Protein Degradation

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Protein Degradation

• BL4010 10.7.05

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Proteins have variable life-spans
EnzymeHalf-life Hours
Ornithine decarboxylase 0.2
RNA polymerase I 1.3
Tyrosine aminotransferase 2.0
Serine dehydratase 4.0
PEPcarboxylase 5.0
Aldolase 118
GAPDH 130
cytochrome c 150
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Life-span factors

• Natural stability ("genetically encoded")
• an inherent biophysical characteristic
• Change in environment
• temperature
• pH
• Active degradation
• specific mechanism
• location
• partners

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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

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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

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Ubiquitin mediates degradation for many but not
all proteins
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Ubiquitination
DEGRADATION
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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

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Ubiquitin

• 76 amino acids
• Highly conserved
• 3 amino acid changes yeast to human
• Thermostable

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Ubiquitin is first activated

• Ubiquitin is adenylated
• Forms bond at Cys of E1 activating enzyme
• E1 transfers Ubq to E2 conjugating enzyme

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Polyubiquitination

• E2 conjugating enzyme is bound by E3 ligase which
  transfers Ubq to the target protein

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Why have a 3-step ubiquitination process?

• Ubiquitin
• E1 (1)
• E2 (12-30)
• E3 (gt200?)
• HECT-type
• RING-type
• PHD-type
• U-box containing

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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 (
  lt30 min.)

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Signals for degradation (degrons)

• PEST sequences (Pro, Glu, Ser, Thr)
• FREQK nonessential under starvation conditions
• DUBS (de-ubiquinating enzymes) provide additional
  regulation

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SUMOylation

• SUMO small ubiquitin related modifier (1996)

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The proteasome

• Alfred Goldberg Martin Rechsteiner in 1980's
• Similar in structure to GroEL chaperone
• Unfolding and proteolysis
• Much more specific
• Why?

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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

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Yeast proteasome
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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

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Thermoplasma proteasome
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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)

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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)

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Htr Protease
serine protease (chymotrypsin family)
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Unlike proteasome, most proteases are specific
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Proteolysis as a regulatory mechanism
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Proteolysis regulates cell death
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Proteolysis as a regulatory mechanism
(sequestration of sterol response element
transcription factor)
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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.