Welcome to the (Extended)Cascio Lab Homepage
Welcome to the (Extended)
Cascio Lab Homepage
STANDARD DISCLAIMER: Page still under construction.
CAST: (From left to right)
The main focus of the laboratory is the correlation between structure and function for membrane proteins, primarily neuroreceptors. Current lab projects include characterization of the glycine receptor, expression of NMDA receptors, as well as computational studies of the T cell receptor, human G-CSF receptor, and zinc metalloendoproteinases. For a more detailed description of each project, select the links below.
Molecular modeling
Glycine Receptor Structure
and Function
In rat spinal cords, the glycine receptor (GlyR) is a heteromeric pentamer
composed of alpha (48kD) and beta (58kD) subunits. Our lab has succesfully
overexpressed a homomeric channel of recombinant human alpha 1 subunits,
utilizing a baculovirus system. Due to its homogeneous composition and our
ability to generate large quantities of pure channels in vesicles, GlyR
alpha 1 is an excellent candidate for structural studies. Also, GlyR has
a high degree of homology to members of the ligand-gated channel (LGC) superfamily,
some of which have been implicated in neuromuscular and psychological disorders
(e.g. nicotinic acetylcholine receptor, glutamate receptor). We feel that
GlyR may serve as a structural paradigm for the LGC superfamily.
In lieu of crystal structure analysis, which is difficult with integral
membrane proteins such as GlyR, alternative methods for structure analysis
must be employed. In particular, limited proteolysis is a useful tool in
elucidating topology and obtaining initial structural information. We are
currently using reverse-phase HPLC, MALDI-TOF MS plus tandem MS to analyze
peptides generated from proteolysis. Our goal is to identify potential "hot-spots"
for proteolytic cleavage as well as regions that cleave poorly. We also
aim to identify regions of GlyR that are membrane integrated.
In addition to elucidating the topology of GlyR alpha 1, we are also currently
identifying peripheral cytoplasmic proteins which mediate channel clustering.
Clustering is an essential process which guides GlyR to the post-synaptic
membrane of neuronal cells. We aim to identify proteins responsible for
the regulation of this process as well identify the binding domains involved.
Publications:
Overexpression and Characterization of
the NMDA Receptor
The glutamate receptor (GluR) plays a key role in brain function. In
the central nervous system (CNS), most rapid excitatory synaptic transmission
is mediated by these channels. Dysfunction of the glutamatergic pathways
has been implicated in progressive degenerative diseases such as Alzheimer's
disease (AD), Huntington's disease, Parkinson's disease, amyotrophic lateral
sclerosis, lathyrism, and AIDS encephalopathy and dementia complex, as well
as in schizophrenia and other psychiatric disorders. Excitatory amino acids
which normally participate in signaling in the CNS, when present at elevated
concentrations are neurotoxic, and have been implicated in both acute injury,
such as caused by epileptic seizure or hypoxia, and during periods of ischemia
and hypoglycemia. Glutamate interacts with at least three related classes
of ionotropic receptor channels, each commonly referred to by their preferred
pharmacological agonists: NMDA, AMPA, and kainate. These receptors have
been implicated in learning and memory acquisition. We have focused our
studies on the NMDA receptor.
The NMDA subtype of the glutamate receptor family is an excitatory neurotransmitter
receptor whose subunits comprise a generally nonselective cation channel
and exhibits voltage dependent blockade by Mg2+. Unlike AMPA and KA receptors,
the NMDA receptor is also permeable to Ca2+, and the resultant increased
intracellular Ca2+ in neuronal cells upon gating is thought to be responsible
for evoking the receptor's role in neuronal plasticity and neurotoxicity.
Glycine is a coagonist, and essential for receptor activation. While it
is known that the pathogenesis of many neurodegenerative conditions and
psychiatric disorders is mediated in some way by the NMDA receptors, little
fundamental information is available at the basic level of receptor function.
There is a desperate need for effective prophylaxis and therapy in acute
and chronic neurodegenerative disorders that involve excitotoxic mechanisms.
Like most mammalian neuroreceptors, the NMDA receptor is produced by the
cells in very low abundance, making biochemical and biophysical studies
difficult. We propose to develop a baculovirus expression system in order
to provide ample material for subsequent studies investigating the structure,
topology, and biochemistry of this physiologically relevant macromolecule.
Modeling of TCR-Mucin
Mucin, a tumor-specific amtigen, interacts with the T Cell Receptor (TCR)
in an MHC-unrestricted manner. In tranformed epithelial cells, mucin is
expressed on the entire cell surface. Underglycosylation exposes a unique
epitope (with the sequence PDTRP) of the tandem repeat of mucin. In collaboration
with the laboratory of Dr.
Olja Finn, we are currently modeling the interaction of mucin with the
TCR.
The figure at left illustrates the interaction of mucin (shown as a stick
figure) with a variable domain of SM3, an antibody specific for mucin expressed
on breast tumor. The electrostatic potential has been mapped to the surface
of the SM3 antibody (with positive charge shown as blue, negative as red).
The knob-like epitope of the tandem repeats of mucin interacts with the
cleft defined by the CDR loops of the antibody. The structure of mucin was
solved by nmr spectroscopy (Fontenot et al. (1995) J. Bimol. Struct. Dynamics
12:245-260) and the antibody was modeled on homologous varaible light and
heavy chains. The energy was minimized and dynamics were run with AMBER
snd this figure was made with GRASP.
Recently, Mark Alter, an M.D./Ph.D. student in the Finn lab, cloned a T
cell receptor specific for mucin from a cytotoxic T lymphocyte line which
shows mucin-specific killing. We are currently modeling the receptor based
on the SM3-mucin interaction as well as the recently published structures
of the T cell receptor.
Modeling of EP 24.15
The mammalian Zn-endoproteinase EP 24.15, is a crtitical regulatory enzyme
of the neuro-endocrine axis since it proteolytically processes many bioactive
peptides. In collaboration with Dr.
Marc Glucksman at the Fishberg Research center for Neurobiology at Mt.
Sinai School of Medicine, we are modeling the active site of this enzyme
using other well characterized Zn-metalloendoproteinases (e.g. thermolysin,
neutral protease) as templates.
The figure at left was generated using MOLSCRIPT and shows a backbone ribbon
model of the active site of the enzyme as well as conserved residues which
are either involved in coordination with the Zn ion or important in catalysis.
Modeling the interaction of Stat3 with
human G-CSF receptor
The granulocyte colony-stimulating factor (G-CSF) receptor is a member
of the cytokine receptor family, and act in signal transduction involved
in differentiation and proliferation. Upon binding of ligand, dimerization
of the receptor results in activation of protein Tyr kinases, and subsequent
recruitment of SH2-containing proteins, such as Stat3. In collaboration
with Dr. Dave Tweardy
we are modeling the interaction of G-CSF phosphotyrosine ligands with Stat3
based on the known structure of many SH2 domains.
The figure at left was generated using GRASP, and was derived from the crystal
structure of v-src with bound ligand.
Wait, there's more...
For a more intimate look at the sordid interests of
the lab, follow this link.