Use of Destabilizing Domains in Protozoa

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Use of Destabilizing Domains in Protozoa

• Nature Methods (2007) Armstrong et al. (P.
  falciparum) and Herm-Götz et al. (T. gondii)

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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.

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Degradation
Stabilization
Stabilized Form
ligand nontoxic?
works with endogenous targets
Yes
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EC50 30 nM
EC50 3 nM
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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

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


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Shld 1 Suppresses Destabilization in a
Dose-dependent Manner
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Kinetics of Upregulation and Downregulation are
Rapid
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Regulation Observed in both Intracellular and
Extracellular Parasites
Shld 1 added at t0
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Effect of DD on MyoA
Unable to to make a ?myoA in presence of DD-MyoA
1 µM Shld1
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Conditional Production of Rab1 Dominant Negative
Mutants on Cell Viability
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Conditional Production of Rab11A Dominant
Negative Mutants and their Effect on Cell
Viability
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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
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Shld1 No Effect on RH Parasites Expressing Wild
Type Rab11a
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Effect of DD on Falcipain 2
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(No Transcript)
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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.