (more than the number of protein kinases)
-SEE DIAGRAM (L21 pg 17)
Why are there more E2’s than E1’s?
To enable substrate specificity and diverse regulation patterns
What is necessary for a target protein to be degraded? (ubiquitin-based degradation)
-For a target protein to be degraded, it must be linked to a chain of at least 4 tandemly linked ubiquitins
-Ubiquitin chains are linked via internal Lys residues
-Lys 48 of Ub forms an isopeptide bond with the C-terminal carboxyl of the next ubiquitin
-The ε-amino group of a Lys residue of one ubiquitin is linked to the terminal carboxylate of another
Polyubiquitin chains (what are they, what generates them, linkage)
-Can be 50+ ubiquitins long
-Generated by E3s, how E3s switch from transferring a Ub to a target protein to synthesize a polyUb chain is unknown
-Lys/amino group linkage determines chain geometry
-Different types of linkages have different signaling outcomes beyond degradation
Degrons (what are they, where are they)
-Specific sequences of amino acids that indicates protein should be degraded
-For many proteins, the amino-terminal amino acid residue (N-degron) is an important degradation signal E3
-Residue may only be exposed afterproteolytic cleavage, may be added after protein synthesis, and may be impacted by modification (like N-terminal acetylation)
-Also called the “N-end rule”
-Other degrons include cyclin destruction boxes
N-end rule (what is it, what does it result from)
-Half-lives of many proteins vary with the identity of N-term AAs (the N-end rule)
-Rule results from actions of a RING E3 called E3a, which recognizes the destabilizing residues for ubiquitination
What do K48 Polyubiquitin chains do?
-Target a protein to the 26S proteasome for degradation
26S proteasome structure
-~2100 kDa
-Complex of three components:
-One 20S catalytic unit arranged as a barrel
-Two 19S regulatory units that control access to the interior of the 20S catalytic subunit
26S proteasome basic characteristics
-Exists in the nucleus and cytoplasm
-Rivals the ribosome in size and complexity
-Exists in all eukaryotes and some archaea
19S subunits of 26S proteasome
-Regulatory units, control access to interior of catalytic subunit (20S)
-Contains ubiquitin receptors that bind specifically to polyubiquitin chains
-Use ATP to unfold polyubiquitinated chains and direct them into the 20S core
-Contain an isopeptidase (a DUB) that cleaves off intact ubiquitin molecules (so it can be reused)
20S subunits of 26S proteasome
-Catalytic unit
-Has three types of active stirs in the β subunits, each with a different specificity
-Substrates are degraded in a processive manner without intermediate release
-Substrates are reduced to peptides ranging from 7 to 9 amino acids before release
What degrades peptides to amino acids?
Cytosolic peptidases
