Ciliary and cytoskeletal functions of an ancient monooxygenase essential for bioactive amidated peptide synthesis
Many secreted peptides used for cell–cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies.
KeywordsActin Amidation Chlamydomonas Cilia Microvilli Peptidylglycine α-amidating monooxygenase
Nearly all cells in the human body (except those of lymphoid and myeloid origin) build a cilium at some point in their life cycle . In addition to the motile sperm flagellum (a modified cilium), multiple motile cilia are present on the apical surfaces of cells lining various structures. The motile cilia on the ependymal cells lining the ventricles of the brain generate the force needed to move cerebrospinal fluid. In the lung, motile cilia play an essential role in mucus clearance which acts as a first line of defense against airborne pollutants and pathogens. Most other cell types possess a solitary, immotile (primary) cilium (Fig. 1a) that acts as a sensory antenna  and in some cases has become highly modified to perform specific tasks, e.g., light detection by the outer segments of rods and cones in the eye and odorant reception by multiciliated olfactory neurons.
Cilia are highly complex: proteomic, transcriptomic and comparative genomics approaches in various organisms have identified many hundreds of proteins associated with these organelles [8, 9]; indeed, a recent estimate suggests that the human “ciliome” consists of approximately 1200 genes . Consequently, perhaps 5% or more of the ~ 21,000 human protein-encoding genes  are involved in ciliary assembly, structure and/or function. Cilia are essential for organismal development and homeostasis; defects result in a wide array of ciliopathies —complex syndromes (e.g., Bardet–Biedl  and Joubert  syndromes) which can have broad phenotypic consequences [15, 16, 17].
The cilium is a discrete cellular compartment; entry into both motile and immotile cilia is controlled in part by a multi-subunit gate termed the transition zone [18, 19] (Fig. 1b). Although the ciliary membrane is contiguous with the plasma membrane it has a very distinct lipid and protein content. Numerous receptors/channels are localized to this compartment, allowing the organelle to both sense the extracellular environment and initiate appropriate signaling cascades that relay information to the cell body in response to external chemical or mechanical signals. Well-known primary cilia-dependent pathways include non-canonical Wnt (planar cell polarity) [20, 21] and Hedgehog [22, 23] signaling as well as G-protein coupled receptor-mediated responses to peptide hormones such as somatostatin  and kisspeptin . Motile cilia also exhibit sensory functions. For example, ciliated tracheal epithelial cells are mechanosensitive, modulating ciliary beat frequency to match the viscosity of the mucus they encounter . In Chlamydomonas, enhanced ciliary power output during viscous loading is mediated by increased trafficking of the dynein regulatory factor Lis1 into cilia . Some cilia also possess chemoreceptors of the bitter taste family that can sense and respond to noxious compounds . In addition, the initial steps of the cAMP-mediated signaling pathway, which are initiated in response to cell–cell contact during sexual reproduction in Chlamydomonas, are confined to its motile cilia .
Protein and membrane trafficking are critical, yet poorly understood, aspects of cilium assembly. For example, soluble protein components must be specifically moved into the growing cilium, along with new membrane and proteins delivered in Golgi-derived vesicles. Although there are roles for ARF family small GTPases  and microtubule motors [31, 32], how the vesicular trafficking process is regulated to control the rate of ciliary membrane addition is uncertain. Ciliary membrane is lost during the budding of ectosomes, a process that involves branched actin filament dynamics [3, 33]. Ciliary integrity and homeostasis require a delicate balance between post-Golgi trafficking and the release of ectosomes. Indeed, ciliary length is a tightly regulated parameter  and, for example, can change in response to hypoxia .
Recent studies have uncovered an unanticipated role for a secretory pathway enzyme involved in bioactive peptide synthesis (peptidylglycine α-amidating monooxygenase; PAM) in building cilia [36, 37, 38]; this connection has been conserved between Chlamydomonas and metazoans (planaria, mice and zebrafish) suggesting that it dates to the last eukaryotic common ancestor and represents an important aspect of ciliogenesis. Here we review the evidence supporting a role for the PAM protein and its amidating activity in ciliary assembly, suggest models for PAM function in this process, and describe how interactions of PAM with the actin cytoskeleton might alter both cilia and microvilli, leading to broad and generalized effects on cytoskeleton-based cellular protrusions. Furthermore, we address the intriguing phylogenetic question of how some organisms that lack PAM can still build cilia and briefly discuss the more clinical implications of merging the fields of ciliogenesis and peptidergic signaling.
General principles underlying ciliary formation and assembly
The trafficking of membrane proteins such as PAM into and out of the cilium poses further challenges , and their movement is aided by an additional IFT-associated multimeric complex—the BBSome [48, 49, 50, 51]. Indeed, current evidence suggests that the BBSome is primarily involved in ciliary protein export . Although some IFT-cargo interactions, including those involved in tubulin and outer arm dynein transport with its associated IFT46 cargo adaptor, are now understood at the structural level e.g., [53, 54], the general principles by which IFT and BBSome complexes recognize the enormous range of ciliary cargoes that must be trafficked remain unclear. Intriguingly, some IFT proteins play non-cilia related roles in the cytoplasm and, for example, have been implicated in cell cycle progression , mitotic spindle orientation  and cytoplasmic microtubule dynamics .
PAM, bioactivation by amidation and the secretion of signaling peptides
PAM (EC 18.104.22.168) is best known for its role in the neuroendocrine system, where it catalyzes a late step (α-amidation) in the synthesis of many secreted bioactive peptides such as oxytocin, vasopressin, gonadotropin-releasing hormone and neuropeptide Y . As peptide precursors traverse the secretory pathway, they are acted on by proprotein convertases that cleave after paired basic amino acid sites, and then by carboxypeptidases, which remove the basic residues. If during this process a glycine residue is exposed at the C-terminus of the processing intermediate, it becomes a potential substrate for amidation by PAM. Amidation plays three major roles: (1) it alters the charge and structure at the C-terminal end of the peptide allowing specific and high affinity recognition by cognate receptors, (2) it affects overall peptide conformation, and (3) it enhances peptide stability, extending lifetime in the extracellular environment. In addition to their numerous roles in metabolism, tissue homeostasis and other aspects of vertebrate physiology, amidated peptides are commonly used by invertebrates. For example, species-specific amidated peptides released by sea urchin eggs allow for sperm chemoattraction , and many of the toxic peptides present in cone snail, bull ant and spider venoms [60, 61, 62, 63] are amidated.
PAM is essential for vertebrate development and edema is a major feature observed in mice and zebrafish lacking PAM. Pam-null mice die by embryonic day (E) 14.5 with ventricular hypertrophy, massive edema and a poorly formed vasculature . Homozygous pam−/− zebrafish embryos exhibit small eyes, cyst-like protrusions associated with the pronephros, hydrocephalus and edema, dying at ~ 10 days post-fertilization (Fig. 4e) . PAM is highly expressed in the mouse heart at these early developmental stages but is not highly expressed in zebrafish heart. Nevertheless, in both cases the edema is first observed in the pericardial region and likely derives from altered hormonal signaling and consequent alterations in fluid homeostasis. In Drosophila, which has separate dPHM and dPAL genes, loss of the dPHM gene, which encodes soluble amidating enzyme, leads to larval lethality during molting due to defective peptidergic signaling .
It is now clear that bifunctional, integral membrane PAM predates evolution of the nervous system . A PAM-like gene is present throughout the metazoa including sponges and placozoans that lack neurons; insects are an exception as they lack integral membrane PAM but rather express separate PHM and PAL proteins. Bifunctional PAM is also found in unicellular and colonial chlorophyte green algae (Chlamydomonas, Gonium and Volvox), strongly suggesting that this enzyme was present in the last eukaryotic common ancestor which is thought to have had a motile cilium that was also used for signaling [36, 66].
Post-Golgi trafficking, ciliary/cellular localization and topology of PAM
Separation of the catalytic domains of PAM from its TMD/CD allows intramembrane proteolysis by γ-secretase, producing a soluble cytosolic fragment (sfCD) that traffics to the nucleus and alters the expression of a subset of genes [70, 71]. Golgi-derived vesicles destined for cilia are known to transport both ciliary membrane proteins and specific lipids [72, 73], and potentially contain PAM (Fig. 5a). Alternatively, ciliary PAM may derive from endosomes recycling PAM from the cell surface; directed exocytosis would allow internalized PAM to enter the cilium, as has been proposed for the Kim1 protein [74, 75]. Indeed, in Chlamydomonas, ~ 7% of the total PHM activity is present in cilia . Disrupting the Golgi with Brefeldin A leads to cell body accumulation of PAM and Arf1, a vesicle trafficking factor, and to a decrease in the modified tubulins normally present in Chlamydomonas cilia. Similarly, Chlamydomonas PAM lacking most of its CD exhibits impaired trafficking; although the mutant protein is still found in the cilia, it tends to accumulate in the secretory pathway . The trafficking determinants in the CD appear to have been well conserved as Chlamydomonas PAM is distributed appropriately when expressed in murine cells (Fig. 5b).
Given PAM’s topology in the secretory pathway, the catalytic domains are predicted to be on the outside of the cilium (Fig. 5a) where they could potentially act catalytically on soluble glycine-extended factors in the environment; i.e., locally generating amidated products on/near the ciliary surface. This domain orientation was directly demonstrated in Chlamydomonas cilia using antibodies against both the PAM luminal domain and the CD; the latter only yielded a signal after the ciliary membrane had been permeabilized . Intriguingly, immuno-electron microscopy revealed that PAM staining exhibits a distinct periodicity of ~ 250 to 300 nm along the Chlamydomonas cilium, and biochemical fractionation demonstrated that PAM associates with the microtubular axoneme as does another ciliary membrane protein—the non-selective cation channel, polycystin 2 ; whether this tethering occurs directly through the PAM CD or via some other axoneme-associated component remains uncertain.
PAM-actin associations and microvillus formation
Further evidence that PAM plays a key role in the formation and/or maintenance of actin-based structures came from observations in mutant zebrafish  (Fig. 6b). In wild-type embryos at 3 days post-fertilization, the lumen of the pronephros (kidney) is almost completely filled by motile cilia surrounded by a dense outer array of microvilli. In zebrafish, many mRNAs are maternally loaded into the early zygote and indeed Pam mRNA is one of the most abundant . Initially, both cilia and microvilli form in pam-null mutant embryos as PAM protein can be made at very early developmental stages from the large maternally derived mRNA stores. However, after several days of development, there is no detectable amidating activity and a dramatic loss of microvilli and motile cilia is observed along almost the entire length of the pronephros, suggesting that these structures either cannot form or be maintained in the absence of PAM. As PAM is absent from the microvilli themselves (Fig. 6a), the essential role this enzyme plays in their assembly and/or maintenance must be indirect.
Thus, although the observed changes are both complex and varied, and the vertebrate and Chlamydomonas PAM-CD sequences are highly divergent, control of actin cytoskeletal dynamics and behavior is a fundamental property of PAM that has been highly conserved across the eukaryotes.
PAM and amidating activity are needed to build cilia
Golgi morphology was altered in these knockdown strains, as the stacks were more curved than in controls, possibly due to the loss of PAM-actin interactions. PAM deficiency also reduced trafficking of Golgi-derived starch metabolic enzymes, leading to changes in starch granule size, and there were defects in both basal and nutrient deprivation-stimulated secretion. Importantly, PAM knockdown strains grew at the same rate as controls (under both photoautotrophic and photoheterotrophic conditions), and exhibited a normal contractile vacuole cycle, which is another process heavily dependent on membrane trafficking. Thus, the ciliary and Golgi defects observed following the loss of Chlamydomonas PAM derive from the disruption of specific cellular processes and do not merely reflect a general decrease in cell fitness or viability.
Demonstration that PAM plays a role in metazoan ciliogenesis came from studies in the planarian Schmidtea mediterranea and two vertebrates (zebrafish and mice) [37, 38]. Planaria have a ventral ciliated epithelium used for gliding locomotion and express three PAM-related proteins: a canonical bifunctional integral membrane PAM as well as separate, soluble PHM and PAL proteins that would reside within the secretory pathway. Knock down of membrane PAM and soluble PHM together, using RNAi constructs, reduced enzyme activity to < 10% of control levels and dramatically reduced the number of motile cilia on the ventral surface (Fig. 7b). The remaining cilia were dyskinetic and often had aberrant axonemal architecture likely due to defective remodeling. The double knockdown animals moved at a much slower rate than controls, consistent with movement driven by contractions of the body musculature rather than ciliary beating against secreted mucus. Analysis of the ciliated epithelial cells in these knockdown animals revealed some basal bodies docked at the plasma membrane lacking axonemal extensions, and numerous morphologically normal motile ciliary axonemes located in the cytoplasm very close to the plasma membrane but with no surrounding ciliary membrane (Fig. 7b, lower panel). Strikingly, these unusual cytosolic axonemes were in general oriented with their distal end towards the head of the animal. This latter observation suggests that lack of PAM affects the docking of at least some basal bodies and/or trafficking of Golgi-derived ciliary membrane components. Intriguingly, this “cytosolic axoneme assembly” phenotype was also seen in cells lining the pronephros of pam-null zebrafish , suggesting that it is a conserved response of multiciliated cells to the lack of PAM.
In mice, lack of PAM is lethal in early development, prior to the formation of multiciliated epithelial cells . However, primary cilia in the developing neuroepithelium of Pam−/− embryos were much shorter than controls (0.5 versus 0.9 μm), suggesting that they represent immature or aberrant forms (Fig. 7c). Zebrafish pam−/− mutant embryos have several cilia-related phenotypes, including small eyes, kidney-associated cysts and hydrocephalus , and exhibit loss of both cilia and microvilli in the pronephros within 5 days after fertilization . Interestingly, some ciliary structures, including those in the olfactory bulb, otic vesicles and on neuromasts, appear generally unaffected.
A key question raised by these observations is whether the ciliary phenotypes are caused by the lack of PAM-mediated protein–protein interactions, by the loss of amidating activity per se, or both. Currently, several lines of evidence provide support for a role for amidating activity. First, planaria exhibited the strongest ciliogenesis phenotype only when both membrane-PAM and soluble PHM were targeted together, thereby reducing amidating activity to very low levels. Furthermore, knockdown of soluble PHM alone resulted in ciliary loss and the slow muscle-driven motility characteristic of animals with defective motile cilia , again suggesting a role for monooxygenase activity. Second, PHM is inhibited by 4-phenyl-3-butenoic acid (PBA) , a suicide inhibitor, and by neocuproine, a copper-specific chelator (PHM has an absolute requirement for copper) . When wild-type Chlamydomonas were deflagellated in the presence of either inhibitor, reciliogenesis was significantly delayed, strongly suggesting that amidating activity is a key ciliogenic parameter. That reciliogenesis was delayed rather than completely inhibited likely reflects the observation that Chlamydomonas maintains sufficient components (potentially including enough amidated products) in the cell body to build two approximately half-length cilia without additional protein synthesis . Third, Chlamydomonas cells expressing CrPAMΔCD, a mutant with functional catalytic cores that lacks ~ 75% of the CD, have enhanced enzyme activity as the truncated protein accumulates in the secretory pathway, and make full-length cilia that are completely motile . Furthermore, insects only express separate PHM and PAL proteins , and while for many (e.g., Drosophila) the PAL protein also has an associated TMD/CD, recent database searches suggest that one insect order—the lepidoptera—express only soluble PAL lacking the TMD/CD again indicating the catalytic domains may be important. Although insects lack multiciliated epithelial cells, they still build ciliated motile sperm and use modified cilia for mechano- and chemo-sensation and in the chordotonal organ [91, 92, 93]. These experimental and bioinformatics observations suggest that the PAM CD may not play a fundamentally essential role in cilia formation and thus that the catalytic domains likely provide a key ciliogenic factor. The ability of PAM to amidate the C-termini of proteins (e.g., ubiquitin and monoclonal antibody heavy chains ), selected lipids [95, 96] and other metabolites, complicates the search for substrates. Site-directed mutants lacking PHM and/or PAL activity will be needed to definitively answer this important question.
Models for the role of PAM in ciliogenesis
What function(s) might ciliary localized PAM perform?
In addition to its role in cilia formation, PAM is also trafficked into these organelles; in Chlamydomonas, it becomes stably associated with the microtubular axoneme . The topology of ciliary-PAM places its catalytic domains on the external face of the organelle. Although PHM has an acidic pH optimum, it retains some activity under neutral conditions; in the presence of adequate copper, ascorbate and molecular oxygen, cilia-localized PAM is expected to be catalytically active. Thus, ciliary PAM could act on glycine-extended proteins present in the extracellular environment or associated with the external face of the ciliary membrane, generating amidated products which might play a role in signaling or some other process. In situations where cilia interact, e.g., during mating in Chlamydomonas, ciliary PAM might function in trans, amidating substrates present on the surface of the interacting cilium. In Chlamydomonas, the ciliary membrane is the only membrane normally directly exposed to the environment (except when the cell wall is shed during the mating reaction). Local generation of amidated factors might play a key role in providing a sufficiently high concentration of modified products for productive downstream signaling. As PAM CD binds actin directly with sub-μM affinity , it is also possible that ciliary PAM alters the formation or dynamics of short branched actin filaments within cilia, a process which is thought to control primary ciliogenesis in animal cells [98, 99].
Intriguingly, PAM is tightly associated with the axoneme and in vitro, unlike most ciliary membrane proteins, PAM is only solubilized when demembranated cilia are treated with increased salt concentrations, as observed for several other axonemal substructures such as the dynein arms. This interaction might occur directly, through association of the intraciliary PAM CD with the axoneme, or indirectly, through an axoneme-associated component (which might even extend across the membrane into the extracellular space). Why is this unusual transmembrane protein tethering necessary? One possible explanation is that it provides a mechanism to avoid the general, non-specific or unregulated loss of membrane-associated PAM from the cilium—either in ectosomes budded from the ciliary membrane or from BBSome/IFT-mediated recycling back to the cell body. A further puzzle is the apparent 250–300 nm spacing for PAM observed by immunogold EM ; this spacing does not directly correlate with other known axonemal repeat distances.
A phylogenetic conundrum—many organisms lacking PAM still build cilia
Genomic studies reveal that numerous other ciliated groups, including excavates (e.g., Trypanosoma), stramenopiles (e.g., diatoms, oomycetes and brown algae), and fornicates (e.g., Giardia), also completely lack PAM. Intriguingly, a more complex pattern of loss is apparent in the ciliated alveolates. Some, such as Tetrahymena and Paramecium, do not encode PAM-like sequences. However, PHM- (EX872387) and PAL-like (EX872386) partial sequences were previously reported in another alveolate—the dinoflagellate Karenia brevis . Furthermore, there are now two examples of PAM-like genes in ciliated haptophytes—unicellular organisms abundant in the marine phytoplankton; both Emiliania huxleyi and Chrysochromulina sp. express a soluble PHM + PAL (XP_005764558 and KOO21218, respectively). Haptophytes have calcite-based coccoliths forming an exoskeleton and two motile cilia. Furthermore, they assemble an additional thin microtubule-based cellular protrusion (the haptonema), and use bidirectional transport along this structure for food/prey retrieval  in a process that, at least superficially, is remarkably reminiscent of IFT-driven bead transport along Chlamydomonas cilia [101, 102]; haptonema may also play a sensory role. Together, these observations suggest that the presence/absence of PAM may define a fundamental dichotomy in the assembly and/or function of cilia and divide the ciliated eukaryotes into two broad groupings. Identification of genes expressed in ciliated organisms encoding PAM but missing in ciliated organisms lacking PAM may provide a path to revealing a unique subset of genes that affect or define this intriguing ciliogenic pathway.
A broad and diverse array of non-ciliated unicellular organisms (e.g., yeasts, amoebae, rhizarians such as Reticulomyxa, and the red alga Cyanidioschyzon merolae) lack PAM. However, that PAM likely plays non-cilia related roles in at least some unicellular organisms is evidenced by its retention in the filasterean C. owczarzaki, which appears to lack all cilia-specific genes . Similarly, the planktonic pico-chlorophyte Ostreococcus lucimarinus, which is entirely missing any IFT machinery, also has PAM; this organism does not build cilia and retains only a single axonemal inner arm dynein that may have been repurposed for a non-ciliary role [104, 105]. Likewise, the presence of PHM sequences in both Coccomyxa sp. and Chlorella variabilis  further suggest a cilia-independent role for amidation in green algae; intriguingly, although both these organisms encode components of the outer dynein arm [106, 107], they lack most of the IFT system and neither has been observed to form cilia.
Implications for cilia-based signaling and ciliopathies
Ciliopathies represent a broad group of multisystemic disorders that derive from defective cilia-based signaling and/or motile behavior [12, 17]. Signaling-associated phenotypes can include severe brain malformations (e.g., Joubert and Meckel Syndromes ), skeletal abnormalities (e.g., juvenile thoracic dystrophy and short rib polydactyly ), polycystic kidney disease (the most common genetic disorder in humans with an incidence of ~ 1/1000) , and other complex syndromes with multiple overlapping clinical features such as rod/cone dystrophy, mental retardation, obesity, anosmia, and insulin resistance (e.g., Bardet–Biedl syndrome ). Recent studies have revealed human genetic variants in PAM affecting insulin resistance , altered risk for diabetes , and hypertension with associated insulin resistance and altered low density lipoprotein levels . As many ciliopathies include endocrine features such as obesity in their pathology, this raises the possibility that ciliary PAM defects contribute to these complex phenotypes.
Defects in motile cilia result in primary ciliary dyskinesia . Phenotypes include inhibition of sperm motility, resulting in male infertility. More generally, defective ciliary motility compromises lung function as secreted mucus, which acts as a protectant, cannot be cleared, and cerebrospinal fluid flow in the brain ventricles is restricted, leading to hydrocephalus. In addition, left–right patterning is disrupted by cilia dysfunction at the embryonic node, resulting in situs inversus or heterotaxy. Lack of cilia-driven flow in the fallopian tubes, which is required to move oocytes to the uterus, causes female infertility. In these latter cases, it is the cilia-driven movement of fluid bathing the ciliated epithelium that is required for normal physiology. Although no obvious motile cilia phenotypes (such as laterality defects) have yet been described in PAM knockout mice, the zebrafish null mutants exhibit hydrocephalus and kidney-associated cysts, both of which can derive from defective ciliary motility in this organism.
Recent studies revealed an unexpected role for the peptide amidating monooxygenase (PAM), a highly conserved copper-, molecular oxygen- and ascorbate-dependent secretory pathway enzyme, in building cilia. This connection has been observed in both vertebrates and algae, suggesting that it dates to the last common ancestor of eukaryotes and represents a fundamental feature of ciliogenesis. Current data support a role for amidating activity in forming the ciliary gate and regulation of the IFT system. Numerous studies have also revealed a connection between PAM and control of the actin cytoskeleton, and lead to the suggestion that PAM might play a central role in trafficking of membrane or other components to these cytoskeleton-based cellular extensions. Key issues for the future include identifying the PAM substrate(s) involved in ciliogenesis and determining the role and fate of ciliary-localized PAM and its potential amidated products.
We thank all our colleagues and collaborators who helped unravel the intriguing connections between PAM, actin and cilia.
This manuscript was prepared, edited and approved by all authors.
This work was supported by Grants DK032949 (to BAE), GM051293 (to SMK) and GM125606 (to SMK and BAE) from the National Institutes of Health. DK is a Jane Coffin Childs postdoctoral fellow.
Compliance with ethical standards
Conflict of interest
We have no competing interests.
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