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Use of Destabilizing Domains in Protozoa

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Title: Use of Destabilizing Domains in Protozoa


1
Use of Destabilizing Domains in Protozoa
  • Nature Methods (2007) Armstrong et al. (P.
    falciparum) and Herm-Götz et al. (T. gondii)

2
New tools are needed for studying the biological
roles of proteins in protozoa
  • Homologous recombination not feasible for
    deleting essential genes.
  • RNA interference is limited to a few protozoa,
    can be nonspecific, slow onset kinetics, and can
    give unpredictable levels of knockdown.
  • In Leishmania tools for inducible expression of a
    gene are limited.
  • The approach described in these papers involves
    attaching a destable fusion protein tag to gene
    of interest.

3
Degradation
Stabilization
Stabilized Form
ligand nontoxic?
works with endogenous targets
Yes
4
EC50 30 nM
EC50 3 nM
5
Mutant form of FKBP12 is rapidly targeted for
degradation
  • Mutations F36V, L106P lead to a destabilized
    fkbp12 protein, that is rapidly degraded
    (t1/2 2h)
  • Degradation predominantly via proteasome, since
    MG132 and lactacystin inhibit degradation.
  • fkbp12 can be stabilized by the addition of
    rapamycin derivative - Shield1

6
Fusion of DD to YFP leads to protein that is
rapidly degraded in the absence of Shld-1 in T.
gondii.
7
.and P. falciparum


8
Shld 1 Suppresses Destabilization in a
Dose-dependent Manner
9
Kinetics of Upregulation and Downregulation are
Rapid
10
Regulation Observed in both Intracellular and
Extracellular Parasites
Shld 1 added at t0
11
Effect of DD on MyoA
Unable to to make a ?myoA in presence of DD-MyoA
1 µM Shld1
12
Conditional Production of Rab1 Dominant Negative
Mutants on Cell Viability
13
Conditional Production of Rab11A Dominant
Negative Mutants and their Effect on Cell
Viability
14
Effect of Rab11A Dominant Negative Mutation on
Invasion and Replication
DN mutants
/- Shld1 20 min
Incubated HFF 3 h
Wash monolayer
Incubate 16 h
Analyze for invasion and replication
15
Shld1 No Effect on RH Parasites Expressing Wild
Type Rab11a
16
Effect of DD on Falcipain 2
17
(No Transcript)
18
Summary
  • DD useful for tool for studying role of protein
    of interest in biology of cell.
  • Kinetics of either upregulation or downregulation
    are fast (h vs. days when using RNAi).
  • Amount of protein produced can be regulated by
    the dose of Shld1 given - on the flipside protein
    stability dependent on a continued source of
    Shld1
  • Technology that could prove useful in Leishmania
    for studying both essential and non-essential
    gene functions.
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