Intracellular Protein Degradation- The lysosome and Ubiquitin Proteasome System

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Intracellular Protein Degradation- The
lysosome and Ubiquitin Proteasome System

• Scott Wilson
• Department Neurobiology
• 5-5573
• Wilson_at_nrc.uab.edu

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Outline

• Sites of proteolysis
• Gastrointestinal tract
• Circulatory system
• Intracellular proteolysis
• Lysosome
• Biogenesis and function
• Degradation of extracellular material
• Degradation of intracelluar components by
  autophagy
• Ubiquitin proteasome pathway
• Components
• Ubiquitin and UBLs
• Ubiquitin conjugating enzymes
• Ubiquitin deconjugating enzymes
• The proteasome- generation and activity

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• Gastrointestinal tract
• Destruction of antigenicity
• Controlled but no specificity- everything that
  enters gut is proteolyzed
• Production of energy
• Remember that destruction of proteins is an
  energy producing process (exergonic)
• Circulatory system
• Blood coagulation
• Conversion of prothrombin to thrombin which
  converts fibrinogen to fibrin and a blood clot is
  formed.
• Process is highly controlled (?1-antitrypsin
  deficiency)

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The questionIs there turnover of cellular
constituents? Or is food intact a function
primarily for energy-providing (fuel for a car),
that is independent from the structural and
functional proteins of the body?
Studies on ?-galactosidase in E. coli indicated
that there was no conclusive evidence that
proteins within cells are in a dynamic state and
that they are likely to be stable and static
Without metabolic labels (ex. 35S cysteine or 3H
leucine) the problem of determining protein
stability was not approachable
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How do you tag proteins to study protein
dynamics?

• 1939 Rittenburg and Urey succeeded in generating
  radiolabeled Nitrogen (15N)
• Schoenheimer found that following administration
  of 15N-labeled tyrosine to rats, they found that
  only 50 of the label was found in excretions.
  Where was the rest?
• The label was found incorporated in body proteins!

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• Therefore the proteins of the body are in a
  dynamic state of synthesis and degradation!
• It is thought that we are degrading and
  resynthesizing 3-5 of our cellular proteins
  daily.
• Paradigm that cellular processes are controlled
  mainly by only transcription and translation must
  be changed.

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Why are proteins degraded?

• Quality control
• Proteins become denature/misfolded/damaged
• Elevated temperatures (37C)
• Proteins being synthesized are folded incorrectly
• Regulation of biological pathways
• Cell cycle
• Receptor mediated endocytosis
• Synaptic remodeling

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Now that we know proteins are in a dynamic
state in cells.

• How are proteins degraded within cells?
• Is protein degradation regulated?
• Selective?
• Compartmentalized?

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The discovery of the lysosome

• De Duve discover the lysosome in the 1950s
• Vacuolar structure that contains hydrolytic
  enzymes that are optimal at acidic pH.
• Latency of of enzymatic activity- researcher
  found that hydrolyase fractionated from rat liver
  were more active after they were stored in the
  refrigerator for several days?
• The latency was due to the slow breakdown of the
  lysosomal membrane which protected the cells from
  the destructive forces of the acid hydrolyases.
• This compartmentalization of the peptidases by a
  membrane protects cellular components from
  inappropriate degradation.

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Generation of a functional lysosome

• Lysosomal proteases belong to the aspartic,
  cysteine, or serine proteinase families of
  hydrolytic enzymes.
• contain about 40 types of hydrolytic enzymes,
  including proteases, nucleases, glycosidases,
  lipases, phospholipases, phosphatases, and
  sulfatases. All are acid hydrolyase that have
  optimal activity at pH 5.0

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Sorting acid hydrolyases to the lysosome is
accomplished by post-translation modification

• Soluble lysosomal enzymes are synthesized as
  N-glycoslyated precursors in the ER and
  trafficked to the Golgi
• mannose 6-phosphate (M6P) groups are added to
  the hydrolyases
• The M6P groups are recognized by transmembrane
  M6P receptor proteins, which are present in the
  trans Golgi network
• M6P receptors release hydrolyases when pH is
  below 6.0 and the M6P is removed

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Lysosomes use an H ATPase pump in the membrane
to generate acidic pH
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Overview of lysosomal trafficking
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Proteases in the lysosome

• Cysteine protease- cathepsins A, B
• Aspartate protease- cathepsin D
• Zinc protease-?
• Activation of protease by removal of inhibitory
  segment- conversion of proprotein to protein

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Pathways into the Lysosomal/vacuolar System
1
3
2
4
4
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Model of the mechanism for multivesicular
endosome formation
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How do proteins get into the lysosome for
degradation?

• Microautophagy- cytoplasm is segregated into
  membrane -bound compartments and are then fused
  to lysosome
• Maroautophagy- entire organelles such as
  mitochondria, ER and other large cytoplamic
  entities are engulfed and then fused with the
  lysosome

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Autophagy pathway
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Problems that still remain

• Proteins vary greatly in their stability - from
  minutes to days!
• Rates of protein degradation of specific proteins
  changes with physiological conditions (nutrients
  and hormones)
• How could this happen by microautophagy
• Lysosomal inhibitors have differential affects on
  different populations of protein
• If lysosomal proteases degrade proteins in an
  exergonic manner, how could you explain evidence
  that the proteolytic machinery required energy?

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Still more data suggesting another pathway for
degradation of intracellular proteins

• Poole et al were studying the mode of action
  anti-malaria drugs
• Chloroquine and other lysosomotropic (weak bases)
  block the activity of lysosomal proteases by
  neutralizing the low pH of the lysosome.
• Treat macrophages labeled with 3H-leucine with
  chloroquine and then feed them protein extracts
  that were labeled with 14C-leucine
• This allowed them to monitor the stability of
  phagocytosed extracellular and intracellular
  proteins when the lysosome is blocked

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What did they find?

• Lysosomotropic drugs only affected the stability
  of the engulfed extracellular proteins and not
  the intracellular proteins.
• This indicated that there must be a second
  pathway for the degradation of intracellular
  proteins and that the lysosome was the primary
  site of degradation of internalized extracellular
  proteins

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The search for a new proteolytic pathway

• The new pathway must explain several things-
• Requirement for metabolic energy
• ATP depletion inhibits proteolysis
• Why do you need ATP?
• Need phosphorylation of substrates or enzymes?
• Remember proteolysis is exergonic
• Differential stability of intracellular proteins
• Example- RNA polymerase I t1/2 1.5 hrs
• RNA polymerase II t1/2 12 hrs
• How stability of proteins can change under
  different environmental conditions

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Cell-free proteolytic system

• Rabbit reticulocyte lysates
• Made from red blood cells (terminally
  differentiated and do not have lysosomes)
• New that for different hemoglobinopathies, the
  blood cells attempt to rid themselves of abnormal
  hemoglobins and therefore must have a proteolytic
  system that was not lysosomal based.
• Found that reticulate lysates were capable of
  degrading proteins in an ATP dependent manner

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A new paradigm for proteolysis

• Biochemical characterization of reticulate
  lysates
• Divided the lysates into two fractions (DEAE
  cellulose, anion exchange resin) Flow thru and
  high salt eluate
• Each fraction did not have proteolytic activity
  on its own.
• Combination of fraction I and II reconstituted
  proteolysis
• Previous work indicated that only a substrate and
  protease were need for degradation.
• This was very important in that it suggested that
  there was not a single protease that mediated
  degradation.
• This new system need a substrate, protease and
  something else
• Activator?

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Characterization of fractions I and II

• Analysis of Fraction I
• Found that fraction I contained only a single
  factor that was heat sensitive and required ATP
• This factor was termed APF-1 for ATP-dependent
  proteolysis factor
• Critical finding was that APF-1 can be covalently
  attached to a target substrate

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APF-1 is shifted to high molecular mass compounds
following addition of ATP to the fraction I.
125I labeled fractions following gel-filtration
chromatography
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SDS PAGE analysis of samples run on
gel-filtration

• Lane 1- Fraction II 125I- APF (no ATP)
• Lane 2- Fraction II 125I-AFP ATP
• Lane 3- Fraction II 125I-AFP ATP unlabeled
  lysosome as substrate
• Lane 4 5 - Increasing conc of lysosome
• Lane 6- Fraction II 125I-lysosome (no ATP)
  unlabeled APF
• Lane 7- Same as lane 6 ATP

These experiments demonstrate that APF is
covalently attached to substrate (explains the
requirement of ATP) Multiple APF-1s can be
added to a substrate
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What is APF-1 ?

• Amino acid analysis and its known molecular mass
  indicated that APF-1 is ubiquitin.
• Ubiquitin is a 76 aa protein found only in
  eukaryotes
• The covalent attachment of ubiquitin to a
  substrate stimulates its proteolysis (but by
  what?)
• Ubiquitin is covalently attached to a substrate
  by is C-terminal glycine to the ?-NH2 group of an
  internal lysine of the substrate

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Studies of fraction II defined the ubiquitin
conjugation machinery
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Substrate recognition
N-end rule On average, a protein's half-life
correlates with its N-terminal residue.
Proteins with N-terminal Met, Ser, Ala, Thr, or
Gly have half lives greater than 20
hours. ?Proteins with N-terminal Phe, Leu, Asp,
Lys, or Arg have half lives of 3 min or less.
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What about the protease?

• Previous studies demonstrated that the activity
  of the protease was ATP dependent (not just
  ubiquitination requires ATP)
• What is it composed of?
• Where is it located?
• How is it selective toward ubiquitinated
  proteins?
• Why does it need ATP?

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Structure of the 26S proteasome

• Tanaka et al discovered a high-molecular mass
  protease that degraded ubiquitinated lysozyme but
  not untagged lysozyme
• Required ATP for activity
• Protease was later called the 26S proteasome
• Similar multi-subunit proteases found in
  prokaryotes

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Subunits of the 26S proteasome

• 19S regulatory particle- composed of
  approximately 20 different proteins
• 20S core particle- composed of 14 different
  subunits (?1-7 and ?1-7)

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19S Regulatory particle (RP)

• Recognition and binding of ubiquitinated proteins
• Unfolding of ubiquitinated substrate to enter 20S
  mediated by AAA ATPases (ATP dependent)
• Removal of ubiquitin side chains to allow entry
  into 20S ( lumen 1.3 nm) by deubiquitinating
  enzymes
• Activation/opening of 20S lumen

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20S Core Particle (CP)

• Contains the endopeptidase activity
• The alpha subunits function is to control the
  opening and closing of the 20S gate (interacts
  with 19S)
• The beta subunits ?1, ?2 and ?5 contain the
  endopeptidase activity of the proteasome.
• Proteins are not degraded into amino acids but
  into short peptides ( very important for immune
  surveillance).

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The UPS is enormous!

• The genes of the UPS constitutes 5 of the
  genome
• E1s- 1-2 activating enzymes
• E2s- 10-20 conjugating enzymes
• E3s- 500-800 ubiquitin ligase- drives
  specificity
• DUBs- 100 ubiquitin specific proteases-
  regulators of pathway

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Pathways controlled by regulated proteolysis
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Diseases of the lysosome and UPS pathways

• Lysosomal
• Neimann Pick Disease- ataxia, brain degeneration
  and spasticity.
• Krabbe Disease- hypertonia, seizures, deafness
  and paralysis
• Tay-Sachs Disease- cognitive disorder, deafness,
  paralysis

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Ubiquitin-dependent regulation of Ubp6
Hanna, J et al Cell 12799-111 2006
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Ubiquitin-dependent regulation of Ubp6 levels
Hanna, J et al Cell 12799-111 2006
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Altered proteasome content in yeast expressing
Ubp6C118A
Hanna, J et al Cell 12799-111 2006
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Cellular responses to ubiquitin deficiency and
proteasomal stress
Hanna, J et al Cell 12799-111 2006
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Proteasome inhibition increases Usp14
ubiquitin-hydrolase activity
Usp14
Uch37
Borodovsky, A et al EMBO J. 205187-96 2001
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The proteasomal DUB Usp14 impairs protein
degradation
Lee, BH et al Nature 467179-84 2010
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Decrease steady-state levels of aggregate prone
proteins in the absence of Usp14
Lee, BH et al Nature 467179-84 2010
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Proteasome activity can be modulated by Uch37,
Rpn11 and Usp14
Proteasomal DUB functions in yeast 1) Rpn11-
cleaves near base of chain to
remove ubiquitin chains en bloc
2) Usp14 - recycling of residual
ubiquitin conjugates from proteins
entering the proteasome,
ubiquitin chain editing and regulation of
proteasome activity 3)
Uch37- ubiquitin chain editing
Mouse models 1- Rpn11-
unknown but likely lethal 2-
Usp14- KO embryonic lethal (E14)
hypomorphic allele viable
3- Uch37 unknown


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Ubiquitin is not the only small peptide to be
covalently attached to proteins and or lipids

• SUMO 1/2
• Nedd8
• ISG15
• ATG8
• FAT10
• Not thought to target proteins for destruction
• Each is thought to have its own conjugation and
  deconjugation system

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