ProSAAS-derived peptides: Processing, sorting, and function in feeding and bodyweight
Wardman, Jonathan Henry
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Neuropeptides are important signaling molecules in the brain and other neuroendocrine tissues, and are involved in regulating many physiological processes. These peptides control diverse functions including food intake and bodyweight regulation. Neuropeptides are specifically processed and secreted in response to peripheral signals as well as signals from other parts of the brain. ProSAAS derived peptides are specifically processed and sorted neuropeptides which play a role in the regulation of feeding. The proper function of neuropeptides is essentially linked to their specific processing by a number of secretory pathway peptidases. These include the endopeptidases prohormone convertase 1 (also known as PC3, PC1/3), prohormone convertase 2 (PC2), carboxypeptidase D (CPD) and carboxypeptidase E (CPE), all of which have specific target cleavage sequences. PC1/3 is one of the major endopeptidases that colocalizes with its substrate peptides in secretory vesicles. PC1/3 has cleavage site specificity which is partially redundant with that of PC2. In order to better understand the function of these two endopeptidases, both PC1/3 and PC2 knockout mice were created in other labs. Studying the PC1/3 knockout mouse using the quantitative peptidomics technique, I was able to learn which neuropeptides are specifically processed by PC1/3 in several mouse tissues, including the hypothalamus, the amygdala and the striatum. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.;Some of the peptides that were specifically processed by PC1/3 in the hypothalamus and amygdala were derived from the precursor proSAAS. ProSAAS derived peptides are broadly distributed throughout the neuroendocrine system. These peptides were found in early studies to specifically inhibit PC1/3. Later studies on proSAAS transgenic and knockout mice indicated that these peptides play an orexigenic role in the control of feeding and bodyweight. In the present studies, immunofluorescent colocalization is used to show that proSAAS derived peptides PEN/LEN, big LEN and PEN colocalize with orexigenic NPY but not with anorexigenic alpha-MSH. However, unlike proNPY mRNA, fasted mice do not show an increase in the levels of proSAAS mRNA. Intracerebroventricular injections of antibodies to two proSAASderived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake.;The distinct cleavage forms of the proSAAS derived peptides distinguished them from one another in their function. Big LEN and little LEN antibodies did not elicit the same effect when injected and little LEN did not colocalize with NPY or big LEN in the arcuate nucleus of hypothalamus. While differential processing of neuropeptides has previously been shown to regulate their function, there are no mammalian examples of peptides from the same precursor being differentially sorted. By comparing the localization of these peptides in both the arcuate nucleus of the mouse hypothalamus, as well as in AtT-20 cells, it is clear that they are being sorted to different vesicles. This sorting of the individual proSAAS derived peptides adds another level of control to their function. Taken together, all of these data show that big LEN is a specifically processed and sorted neuropeptide functioning in the regulation of bodyweight and feeding.