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Hodsdon Laboratory -- Projects |
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Endocytosis is a cellular process that
involves the internalization of portions of the plasma membrane together with
extracellular material via vesicles and tubules that pinch-off from the cell
surface. Endocytosis is of fundamental importance to cell function and is
needed, for example, to maintain steady state area of the plasma membrane, to
internalize and down regulate receptors, to take up nutrients, to internalize
and kill pathogens. In some cases, endocytosis is also used by intracellular
pathogens to gain access to the cytosolic space. Formation of an endocytic
vesicle, and then its progression along the endocytic pathway toward recycling
or toward fusion with lysosomes, requires a complex interplay of peripheral and
intrinsic membrane proteins as well as of membrane lipids. The long-term goal
of Dr. DeCamilli's laboratory is to dissect the molecular mechanism underlying this
process, in particular at nerve terminals, where endocytic recycling is used by
cells to regenerate synaptic vesicles after each cycle of exocytosis.
Dr. DeCamilli has had a major role in
demonstrating that a class of membrane lipids, the phosphorylated metabolites
of phosphatidylinositol, the phosphoinositides, play a particularly important
role in the regulation of traffic along the endocytic pathway (55-61). Following-up on these findings, they are using reverse mouse genetic, cell biology studies in vitro and
structural studies to elucidate the function of specific phosphoinositide
metabolizing enzymes implicated in endocytic traffic. Current structural
projects are focused on two phosphoinositide 5-phosphatases, OCRL and INPP5b.
These two enzymes are proposed to be recruited at endocytic sites and then to
change the lipids identity of newly formed vesicles by the dephosphorylation of
PI(4,5)P2 and PI(3,4,5)P3. However, the precise mechanisms underlying the
recruitment of these enzymes, the regulation of their enzymatic activities and
the control of their lipid substrate specificities are still elusive. This
topic has important medical relevance because mutations in OCRL have been shown
to cause OculoCerebroRenal Syndrome of Lowe, an X-linked disorder characterized
by congenital cataracts, mental retardation, neonatal hypotonia, and renal
Fanconi syndrome. Mutations in the same gene have also been identified in a
subset of patients with a clinical diagnosis of Dent disease, which like Lowe
syndrome is associated with loss of low molecular weight proteins and
electrolytes in the urine.
The Hodsdon Laboratory is assisting in the above effort to characterize the structural properties and interactions of different domains of OCRL and INPP5b using NMR spectroscopy. We have identified novel domains in the NH2-terminal region of both OCRL and INPP5b and solved their tertiary structures using NMR. We discovered that the NH2-terminal region of both OCRL and INPP5b contain a pleckstrin homology (PH) like domain. Since PH domains are typically involved in a complex array of protein-protein and/or protein-lipid interactions, we plan to further characterize their properties using NMR methods. For example, to test whether these domains can interact with lipids, we plan to monitor NMR spectrum changes upon incubation with micelles containing various lipids. With NMR techniques, we also plan to assess interaction of these modules with their protein-binding partnerss. In the future we plan to perform similar studies on protein module of other phosphoinositide metabolizing enzymes implicated in membrane traffic, including other inositol 5-phophatases, as well as on a variety of endocytic proteins.
Figure: Backbone ribbon diagrams of
the N-terminal domains from OCRL
(left) and INPP5b (right), as determined using solution NMR spectroscopy, demonstrating a pleckstrin-homology (PH domain)
topology.
