Betty Eipper, PhD
Professor
Department of Molecular Biology and Biophysics
UCONN Health
(October 31, 2017)
Using Neuropeptides to Communicate – Lessons Learned from A Single Cell Green Alga
Chemical signals, such as neurotransmitters and neuropeptides, are used by neurons for communication. This method must be adaptable and able to respond to different stimuli. Research has made clear that neuropeptides are of key importance, but many questions remain, including what peptides are necessary for what signal, and where and when they are used. In her seminar, Doctor Eipper discussed her work examining one enzyme, PAM, which plays an important role in neuropeptide function in single-celled green alga, as well as in mammals.
Neuropeptides (α-endorphin, vasopressin and cholecystokinin, for example) far outnumber the classical or small molecule neurotransmitters (glutamate, GABA, acetylcholine, biogenic amines and adenosine,
for example). Flies, sea anemones and human neurons synthesize bioactive peptides in much the same way, utilizing the secretory pathway machinery used to produce membrane proteins and components of the extracellular matrix. It is now clear that most neurons utilize both classical transmitters and neuropeptides to communicate with their target tissues. Neuropeptides, acting almost entirely through G Protein Coupled Receptors, provide a great deal of plasticity to the nervous system: different neuropeptides can be targeted to axons vs dendrites; firing frequency and an array of environmental inputs can alter the transmitter mixture released. With the ability to activate specific subsets of peptidergic neurons, their role in circuit formation and function has become clear.
The ability of many neuropeptides to bind to their receptors requires that the readily ionizable carboxyl group at their C-terminus be amidated, stabilizing the peptide and reducing any effect of modest changes in pH on its ionization. Following up on the observation that pituitary cells maintained in medium lacking serum retained the ability to produce peptide precursors and cleave them into the expected products, but were unable to amidate them, the Eipper lab purified and characterized the only enzyme known to catalyze this reaction, peptidylglycine α-amidating monooxygenase (PAM). This bifunctional type I integral membrane enzyme catalyzes the sequential conversion of peptidylglycine substrates into α-hydroxylated intermediates. Peptidylglycine α-hydroxylating monooxygenase (PHM), a copper, ascorbate (the component missing from serum-
free media) and molecular oxygen dependent enzyme, first catalyzes the stereospecific hydroxylation of the α-carbon of the C-terminal glycine. Cleavage by peptidyl-α-hydroxyglycine α-amidating lyase (PAL) then releases the amidated peptide and glyoxylate. Its intrinsically disordered cytosolic domain governs PAM trafficking as it moves through the biosynthetic pathway, onto the plasma membrane and into the endocytic pathway, where it can be recycled to secretory granules or degraded. Strikingly, PHM is as sensitive to levels of molecular oxygen as the prolyl hydroxylases that control the stability of hypoxia inducible factor 1-α (HIF1-α), suggesting a cell non-autonomous role for peptide amidation in the response of different cells to hypoxia.
In both mice and flies, genetic elimination of PAM is lethal. The discovery of a highly homologous PAM gene in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene in the last eukaryotic common ancestor. Other genes known to play an essential role in nervous system function have also been found in organisms totally lacking a nervous system. CrPAM localizes to the Golgi region, as in mammalian neurons and endocrine cells. In addition, and unexpectedly, CrPAM also localizes to ciliary membranes; consistent with this observation, PAM activity is readily detectable in lysates of purified cilia. Never having looked before, they were quickly able to demonstrate the presence of PAM in both motile and sensory cilia in vertebrate systems.
Intrigued by a role for this copper, ascorbate and molecular oxygen dependent enzyme in cilia, they evaluated the effect of reducing the expression of CrPAM. Two independent CrPAM knockdown lines were unable to form cilia! No axoneme extended beyond the transition zone, which lacked Y-linkers. A conserved, but species-specific role for PAM in ciliogenesis was confirmed by examining the effects of reducing PAM expression in Schmidtea mediterranea, zebrafish and mice. Increased expression of CEP290 and NPHP4, components of the transition zone, in the CrPAM knockdown lines suggests changes in the signaling events involved in ciliogenesis. In addition, cargo-specific alterations in protein secretion were observed, consistent with the effects of PAM expression on secretory pathway function in mammalian cells.