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Title: In vivo model for proteasome activity studies


1
In vivo model for proteasome activity studies
  • Olli Matilainen
  • Tartu C. elegans course
  • 28.8.2008

2
Holmberg-Still LabHow is proteasome activity
regulated?
Molecular and Cancer Biology Program and
Institute of Biomedicine University of Helsinki
3
Father of C. elegans research
Sydney Brenner The Molecular Sciences Institute
Berkeley, CA, USA
4
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
5
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
6
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

7
Ubiquitin-proteasome system
  • Programmed protein degradation is mainly managed
    by ubiquitin-proteasome system
  • Able to irreversibly shutdown function of a
    selected protein molecule

8
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
9
Proteasome
In C. elegans 14 conserved 20S subunits 18
conserved 19S subunits
Modified from Wolf Hilt, Biochim. Biophys.
Acta, 2004
10
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)
11
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

12
Holmberg-Still Lab Our project
  • To study how proteasome activity is regulated
    during the life time of multicellular organism
  • aging
  • genetic factors
  • environmental stress

13
Methods to study proteasome activity
  1. Fluorescent reporters
  2. Affinity purification of proteasome
  3. In vitro proteasome activity assays

14
How to study proteasome activity in vivo?
  • Fluorescence protein based live imaging system
  • YFP
  • Dendra2
  • Polyubiquitin binding reporter

15
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
16
Microinjection for making transgenic animals
agar pad on glass slide
Look for transgenic progeny
Axiovert 200
17
Transgenic worm expressing YFP-CL1 reporter
construct
200 µm
Pdpy-30YFP-CL1 Pmyo2CFP
Normal
18
RNAi in C. elegans
19
RNA interference targeting proteasome ß5 subunit
induces YFP-CL1 accumulation
L4440
pbs-5 (RNAi)
L4440
Pbs-5 (RNAi)
Poly-ubiquitinated proteins
a-tubulin
20
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
21
How to study proteasome activity in vivo?
  • Fluorescence protein based live imaging system
  • YFP
  • Dendra2
  • Polyubiquitin binding reporter

22
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
23
Measuring degradation (proteasome activity) and
synthesis of short-living Dendra2 construct
degradation
synthesis
tconversion
24
Dendra2 activation in C. elegans muscle cells
5 agar pad on glass slide
Zeiss LSM 5 Duo
25
Photoconversion of Dendra2 in muscle cells
before conversion
after conversion
Photoconverted with 405 nm laser
green channel
overlay after conversion
red channel
Punc-54Dendra2
26
How to study proteasome activity in vivo?
  • Fluorescence protein based live imaging system
  • YFP
  • Dendra2
  • Polyubiquitin binding reporter

27
Polyubiquitinated proteins accumulate in cells
NATURE VOL 417 9 MAY 2002
Red Polyubiquitinated proteins Blue Hoechst
HeLa cells
28
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
29
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
30
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
31
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
32
Proteasome inhibition lead to accumulation of
short-living S5aUIM2-ZsGreen reporter
  • 2 uM MG132 8h

MG132 free medium 10h
Before bleach
33
FRAP (Fluorescence Recovery After Photobleaching)
Methodology
Nature Reviews Molecular Cell Biology 2
898-907 (2001) KINETIC MODELLING APPROACHES TO IN
VIVO IMAGING
34
UIM2-ZsGreen reporter is soluble in mammalian
cells when interacting with polyubiquitinated
proteins
Before bleach
Bleach
11s recovery
35
UIM2-ZsGreen reporter interacts with accumulated
polyubiquitin in C. elegans muscle cells
Red FK1 Alexa594 Green UIM2-ZsGreen
Punc-54UIM2-ZsGreen-MODC
36
UIM2-ZsGreen reporter is soluble in C. elegans
muscle cell when interacting with
polyubiquitinated proteins
Before bleach
After bleach
10 s recovery
37
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.

38
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
39
Holmberg-Still laboratory
Jin Congyu
Geert Hamer
Carina Holmberg-Still
Olli Matilainen
Yuehong Yang
Leena Arpalahti
40
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
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