Transcript Lecture 20 Protein degradation

QUESTION
DYSFUNCTIONAL
PROTEIN DEGRADATION
?
NEURODEGENERATION
NEURODEGENERATION
associated with
Alzheimer’s Disease
Parkinson’s Disease
Huntington’s Disease
Amyotrophic Lateral Sclerosis
Neurodegenerative Disorders
Disease
Parkinson’s Disease
Alzheimer’s Disease
Huntington’s Disease
Amyotrophic lateral
sclerosis
Spinocerebellar Ataxia
accumulation of
misfolded
proteins
Cell
Death
Ubiquitin-Protein Aggregates
HUNTINGTON’S
ALZHEIMER’S
c
d
AD: tau
AD: ubiquitin
f
PD: ubiquitin
PARKINSON’S
LOU GEHRIG’S
Protein Degradation
Turnover of protein is NOT constant
Half lives of proteins vary from minutes to infinity
“Normal” proteins – 100-200 hrs
Short-lived proteins
regulatory proteins
enzymes that catalyze committed steps
transcription factots
Long-lived proteins
Special cases (dentin, crystallins)
Protein Degradation
Proteins are not degraded at the same rate
ENZYME
Ornithine decarboxylase
-Aminolevulinate synthetase
Catalase
Tyrosine aminotransferase
Tryptophan oxygenase
Glucokinase
Lactic dehydrogenase
HMG CoA reductase
half-life
11 minutes
70 minutes
1.4 days
1.5 hours
2 hours
1.2 days
16 days
3 hour
Protein Degradation
• May depend on tissue distribution
Example: Lactic Acid Dehydrogenase
Tissue
Half-life
Heart
1.6 days
Muscle
31 days
Liver
16 days
• Protein degradation is a regulated process
Example: Acetyl CoA carboxylase
Nutritional state Half-life
Fed
48 hours
Fasted
18 hours
Protein Degradation
 Ubiquitin/Proteasome Pathway
80-90%
Most intracellular proteins
• Lysosomal processes
10-20%
Extracellular proteins
Cell organelles
Some intracellular proteins
Two Sites for Protein Degradation
Proteasomes
Large (26S) multiprotein complex (28 subunits)
Degrades ubiquitinated proteins
Lysosomes
Basal degradation – non-selective
Degradation under starvation – selective for “KFERQ”
proteins
The Ubiquitin/Proteasome
PATHWAY
UBIQUITIN
 Small peptide that is a “TAG”
 76 amino acids
 C-terminal glycine - isopeptide
bond with the e-amino group of
lysine residues on the substrate
 Attached as monoubiquitin or
polyubiquitin chains
 Three genes in humans:
Two are stress genes (B and C)
One, UbA as a fusion protein
K
G
Tetra-Ubiquitin
Cook, W.J. et al. (1994) J. Mol. Biol. 236, 601-609
UBIQUITIN GENES
Ubiquitin/Proteasome Pathway
Ubiquitination
Ubiquitination
Degradation by the
26S PROTEASOME
The Ubiquitin/Proteasome
Pathway
Four Main Steps:
UBIQUITINATION
RECOGNITION
DEGRADATION
DEUBIQUITINATION
UBIQUITINATED PROTEINS
UBIQUITIN CHAINS
6
11
27 29 33
MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQ
QRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG
48
63
Functions of Ubiquitination
•Mono-ubiquitination
 Receptor internalization
 Endocytosis – lysosome
 Transcription regulation
• Poly-ubiquitination
 Targets proteins from
Cytoplasm, Nuclear & ER for
degradation by the PROTEASOME
 DNA repair
Ubiquitination of proteins is a FOUR-step process
 First, Ubiquitin is activated by forming
a link to “enzyme 1” (E1).
 Then, ubiquitin is transferred to one
of several types of “enzyme 2” (E2).
 Then, “enzyme 3” (E3) catalizes the
transfer of ubiquitin from E2 to a Lys
e-amino group of the “condemned”
protein.
 Lastly, molecules of Ubiquitin are
commonly conjugated to the protein to
be degraded by E3s & E4s
AMP
UBIQUITIN ACTIVATION
E1
UBIQUITIN
ADENYLATE
THIOL
ESTER
UBIQUITIN CONJUGATION
E1-s-co-Ub + E2-SH ----->
-----> E2-s-co-Ub + E1
N UBC domain C
CLASS 1 – UBC domains only; require E3s for Ub; target
substrates for degradation
CLASS 2 – UBC domains & C-terminal extensions;
UBC2 = RAD6 – DNA repair not degradation; no E3s
CLASS 3 – UBC domains & N-terminal extensions;
function not known
UBIQUITIN LIGATION
E3
“recognins” = recognize a
motif (DEGRON) on a
protein substrate
E2-s-co-Ub + Protein-NH2 ------->
E2-SH + Protein-NH-CO-Ub
(ubiquitin = polyubiquitin chains)
Three Major Classes of E3
3) multi-subunit
cullin based E3s
1) HECT-domain
E3s
2) RING fingerdomain E3s
Ubiquitin Ligases (E3)
1) HECT-domain containing a conserved Cys
2) RING finger-domain
Cys & His residues are ligands to two Zn++ ions
stabilizes a molecular scaffold
Ubiquitin Ligases (E3) (cont.)
3) Complex E3s: Multiple subunits
Ex: SCF-type E3, VBC-Cul2 E3 and
other cullin based E3s,
Anaphase promoting complex (APC)
-they provide a Scaffold
for Ub transfer
-F-box – substrate
recognition
ELONGATION = E4
1) U box = CHIP (+parkin)
2) Non-U box = p300 (p53)
3) E3-E4 complex =
C. elegans
ACTIVATION OF A UBIQUITIN-LIGASE
RECOGNITION
DEGRADATION SIGNALS
substrates
N-end RULE
N-end RULE
N-degron - signal
N-recognin - E3
DEGRADATION
PROTEASOME COMPONENTS
20S
Proteasome
19S
Particle
ATP
26S
Proteasome
19-3
The 26S proteasome
Ubiquitinated proteins are degraded
by the proteasome
 Ubiquitinated proteins are degraded in the
cytoplasm and nucleus by the proteasome.
 Proteasomal protein degradation consumes
ATP.
 The proteasome degrades the proteins to ~8
amino-acid peptides.
 Access of proteins into the proteasome is
tightly regulated.
 The peptides resulting from the proteasome
activity diffuse out of the proteasome freely.
Hydrolysis peptide bonds after:
hydrophobic a.a. =
CHYMOTRYPSINLIKE - 5
acidic a.a. = (-)
CASPASE-LIKE -1
basic a.a. = (+)
TRYPSIN-LIKE -2
DEUBIQUITINATION
De-ubiquitinating
Ubiquitin – like proteins
“UBP”
Small
Ubiquitin-like
Modifier
Ubiquitin – like modifiers
LYSOSOMES
Digestive System of the Cell
• Digests
– ingested materials
– obsolete cell components
• Degrades macromolecules of all types
–
–
–
–
Proteins
Nucleic acids
Carbohydrates
Lipids
• Heterogeneous
Lysosomal
Enzymes
• 50 different degradative
enzymes
• Acid hydrolases
– Active at pH 5
(inside lysosome)
– Inactive if released
into cytosol (pH 7.2)
• Acidic pH of lysosomes
maintained by a proton
pump
in the lysosomal membrane
– Requires ATP,
thus mitochondria
Different pathways lead to the lysosome
1) Phagocytosis
–Cell “eating” of material
> 250nm
2) Pinocytosis
–Cell “drinking”
< 150nm
3) Receptor Mediated
Endocytosis
-clathrin-coated pits
4) Autophagy
–“self eat” of old worn out organelles,
– important in cell degradation during apoptosis
Protein degradation in the lysosomes
 Lysosomes degrade extracellular proteins that the cell
incorporates by endocytosis.
 Lysosomes can also degrade intracellular proteins that
are enclosed in other membrane-limited organellas.
 In well-nourished cells, lysosomal protein degradation is
non-selective (non-regulated).
 In starved cells, lysosomes degrade preferentially
proteins containing a KFERQ “signal” peptide.
 The regression of the uterus after childbirth is
mediated largely by lysosomal protein degradation
AUTOPHAGY
- Macroautophagy – inducible (mTOR)
(autophagy)
- Microautophagy - constitutive
- Chaperone-mediated autophagy
(CMA) – KFERQ motif
AUTOPHAGY
AUTOPHAGY (MACRO) PATHWAY
Oxidative stress
Infection
Protein aggregates
AUTOPHAGY (MACRO) PATHWAY
Lysosome
AUTOPHAGY PATHWAY
17 genes = Atg
1) INDUCTION
TOR
target of Rapamycin
Stress
Tight association
AUTOPHAGY PATHWAY
2) AUTOPHAGOSOME FORMATION
NEW MEMBRANE = ER
LIPID KINASES
(PHOSPHATIDYL
INOSITOL)
SIGNALING COMPLEX
AUTOPHAGY PATHWAY
3) DOCKING & FUSION
MEMBRANE ASSOCIATION
phosphatidylethanolamine
PROTEIN CONJUGATION SYSTEM
~ TO THE UBIQUITIN SYSTEM
AUTOPHAGY PATHWAY
4) BREAKDOWN
RECYCLING
&
RETRIEVAL
Only Atg19 & Atg8-PE
remain associated with the
Autophagosome;
Others are re-cycled
AUTOPHAGY PATHWAY

Neurodegenerative diseases
- PD, AD, HD and TSE
- aggregate removal
 Infectious diseases
- remove pathogens
 Cancer
- sequester damaged organelles
- promote autophagic death
Degradation of Proteins
80-90%
26S PROTEASOME
10-20%
AUTOPHAGY/LYSOSOME
PATHWAY
Ubiquitin-Protein Aggregates
Why?  26S Proteasome
Environmental &
Genetic Insults
Inflammation
Aging
Ub
Ub
U
b
Ub
Ub
Ub
Ub
U
b
U
b
Ub U
b
Ub
AGGREGATES
SUBSTRATE
Ub
AUTOPHAGY
INDUCTION
Ub Ub
Ub
CROSS-TALK
?
Lysosome
26S Proteasome
The End
AUTOPHAGY & UPP
Human neuroblastoma SK-N-SH cells
&
Rat Spinal Cord Organotypic Cultures
Model for ALS
ENVIRONMENTAL &
GENETIC INSULTS
Other ?
CELL
DEATH
UPP 
 Protein
Aggregates
?
 AUTOPHAGY
p62/SQSTM1
HDAC6
 Protein
Degradation
CELL RECOVERY
Neurodegeneration = ubiquitin inclusions
ALZHEIMER’S Disease
HUNTINGTON’S Disease
c
d
AD: tau
AD: ubiquitin
f
PD: ubiquitin
PARKINSON’S Disease
Amyotrophic Lateral Sclerosis