Transcript Paul McCain Presentation

Broad activation of the
ubiquitin–proteasome system by
Parkin is critical for mitophagy
Nickie C. Chan, Anna M. Salazar, Anh H. Pham, Michael J. Sweredoski,
Natalie J. Kolawa1, Robert L.J. Graham, Sonja Hess, and David C. Chan
Human Molecular Genetics, 2011, Vol. 20, No. 9, pgs. 1726-1737
A review by Paul McCain
Discussion Topics
•
Experimental Background
•
Focus of the Study
•
SILAC Analysis
•
Mitochondrial Membrane Degradation
•
Conclusion
•
Future Research
Experimental Background
Parkin is a Parkinson’s disease (PD) related protein.
When mitochondria malfunction Parkin translocates from the cytosol
in order to aid in degrading them via mitophagy.
Parkin is an E3 ubiquitinase that attaches the protein ubiquitin to
malfunctioning mitochondria in a chain linked fashion marking them
for degradation.
Two major pathways of Parkin mediated polyubiquitization and
degradation are reported.
I.
The ubiquitin-proteasome system (UPS), which uses the K48-linked
polyubiquitization of substrate proteins, whose malfunction would lead to an
accumulation of misfolded proteins (Lewy bodies)
II.
The K63-linked polyubiquitization of mitochondrial proteins, which recruits
ubiquitin adaptors of the autophagic machinery.(HDAC6 and p62/SQSTM1)
What was the study’s focus?
This study was focused on better understanding the
function of Parkin and the associated changes to the
mitochondrial proteome in response to Parkin.
Key Concepts
•
K48-mediated UPS pathway’s association with mitophagy
•
26S proteasome’s involvement in mitochondrial outer membrane
protein degradation by the UPS
•
UPS is a necessity of mitophagy and is activated prior
•
UPS inhibition prevents mitophagic function
SILAC
Stable isotope labeling by amino acid culture analysis
(Mass Spec Approach)
•
2 hours of treatment with CCCP depletes mitochondrial
membrane potential in Parkin-expressing HeLa S3 clones.
•
Parkin-dependent mitophagy results
•
2979 different protein groups identified. 1013 mitochondrial
proteins
SILAC Interpretation
•
Parkin increased 13 fold
•
P62/SQSTM1, NBR1, LC3 and GABARAPL2 autophage related proteins
highly elevated
•
V-type proton ATPase increased (component of endosomes and
lysosomes)
•
Drp1 had increased output (involved in mitochondrial
fragmentation)
•
UPS system induced: 9 fold increase in ubiquitin and increased
proteosome subunits 19S and 20S of the 26S proteosome
•
Outer membrane proteins reduced (Mfn1, Mfn2, Miro1 and Miro2)
•
Tom70 substantially reduced
Mitochondrial Outer Membrane Degradation
Figure 1A:
Immunoblotting revealed rapid
degradation of Mfn1, Mfn2 and
Tom70 after CCCP treatment of
Parkin-expressing cells after
approximately one hour
VDAC1 (solute transport), Fis1
(mitochondrial fission), Bak
(cellular apoptosis) and Tom20
(protein import) also reduced but
over a longer time period
Intermembrane and matrix proteins
of mitochondria showed no
significant changes
Opa1 has short and long isoforms. The long isoform (outer membrane) is reduced while the short isoform (inner
membrane) is nearly unchanged
Figure 1B and 1C: Treatment with MG132 or epoxomicin proteosome inhibitors before CCCP treatment inhibited
membrane degradation
These results indicate that Parkin is activated by mitochondrial membrane depolarizations and mediates
proteosome-dependent outer membrane degradation.
Mitochondrial Membrane is Degraded Independent of Autophagy
Atg3 is similar to the E2 enzyme
In Atg3-null MEFs do not show conjugation of AT8/ LC3 to phosphatidylethanolamine, indicating the autophagic pathway
has failed to activate.
The addition of epoxomicin blocked membrane degradation
This indicates that Parkin degrades the outer-membrane proteins via the UPS
UPS Up Regulation
Figure A:
SILAC ratio results for K48 and K63linked polyubiquitinization
Figure B:
Anti-polyubiquitin K48 and K63
antibody expression is prevalent in
mitochondria of Parkin expressing
cells upon CCCP treatment
Figure C:
26S β subunits of the proteasome
stained. Upon CCCP treatment the
proteosame is centered upon
mitochondria.
Figure D:
Same as Figure C only using the α
subunit.
Temporal Relationship: Outer Membrane Degradation and Mitophagy
Tom20 outer membrane protein is compared
with Hsp60 of the mitochondrial matrix.
Figure A-B:
Parkin-expressing cells treated with CCCP
segregated into two distinct populations.
Perinuclear mitochondria were aggregated in
nuclear exterior and retained both Tom20 and
Hsp60. Peripheral mitochondria showed no
aggregation and no change in Hsp60 but a loss
off Tom20.
Figure C:
% of Tom20 negative cells that were Hsp60
positive after 24 hours.
Figure D:
Outer membrane proteins were measured in
wild-type MEFs and atg-null MEFs after CCCP
treatment and were shown to be degraded in a
similar fashion while Hsp60 was virtually
unaltered.
Parkin- Mediated Mitophagy Requires the UPS
LC3B is an autophagosome marker
Figure A:
Dispersed mitochondria stained for LC3B
show and Hsp60
Figure B:
MG132 proteasome inhibitor prevents the
loss of Tom20 as a result of CCCP
treatment..
Figure C- D:
12 and 24 hr recovery after 100min pulse
of CCCP
Figure E:
Parkin expressing human neuroblastoma
SH-HY5Y cells treated with CCCP for 4
hours
Figure F:
Epoxomicin prevents mitophagy due to
CCCP treatment .
Epoxomicin blocks mitophagy in CCCP treated cells
EGFP marked cells used to indicating mitofusins may help in mitochondrial
segregation but more proteasome remodeling, including proteolysis, is necessary
for mitophagy.
Conclusion
Parkin is part of a mitochondrial quality control
mechanism
Parkin is part of two distinct degradative mechanisms,
the UPS and autophagy which are linked in
mitochondrial control
Outer membrane degradation by Parkin via the UPS is a
signal for autophagosomes to target mitochondria
Outer membrane degradation by the UPS causes
dispersal of mitochondria which leads to
autophagosome targeting and engulfment
Future Research
Authors suggestions:
Future research on mitophagy will require a matrix protein marker for
accurate assessment.
Investigate the UPS role in other forms of autophagy.
Does Parkin mediated activation of UPS have other functions for mitochondria
besides autophagy?
Other questions:
Why were the Atg-null mutants not tested with the antibodies for K-48 and K63?
What signals causes mitochondrial aggregation and dispersal? Tom20
degradation or another protein?