Transcript PowerPoint

Protein Degradation
BL4010 10.7.05
Proteins have variable life-spans
EnzymeHalf-life
Ornithine decarboxylase
RNA polymerase I
Tyrosine aminotransferase
Serine dehydratase
PEPcarboxylase
Aldolase
GAPDH
cytochrome c
Hours
0.2
1.3
2.0
4.0
5.0
118
130
150
Life-span factors
• Natural stability ("genetically encoded")
– an inherent biophysical characteristic
• Change in environment
– temperature
– pH
• Active degradation
– specific mechanism
– location
– partners
Terminology
• Half-life - Average time for half of the protein pool to
become denatured or degraded (depends on what you
measure)
• Turnover - Lifespan of a protein from synthesis to
degradation
• Stability - Subjective property of a proteins natural
tendency to denature under certain conditions
• Denaturation - Unfolding, partial or total of a polypeptide
• Degradation - Proteolysis of a peptide
• Ubiquitination = Ubiquitylation
• Protease = peptidase
Two routes to digest proteins
• Lysosomes
– Receptor mediated endocytosis & phagocytosis
• Proteasomes: for endogenous proteins
–
–
–
–
–
transcription factors
cell cycle cyclins
virus coded proteins
improperly folded proteins
damaged proteins
Cystic fibrosis is due to the accelerated degradation of
chloride transporter
Ubiquitin mediates degradation
for many but not all proteins
Ubiquitination
DEGRADATION
Ubiquitination
• Ubiquitinating enzymes E1,E2,
E3 - thiol ester bond
• Final target - isopeptide bond
between a lysine residue of the
substrate (or the N terminus of
the substrate) and ubiquitin
Ubiquitin
• 76 amino acids
• Highly conserved
– 3 amino acid changes yeast to human
• Thermostable
Ubiquitin is first activated
• Ubiquitin is
adenylated
• Forms bond at Cys
of E1 activating
enzyme
• E1 transfers Ubq to
E2 conjugating
enzyme
Polyubiquitination
• E2 conjugating enzyme is bound by E3 ligase which
transfers Ubq to the target protein
Why have a 3-step ubiquitination
process?
•
•
•
•
Ubiquitin
E1 (1)
E2 (12-30)
E3 (>200?)
–
–
–
–
HECT-type
RING-type
PHD-type
U-box containing
N-termini
• Acidic N-termini
– Arg-tRNA protein transferase
– conversion of acidic N-terminus to basic!
• VAST MaGiC (Val, Ala, Ser, Thr, Met, Gly,
Cys) resistant to Ubiquinitation
• WHEN sQuiDs FLY tend to have short
half-lives ( <30 min.)
Signals for degradation (degrons)
• PEST sequences (Pro, Glu, Ser, Thr)
• FREQK nonessential under starvation
conditions
• DUBS (de-ubiquinating enzymes) provide
additional regulation
SUMOylation
• SUMO = small ubiquitin related modifier (1996)
The proteasome
• Alfred Goldberg &
Martin Rechsteiner
in 1980's
• Similar in structure
to GroEL chaperone
• Unfolding and
proteolysis
• Much more specific
– Why?
Eukaryotic Proteasome
• 26S (200 kD) complex
– 2OS (673 kD) proteasome or multicatalytic protease complex
(MCP) as the key proteolytic component
– 19S complex containing several ATPases and a binding site for
ubiquitin chains.
• 19S particle "caps" each extremity of the 20S proteasome
– Unfolds the protein substrates
– Controls entry into the 20S proteasome
– Stimulates proteolytic activity
• In yeast, only 3 out of 7 subunits are proteolytically active
Yeast
proteasome
Bacterial proteasomes do not
require ubiquitin
• T. acidophilum 20S proteasome
• 14 -subunits and 14 -subunits in a four stacked ring
– 2 outer rings of seven  subunits/2 inner of seven  subunits
• Central channel with three chambers
–
–
–
–
2 antechambers located on opposite sides of a central chamber.
14 catalytic sites within the central chamber
N-terminal threonine is catalytic residue
Covalent modification of Thr by lactacystin, a natural inhibitor of
the proteasome.
• Unspecific proteolysis but products always 6 to 9 residues.
This corresponds to the length between adjacent catalytic
sites in the central chamber
Thermoplasma proteasome
Other Proteases
• Cell cycle control/stress response proteases
– Proteasome
– HtrA
• Calcium activated proteases (Calpains)
• Apoptotic proteases
– ICE family (caspases)
• Autocatalytic proteases
• Nutrient regulated proteolysis (lysosome)
• Intramembrane cleave protease (ICLiPs)
What is proteolysis?
Proteolysis = peptide hydrolysis (facilitated
nucleophilic attack of water on peptide bond)
Four mechanistic categories of protease
– serine proteinases
• chymotrypsin family
• subtilisin family
– cysteine proteinases (e.g. papain, caspsases)
– aspartic proteinases (e.g. pepsin)
– metallo proteinases (e.g. thermolysin)
Htr Protease
serine protease
(chymotrypsin family)
Unlike proteasome, most
proteases are specific
Proteolysis as a regulatory
mechanism
Proteolysis regulates cell death
Proteolysis as a regulatory mechanism
(sequestration of sterol response element transcription factor)
Why make proteins that have
short half-lives?
• It seems wasteful to try to maintain the concentration of a
protein while it is simultaneously being degraded.