Intracellular Protein DegradationThe lysosome and Ubiquitin Proteasome
System
Scott Wilson
Department Neurobiology
5-5573
Wilson@nrc.uab.edu
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
 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)
The question:
Is there turnover of cellular constituents? Or is
food intact a function primarily for energyproviding (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
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!
 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.
Why are proteins degraded?
 Quality control
 Proteins become denature/misfolded/damaged
 Elevated temperatures (37°C)
 Proteins being synthesized are folded incorrectly
 Regulation of biological pathways
 Cell cycle
 Receptor mediated endocytosis
 Synaptic remodeling
Now that we know proteins are in
a “dynamic state” in cells….
 How are proteins degraded within cells?
 Is protein degradation regulated?
 Selective?
 Compartmentalized?
The discovery of the lysosome
 De Duve discover the lysosome in the 1950’s
 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.
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
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
Lysosomes use an H+ ATPase pump in the
membrane to generate acidic pH
Overview of lysosomal trafficking
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
Pathways into the Lysosomal/vacuolar System
1
3
2
4
4
Model of the mechanism for
multivesicular endosome formation
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
Autophagy pathway
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?
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
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
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
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
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?
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 ATPdependent proteolysis factor
 Critical finding was that APF-1 can be
covalently attached to a target substrate
APF-1 is shifted to high molecular
mass compounds following
addition of ATP to the fraction I.
125I labeled fractions following
gel-filtration chromatography
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-1’s can be added to a substrate
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
Studies of fraction II defined the ubiquitin conjugation
machinery
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.
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?
Structure of the 26S proteasome
 Tanaka et al discovered a highmolecular 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
Subunits of the 26S proteasome
 19S regulatory particlecomposed of
approximately 20 different
proteins
 20S core particlecomposed of 14 different
subunits (1-7 and 1-7)
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
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).
The UPS is enormous!
The genes of the UPS constitutes ~5% of the genome




E1’s- 1-2 activating enzymes
E2’s- 10-20 conjugating enzymes
E3’s- 500-800 ubiquitin ligase- drives specificity
DUBs- 100 ubiquitin specific proteases- regulators of pathway
Pathways controlled by regulated proteolysis
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
Ubiquitin-dependent regulation
of Ubp6
Hanna, J et al
Cell 127:99-111
2006
Ubiquitin-dependent regulation
of Ubp6 levels
Hanna, J et al
Cell 127:99-111
2006
Altered proteasome content in
yeast expressing Ubp6C118A
Hanna, J et al
Cell 127:99-111
2006
Cellular responses to ubiquitin
deficiency and proteasomal stress
Hanna, J et al
Cell 127:99-111
2006
Proteasome inhibition increases Usp14
ubiquitin-hydrolase activity
Usp14
Uch37
Borodovsky, A et al
EMBO J. 20:5187-96
2001
The proteasomal DUB Usp14
impairs protein degradation
Lee, BH et al
Nature 467:179-84
2010
Decrease steady-state levels of aggregate
prone proteins in the absence of Usp14
Lee, BH et al
Nature 467:179-84
2010
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
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
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