Areas of Interest
Peptides; membrane protein trafficking; cuproenzymes; neurons;
pituitary
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 and in the whole animal.
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 often turns out to be essential for
their biological activity. 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, this 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, zinc, for activity. With its need for copper, zinc
and ascorbate, PAM function is sensitive to genetic and
environmental factors.
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 Dr. Mario Amzel,
we were able to deduce the crystal structures for PHM and for
PAL. One copper binds to the N-terminal domain of the PHM
catalytic core and the other to the C-terminal domain; how both
sites contribute to the reaction is not yet clear. The
beta-propeller structure of PAL constrains PHM to a location
near the granule membrane, with important functional
implications.
Along with structure function studies, our current efforts
are aimed at understanding what the cells that use PAM have to
do in order to provide copper to the enzyme. Copper is an
extremely toxic metal and specific pumps and chaperones are used
to deliver it to the proteins that need it. PAM knockout mice do
not survive beyond mid-gestation; PAM heterozygous mice are
viable, but exhibit increased anxiety-like behavior, an
inability to thermoregulate and increased seizure sensitivity.
Many of these deficits are mimicked in mildly copper-deficient
wildtype mice and ameliorated by providing supplementary dietary
copper to PAM heterozygous mice. We are using these animal
models to understand the role of PAM in coping with copper
availability and are collaborating with our clinical colleagues
to see if mild copper deficiency occurs in patient populations.
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. We recently found
that a gamma-secretase-like cleavage releases a soluble
cytosolic domain fragment of PAM that enters the nucleus and
alters gene expression. This adds a new dimension to our studies
of PAM and we are in seach of the underlying mechanism.
A PAM cytosolic domain interactor protein of great interest
is kalirin, 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. Mice engineered to lack
expression of Kalirin globally or only in pituitary
corticotropes are being used to identify the major roles of
Kalirin in the pituitary and in the nervous system.
Neuropeptide
Laboratory
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: Routing of PAM in neurons.
PAM is a 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.
Although the trafficking of PAM in endocrine cells has been
studied in detail, little is know about how PAM travels to
dendrites and to axon terminals. Primary neuronal cultures,
fluorescence microscopy, enzyme assays.
Project 3: Search for Kalirin-7 interactors.
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 and localization of Kalirin-7
are poorly understood. Structure/function studies have revealed
an important role for the spectrin-repeat region but the key
interactors have not yet been identified.
Publications
Selected Publications
Bousquet-Moore D, Ma X-M, Nillni EA, Czyzyk TA, Pintar FE,
Eipper BA and Mains RE (2009) Reversal of Physiological Deficits
Caused by Diminished Levels of Peptidylglycine a-Amidating
Monooxygenase by Dietary Copper. Endo 150:1739-1747.
Xin X, Rabiner CA, Mains RE and Eipper BA (2009) Kalirin12
Interacts with Dynamin. BMC Neuroscience Jun 17;10:61. PMID:
19534784
Sobota JA, Back N, Eipper BA and Mains RE (2009) Inhibitors
of the Vo Subunit of the Vacuolar H+ATPase Prevent Segregation
of Lysosomal and Secretory Pathway Proteins. J Cell Sci, in
press.
Chufan EE, De M, Eipper BA, Mains RE and Amzel LM (2009)
Amidation of Bioactive Peptides: The Structure of the Lyase
Domain of the Amidating Enzyme. Structure 17:965-973l
Rajagopal C, Stone KL, Francone VP, Mains RE and Eipper BA
(2009) Secretory Granule to Nucleus: Role of a Multiply
Phosphorylated Intrinsically Unstructured Domains. J Biol Chem,
Jul 27. [Epub ahead of print]PMID: 19635792.
Ma X-M, Wang Y, Ferraro F, Mains RE, Eipper BA (2008)
Kalirin-7 Is an Essential Component of both Shaft and Spine
Excitatory Synapses in Hippocampal Interneurons, J Neurosci
28:711-724.
Schiller MR, Ferraro F, Wang Y, Ma X-M, McPherson CE, Sobota
JA, Schiller NI, Eipper BA (2008) Autonomous functions for the
Sec14p/spectrin-repeat region of Kalirin. Expt Cell Res 314:
2674-2691.
Xin X, Wang Y, Ma X-M, Rompolas P, Keutmann HT, Mains RE,
Eipper BA (2008) Regulation of Kalirin by Cdk5, J Cell Sci
121:2601-2611.
Ma, X-M, Kiraly DD, Gaier ED, Wang Y, Kim E-J, Levine ES,
Eipper BA, Mains RE (2008) Kalirin7 is required for synaptic
structure and function. J Neurosci 28: 12368-12382.
Ferraro F, Ma XM, Sobota JA, Eipper BA, Mains RE 2007 Kalirin/Trio
RhoGEFs Regulate a Novel Step in Secretory Granule Maturation.
Mol Biol Cell 18:4813-4825.
Linz R, Barnes N, Zimnica A, Eipper B, Kaplan J, Lutsenko S
(2007) The Intracellular Targeting of Copper-Transporting ATPase
ATP7A in a Normal and ATP7B-/- Kidney, Am J Physiol (Renal
Physiol), PMID: 17928409.
 View more publications, see
Pubmed listing. |
