Non-apoptotic role of caspase 3 in endosomal remodeling

  • Santambrogio, Laura (PI)
  • Hendricks, Marvin (PI)

Project: Research project

Project Details

Description

In immature bone marrow derived dendritic cells (BMDC) caspase 3 acting in a non-apoptotic pathway
controls the processing of molecules required for endosomal remodeling into tubular structures. This
activity allows delivery of proteins involved in antigen presentation to the cell membrane. The importance of
caspase 3 in this process has been previously analyzed using caspase inhibitors. In this application it will
be further characterized by analyzing DC from caspase 3-/- mice or after "in vitro" silencing of caspase 3 in
immature BMDC using Lentiviral vectors. "In vivo" experiments are also proposed where caspase 3 will be
silenced in CD11c positive cells only and the impact on the immune response analyzed. Levels of active
caspase 3 in immature BMDC are very low (5-8% of what observed during apoptosis). The hypothesis that
the low amount of caspase 3 activity is selectively contained at sub-cellular locations will also be
determined using confocal and electron microscopy as well as sub-cellular fractionation.
"In situ" trapping will be performed to pull down the molecular form of active caspase 3 since western
blotting indicates that in bone marrow dendritic cells a 29 kDa form of caspase 3 is present in addition to
the 32 kDa caspase 3 pro-enzyme. The hypothesis that the 29-kDa form is either functionally active, per se,
or more easily cleavable will be evaluated using in situ trapping and functional overexpression. Finally the
biochemical characterization of caspase 3 activity on target proteins (AP-1 and dynamin) will be evaluated
using gel filtration, site-directed mutagenesis and overexpression experiments with the ultimate goal of
understanding how truncated or full length proteins are functionally involved on endosomal remodeling in
maturing BMDC.
Mature dendritic cells are the most powerfull initiator of an immune response. Thus any molecule interfering
with the maturation process will have a profound impact on their immunological function. We previously
found that low levels of caspase 3 activity is important in maintaining dendritic cells in an immature state.
The major focus of this proposal will be to further our knowledge on caspase 3 control of dendritic cell
maturative processes.
StatusFinished
Effective start/end date6/25/873/31/09

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)