Project Details
Description
The broad, ling-term objectives of this research proposal are to develop
general methods for protein structure analysis by NMR and to expand
our understanding of the details of molecular recognition in protein:
RNA complexes. This is a long-term project combining expertise in
moleculr biology, molecular biophysics, and modern NMR
spectroscopy. The work focuses on NMR studies of proteins and
protein: RNA complexes in the 15 - 30 kD range. Technology for
determining high resolution structures in this size range will be
developed primarily o domains os a biomedically-important RNA-
binding protein, the nonstructural protein 1 (NS1 protein) from
influenza virus and complexes between the RNA-binding domain of the
NS1 protein and its specific RNA target molecules. Influenza virus
infection is a major human health problem in the United States and
throughout the world. The NS1 protein plays a central role in the life
cycle of the influenza virus and is potentially an important target for
the rational design of antiviral drugs. We will characterize the three-
eimensional structures and internal dynamics of the RNA-binding
domains from two types (A and B) of human influenza virus. Specific
RNA oligonucleotide molecules will be produced, and their binding
epitopes on the surfaces of these NS1 proteins will be mapped by
heteronuclear NMR. In addition, we will extend this work to dtudies
of the 18kD effector domain of the influenza A virus NS1 protein,
which will be expressed in E.coli and studies by NMR. This domain
is responsible for regulating the translocation of mRNA from the
nucleus into the cytosol, thereby regulating the expression of viral and
cellular proteins during the infection cycle. This work will provide
new insights into the mechansims by which proteins bind to RNAm and
details of structure-function relationships in the NS1 protein from
influenza virus.
Neuropeptide Y (NPY), a 36, residue peptide amide initially isolated
from porcine brain, is a member of a family of homologous hormones
including peptike YY (PYY) and pancreatic polypeptide. It is the most
abundant and widely distributed neuropeptide present in the mamalian
central and peripheral nervous system. NPY has been implicated in a
wide variety of central and peripheral functions. Of these, the most
striking are its effects on feeding, blood pressure and cardiac
contractility. Investigations to date suggest that NPY is the most potent
orexigenic substance known. Its potent vasoconstrictor effects have
also led to the classification of NPY among the most potent
vasocpressor peptides isolated to date. While hypothalamic NPY levels
and/or its mRNA are increased in obese rats, it is decreased in
anorectic tumor bearing rats. Also, plasma NPY levels are elevated in
patients with congestive heart failure (CHF) and pheochromocytoma,
and in hypertensive rats. Moreover, NPY actions on feeding, blood
pressor and contractility are mediated by Y-1-like, Y-1 and Y-3
subtypes, respecitively. These obsevations indicate that highly selective
NPY analogs could be designed and synthesized to treat a variety of
disorders. Our systematic investigations dircted towards these goals
during the past none years have led to the identification of lower
molecular weight strucyural moieties, T-54 and T-190 using classical
SAR strategy. Our approach will include: 1) altteration of the
hydrophobic/ hydrophilic / steric/ionic characteristics of each amino
acid; 2) substitution with unnatural/D-amino acids, and 3) imposing
structural constraints via cyclization, cyclic amino acids and backbone
modifications. T-54 and T-190 analogs will be screened for their
effects on feedings (Y-1 like) and on SK-N-MC cells (Y-1) ,
respectively. Similarly, we have already determined that the
introduction of pseudobond in the C-terminal region of our cardiac
NPY receptor antagonist, NPY (18-36), can impart high affinity and
selectivity. Therefore, it is also proposed to continue to synthesize and
investigate the receptor affinities and the effects on cardiac adenylate
cyclase activity of NPY (18-36) analogs. These investigations are
expected to result in the development of highly selective and long
acting analogs for a variety of NPY receptors. These analogs will have
great significance both in fundamental studies as well as in treating (or
in developing drugs for) obesity, hypertension and failing heart.
Status | Finished |
---|---|
Effective start/end date | 2/1/92 → 3/31/03 |
Funding
- National Institute of General Medical Sciences
ASJC
- Structural Biology
- Spectroscopy
- Virology
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