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Faculty
Betty Eipper
Professor, Neuroscience and Molecular, Microbial & Structural Biology
eipper@nso.uchc.edu
Areas of Interest:
Peptides; trafficking; enzymes; tissue culture;
development; pituitary; heart.
Peptides seem to have preceded the 'classical' transmitters as the
nervous system developed - creatures like Hydra and Drosophila utilize
peptides to control key developmental decisions. Beginning with our work
on proopiomelanocortin and the coordinate biosynthesis of ACTH and the
opioid peptide beta-endorphin, I have been fascinated with the effort
that neurons and endocrine cells devote to the biosynthesis, storage and
regulated secretion of peptides. As we have learned more about the
specific enzymes involved in the biosynthesis of peptides, we have
learned important questions to ask about how these enzymes function in
cells.
In particular, we have focused a great deal of our effort on the
process of peptide amidation. This seemingly trivial modification to the
COOH-terminus of peptides turns out to be essential for the biological
activity of many peptides. Hypothalamic peptides like oxytocin and
vasopressin along with neuropeptides like substance P and
gastrointestinal mediators like gastrin must be amidated in order to
affect their target tissues. By purifying an enzyme capable of
converting peptidylglycine precursors into amidated products, we were
then able to clone a cDNA encoding this enzyme.
To our surprise, the modification requires the sequential action of
two enzymes, a monooxygenase and a lyase. The enzyme has been named PAM,
short for peptidylglycine alpha-amidating monooxygenase. The
monooxygenase itself is called PHM, short for peptidylglycine alpha-hydroxylating
monooxygenase, and the lyase is called PAL, short for
peptidyl-alpha-hydroxylglycine alpha-amidating lyase. PHM uses ascorbic
acid (vitamin C) to reduce the two copper atoms that are bound to its
catalytic core and molecular oxygen is the final component of the
reaction. PAL also requires a metal ion for activity, with zinc the most
likely ion.
We have expressed the bifunctional PAM protein in soluble and
membrane forms and have purified milligram amounts of the two separate
catalytic domains, PHM and PAL. With Drs. Mario Amzel and Sean Prigge,
we were able to deduce the crystal structure of PHM. One copper binds to
the N-terminal domain of the PHM catalytic core and the other to the
C-terminal domain.
Along with structure function studies, our current efforts are aimed
at understanding what the cells that use PHM have to do in order to
provide copper to the enzyme. Copper is an extremely toxic metal and
specific pumps and chaperones are generally used to deliver copper to
the proteins that need it. Mottled mice lack ATP7A, one of the copper
transporting ATPases. We are using these mutant mice to better
understand how neurons and endocrine cells get copper into the lumen of
the secretory pathway so that it can be loaded onto PHM.
PHM is similar in sequence to dopamine beta-monooxygenase, a key
enzyme in catecholamine biosynthesis. Despite their similarities, PHM
and DBM have distinctly different features. We are using insights gained
from our studies of PHM to understand better the unique features of DBM.
In addition to its catalytic domains, PAM has non-catalytic regions.
In particular, the transmembrane domain and cytosolic domain of PAM need
not be present for the enzyme to function. The role of these
non-catalytic domains seems to be in getting PAM to the right place in
the cell so that it can do its job. In particular, the cytosolic domain
is essential for targeting PAM to the secretory granules of pituitary
endocrine cells and for guiding PAM protein that has reached the cell
surface back into secretory granules following internalization.
After mapping several key determinants in the rather short cytosolic
domain of PAM, we identified proteins that interact with it. One of
these proteins, PCIP-2, is a protein kinase that is highly selective for
PAM. The Ser residue phosphorylated by PCIP-2 plays a role in secretory
granule targeting and in trafficking from late endosomes into the TGN
and secretory granules. Current studies are directed to understanding
the structure and function of PCIP-2.
Another PAM cytosolic domain interactor protein, kalirin, is a member
of the Dbl family of GDP/GTP exchange factors for small GTP binding
proteins of the Rho sub-family. The cytosolic domain of PAM binds to the
spectrin-like repeat region of kalirin. This region of Kalirin is
followed by a Dbl homology or DH domain, and a PH domain. Kalirin occurs
naturally in a variety of isoforms and this first DH/PH domain can be
followed by a PDZ-binding motif (Kalirin-7), an SH3 motif (Kalirin-8),
another DH/PH domain (Kalirin-9) or another DH/PH domain and a putative
serine/threonine protein kinase (Kalirin-12). The various isoforms of
kalirin are expressed at different times during development and are
localized to different regions of the cell. Over-expression of kalirin
or its subdomains dramatically alters growth of axons and formation of
synapses and current studies are aimed at understanding the structure
and function of kalirin.
http://neuropeptidelab.uchc.edu
Lab Rotation Projects:
Project 1: Isolating multivesicular bodies/recycling endosomes.
Following exocytosis, secretory granule membrane proteins can be
re-used or degraded; the choice is regulated by ubiquitination and
phosphorylation. Pituitary cells incubated with antibody to a secretory
granule membrane protein (PAM) will be stimulated with secretagogue and
the subcellular compartments containing the internalized antibody (and
thus PAM that has visited the cell surface) will be isolated and
characterized. Cell culture, Western analyses, subcellular fractionation
techniques.
Project 2: Modifications of routing determinants for PAM.
PAM is the large dense core vesicle integral membrane protein which
amidates bioactive peptides. The intracellular routing of PAM is
controlled largely by its 80-residue cytosolic domain, which is known to
undergo phosphorylation at several residues, and possibly ubiquitination.
The precise residues modified under a number of physiological states
(basal secretion, rapid stimulated secretion) will be determined for
wildtype and existing mutant forms of PAM. Cell culture,
immunocytochemistry, Western analyses, peptide analyses, isoelectric
focusing.
Project 3: Identification of phosphorylation sites in Kalirin-7.
Rho GDP-GTP exchange factors (GEFs) play critical roles in regulating
the actin cytoskeleton. Kalirin, a GEF specific for RhoG and for Rac1,
interacts with secretory granule membrane proteins and with components
of the post-synaptic density. Factors regulating the activity of
Kalirin-7 are poorly understood. To explore the effects of Protein
Kinase A, PKC and Cdk5 on Kalirin, recombinant protein will be incubated
with purified kinase and the major sites of phosphorylation will be
identified following proteolytic digestion, RP-HPLC fractionation and
mass spec analysis in collaboration with Dr. Han.
Project 4: Defining the role of an E3 ubiquitin ligase in
the exoctyosis of secretory granule membrane proteins.
P-CIP2, a Ser/Thr kinase identified as an interactor with the
cytosolic domain of PAM, also interacts with PAUL, a putative Ariadne-like
E3 ubiquitin ligase. The regions of PAUL essential for its interaction
with P-CIP2 will be identified. The effects of over- and
under-expressing PAUL on hormone secretion by corticotrope tumor cells
will be evaluated. Cell culture, vector construction, transfection
techniques, radioimmunoassays.
More Information
Publications
Selected Publications:
McPherson CE, Eipper BA, Mains RE. Kalirin expression is regulated by
multiple promoters. J Mol Neurosci 22:109-120, 2003.
Ma XM, Huang J, Wang Y, Eipper BA, Mains RE. Kalirin, a
multifunctional Rho guanine nucleotide exchange factor, is necessary for
maintenance of hippocampal pyramidal neuron dendrites and dendritic
spines. J Neurosci 2003 Nov 19;23(33):10593-603.
Chei FY, Eipper BA, Mains RE, Fricker LD. Quantitative peptidomics of
pituitary glands from mice deficient in copper transport. Cell Mol Biol
(Noisy-le-grand). 2003 Jul;49(5):713-22.
Bell J, El Meskini R, D'Amato D, Mains RE, Eipper BA. Mechanistic
investigation of peptidylglycine alpha-hydroxylating monooxygenase via
intrinsic tryptophan fluorescence and mutagenesis. Biochemistry. 2003
Jun 17;42(23):7133-42.
Penzes P, Beeser A, Chernoff J, Schiller MR, Eipper BA, Mains RE,
Huganir RL. Rapid induction of dendritic spine morphogenesis by
trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin.
Neuron. 2003 Jan 23;37(2):263-74.
El Meskini R, Culotta VC, Mains RE, Eipper BA. Supplying copper to
the cuproenzyme peptidylglycine alpha-amidating monooxygenase. J Biol
Chem. 2003 Apr 4;278(14):12278-84. Epub 2003 Jan 14.
Steveson TC, Ciccotosto GD, Ma XM, Mueller GP, Mains RE, Eipper BA.
Menkes protein contributes to the function of peptidylglycine alpha-amidating
monooxygenase. Endocrinology. 2003 Jan;144(1):188-200.
Jaron S, Mains RE, Eipper BA, Blackburn NJ. The catalytic role of the
copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase
(PHM): a spectroscopic study of the H172A mutant. Biochemistry. 2002 Nov
5;41(44):13274-82.
Kolhekar AS, Bell J, Shiozaki EN, Jin L, Keutmann HT, Hand TA, Mains
RE, Eipper BA. Essential features of the catalytic core of
peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Biochemistry. 2002
Oct 15;41(41):12384-94.
May V, Schiller MR, Eipper BA, Mains RE. Kalirin Dbl-homology guanine
nucleotide exchange factor 1 domain initiates new axon outgrowths via
RhoG-mediated mechanisms. J Neurosci. 2002 Aug 15;22(16):6980-90.
Ma XM, Mains RE, Eipper BA. Plasticity in hippocampal peptidergic
systems induced by repeated electroconvulsive shock.
Neuropsychopharmacology. 2002 Jul;27(1):55-71. |
