In vivo model for proteasome activity studies

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In vivo model for proteasome activity studies

• Olli Matilainen
• Tartu C. elegans course
• 28.8.2008

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Holmberg-Still LabHow is proteasome activity
regulated?
Molecular and Cancer Biology Program and
Institute of Biomedicine University of Helsinki
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Father of C. elegans research
Sydney Brenner The Molecular Sciences Institute
Berkeley, CA, USA
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The C. elegans hermaphrodite cell lineage
Kipreos, Nat.Rev. Mol.Cell Biol. 2005
The cell number (959 somatic cells) the
positions of cells are constant
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Model organism C.elegans

• life span of 2-3 weeks
• generation time of 2,5 d (25 ?C)

• -around 1mm long
• 959 cells
• mostly hermaphrodites

pictures modified from Worm Anatomy
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Why we use C.elegans

• Ubiquitin-proteasome system and many other
  signalling pathways are conserved
• Possibility to study ubiquitin-proteasome pathway
  in whole animal level
• Short lifetime
• Transparent
• 959 cells
• genetics
• RNAi libraries

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Ubiquitin-proteasome system

• Programmed protein degradation is mainly managed
  by ubiquitin-proteasome system
• Able to irreversibly shutdown function of a
  selected protein molecule

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Ubiquitin-proteasome system
Human
C.elegans

• 1 E1
• 3 dozen E2s
• gt 500
• E3s

• 1 E1
• 22 E2s
• gt 200
• recognized

• There are four major classes of ubiquitin ligases
  (E3s) HECT-domain proteins U-box proteins
  monomeric RING finger proteins and multisubunit
  complexes that contain a RING finger protein

Nalepa G. et al., 2006
Wolf DH. Hilt W., 2004
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Proteasome
In C. elegans 14 conserved 20S subunits 18
conserved 19S subunits
Modified from Wolf Hilt, Biochim. Biophys.
Acta, 2004
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C. elegans proteasome subunit interaction network
A and B Two-hybrid protein network of the C.
elegans 26S proteasome C Model for the 26S
proteasome
Davy A. et al. EMBO reports (2001)
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Proteasome regulation still mainly unknown

• Proteasome is dynamic and not a static molecular
  machine as previously thought
• It is known that proteasome activity fluctuates,
  but it is still mainly unknown how these changes
  are regulated
• Amount of active proteasomes
• Subunit composition
• Peptidase activity

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Holmberg-Still Lab Our project

• To study how proteasome activity is regulated
  during the life time of multicellular organism
• aging
• genetic factors
• environmental stress

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Methods to study proteasome activity

1. Fluorescent reporters
2. Affinity purification of proteasome
3. In vitro proteasome activity assays

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How to study proteasome activity in vivo?

• Fluorescence protein based live imaging system
• YFP
• Dendra2
• Polyubiquitin binding reporter

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Principle of the proteasome activity live imaging
Ubiquitin-dependent degradation
enhanced
CL1
FP
YFP
normal
Ubiquitin-independent degradation
impared
MODC
FP
Unique opportunity to study proteasome activity
in specific tissues and individual cells over
time throughout the lifetime of individual worms
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Microinjection for making transgenic animals
agar pad on glass slide
Look for transgenic progeny
Axiovert 200
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Transgenic worm expressing YFP-CL1 reporter
construct
200 µm
Pdpy-30YFP-CL1 Pmyo2CFP
Normal
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RNAi in C. elegans
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RNA interference targeting proteasome ß5 subunit
induces YFP-CL1 accumulation
L4440
pbs-5 (RNAi)
L4440
Pbs-5 (RNAi)
Poly-ubiquitinated proteins
a-tubulin
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Heat stress induces transient YFP-CL1 accumulation
37C, 2h
recovery
20C
2h
6h
24h
0h
Poly-ubiquitinated proteins
a-tubulin
(made by Yuehong Yang)
Before HS
After HS
21 h Recovery
2 h HS at 37 oC
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How to study proteasome activity in vivo?

• Fluorescence protein based live imaging system
• YFP
• Dendra2
• Polyubiquitin binding reporter

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Dendra2 based reporter system

• Sensitivity
• Possibility to follow a subset of proteins in
  single cell or in whole animal
• Newly synthesized proteins are not affecting
• Dual-color photoswitchable monomeric fluorescent
  protein
• Gurskaya NG, et al. Nat Biotechnol. 2006
• Irreversible photoconversion from green to red
  fluorescent form with intense 405 nm or 488 nm
  light irradiation

Normal proteasome function
photoconversion
Proteasome impairment
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Measuring degradation (proteasome activity) and
synthesis of short-living Dendra2 construct
degradation
synthesis
tconversion
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Dendra2 activation in C. elegans muscle cells
5 agar pad on glass slide
Zeiss LSM 5 Duo
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Photoconversion of Dendra2 in muscle cells
before conversion
after conversion
Photoconverted with 405 nm laser
green channel
overlay after conversion
red channel
Punc-54Dendra2
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How to study proteasome activity in vivo?

• Fluorescence protein based live imaging system
• YFP
• Dendra2
• Polyubiquitin binding reporter

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Polyubiquitinated proteins accumulate in cells
NATURE VOL 417 9 MAY 2002
Red Polyubiquitinated proteins Blue Hoechst
HeLa cells
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UIM2
UIM2
FP
FP
UIM2
FP
UIM2
FP
Interaction between the reporter construct and
polyubiquitinated proteins stabilizes reporter
following increased fluorescence ? in vivo
reporter system to detect accumulation of
polyubiquitinated proteins
FP
UIM2
FP
FP
FP
FP
UIM2
UIM2
UIM2
UIM2
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Ubiquitin interactors

• Lys48-linked polyubiquitin conjugates are the
  proximal substrates of proteasomal proteolysis,
  so their steady-state abundance in the cell
  should reflect proteasome function in cells or
  tissues without the use of artificial reporters
• Bennet, EJ. et al. Nature (2007).

Raasi, S. et al. Nature Structural Molecular
Biology (2005).
hRAD23A UBA2
RPN-10 and S5a UIMs
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Ubiquitin interactor fusions in cell cultures
S5a UIM2-ZsGreen
S5a UIM1-ZsGreen
ZsGreen
ZsGreen
UIM
UIM
NH2
CO2H
UIM
CO2H
NH2
hRAD23A UBA2-ZsGreen
ZsGreen
NH2
CO2H
UBA2
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S5a-ZsGreen reporter recognizes polyubiquitin
accumulations
S5a UIM2-ZsGreen
ZsGreen
1 uM MG132 8h
1 uM MG132 8h
Green S5aUIM2-ZsGreen /ZsGreen Red
Polyubiquitinated proteins Blue Hoechst
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Proteasome inhibition lead to accumulation of
short-living S5aUIM2-ZsGreen reporter

• 2 uM MG132 8h

MG132 free medium 10h
Before bleach
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FRAP (Fluorescence Recovery After Photobleaching)
Methodology
Nature Reviews Molecular Cell Biology 2
898-907 (2001) KINETIC MODELLING APPROACHES TO IN
VIVO IMAGING
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UIM2-ZsGreen reporter is soluble in mammalian
cells when interacting with polyubiquitinated
proteins
Before bleach
Bleach
11s recovery
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UIM2-ZsGreen reporter interacts with accumulated
polyubiquitin in C. elegans muscle cells
Red FK1 Alexa594 Green UIM2-ZsGreen
Punc-54UIM2-ZsGreen-MODC
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UIM2-ZsGreen reporter is soluble in C. elegans
muscle cell when interacting with
polyubiquitinated proteins
Before bleach
After bleach
10 s recovery
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Summary

• Protein degradation is mainly managed by
  ubiquitin proteasome system
• Ubiquitin-proteasome pathway is very complex, but
  it is a well conserved system
• We have generated fluorescent reporters which can
  be used to study ubiquitin-proteasome system in
  vivo.

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Ongoing things and future plans

• Establish and characterize stable transgenic
  lines and to develop more sensitive live imaging
  system to study proteasome activity
• Genome-wide RNAi screens to identify genetic
  factors that affect proteasome function
• Mechanism of function
• single genes
• signalling pathways

2-3 days
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Holmberg-Still laboratory
Jin Congyu
Geert Hamer
Carina Holmberg-Still
Olli Matilainen
Yuehong Yang
Leena Arpalahti
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Acknowledgements

• Holmberg-Still lab
• Marikki Laiho lab
• Risto Renkonen lab and Medicel
• Molecular Imaging Unit
• Funding
• Helsinki Biomedical Graduate School
• Nordforsk Nordic C.elegans Network

Biomedicum Helsinki