BBS Proteins Research Articles

Neofunctionalization of ciliary BBS proteins to nuclear roles is likely a frequent innovation across eukaryotes

The eukaryotic BBSome is a transport complex within cilia and assembled by chaperonin-like BBS proteins. Recent work indicates nuclear functions for BBS proteins in mammals, but it is unclear how common these are in extant proteins or when they evolved. We screened for BBS orthologues across a diverse set of eukaryotes, consolidated nuclear association via signal sequence predictions and permutation analysis, and validated nuclear localization in mammalian cells via fractionation and immunocytochemistry. BBS proteins are-with exceptions-conserved as a set in ciliated species. Predictions highlight five most likely nuclear proteins and suggest that nuclear roles evolved independently of nuclear access during mitosis. Nuclear localization was confirmed in human cells. These findings suggest that nuclear BBS functions are potentially not restricted to mammals, but may be a common frequently co-opted eukaryotic feature. Understanding the functional spectrum of BBS proteins will help elucidating their role in gene regulation, development, and disease.

Lymphocyte Polarization During Immune Synapse Assembly: Centrosomal Actin Joins the Game.

Interactions among immune cells are essential for the development of adaptive immune responses. The immunological synapse (IS) provides a specialized platform for integration of signals and intercellular communication between T lymphocytes and antigen presenting cells (APCs). In the T cell the reorganization of surface molecules at the synaptic interface is initiated by T cell receptor binding to a cognate peptide-major histocompatibility complex on the APC surface and is accompanied by a polarized remodelling of the cytoskeleton and centrosome reorientation to a subsynaptic position. Although there is a general agreement on polarizing signals and mechanisms driving centrosome reorientation during IS assembly, the primary events that prepare for centrosome repositioning remain largely unexplored. It has been recently shown that in resting lymphocytes a local polymerization of filamentous actin (F-actin) at the centrosome contributes to anchoring this organelle to the nucleus. During early stages of IS formation centrosomal F-actin undergoes depletion, allowing for centrosome detachment from the nucleus and its polarization towards the synaptic membrane. We recently demonstrated that in CD4+ T cells the reduction in centrosomal F-actin relies on the activity of a centrosome-associated proteasome and implicated the ciliopathy-related Bardet-Biedl syndrome 1 protein in the dynein-dependent recruitment of the proteasome 19S regulatory subunit to the centrosome. In this short review we will feature our recent findings that collectively provide a new function for BBS proteins and the proteasome in actin dynamics, centrosome polarization and T cell activation.

Composition and function of the C1b/C1f region in the ciliary central apparatus

Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.

A mouse model of BBS identifies developmental and homeostatic effects of BBS5 mutation and identifies novel pituitary abnormalities.

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss.

Genetic interaction of mammalian IFT-A paralogs regulates cilia disassembly, ciliary entry of membrane protein, Hedgehog signaling, and embryogenesis.

Primary cilia are sensory organelles that are essential for eukaryotic development and health. These antenna-like structures are synthesized by intraflagellar transport protein complexes, IFT-B and IFT-A, which mediate bidirectional protein trafficking along the ciliary axoneme. Here using mouse embryonic fibroblasts (MEF), we investigate the ciliary roles of two mammalian orthologues of Chlamydomonas IFT-A gene, IFT139, namely Thm1 (also known as Ttc21b) and Thm2 (Ttc21a). Thm1 loss causes perinatal lethality, and Thm2 loss allows survival into adulthood. At E14.5, the number of Thm1;Thm2 double mutant embryos is lower than that for a Mendelian ratio, indicating deletion of Thm1 and Thm2 causes mid-gestational lethality. We examined the ciliary phenotypes of mutant MEF. Thm1-mutant MEF show decreased cilia assembly, increased cilia disassembly, shortened primary cilia, a retrograde IFT defect for IFT and BBS proteins, and reduced ciliary entry of membrane-associated proteins. Thm1-mutant cilia also show a retrograde transport defect for the Hedgehog transducer, Smoothened, and an impaired response to Smoothened agonist, SAG. Thm2-null MEF show normal ciliary dynamics and Hedgehog signaling, but additional loss of a Thm1 allele impairs response to SAG. Further, Thm1;Thm2 double-mutant MEF show enhanced cilia disassembly, and increased impairment of INPP5E ciliary import. Thus, Thm1 and Thm2 have unique and redundant roles in MEF. Thm1 regulates cilia assembly, and alone and together with Thm2, regulates cilia disassembly, ciliary entry of membrane-associated protein, Hedgehog signaling, and embryogenesis. These findings shed light on mechanisms underlying Thm1-, Thm2- or IFT-A-mediated ciliopathies.

Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.

Bardet-Biedl Syndrome proteins regulate cilia disassembly during tissue maturation.

Primary cilia are conserved organelles that mediate cellular communication crucial for organogenesis and homeostasis in numerous tissues. The retinal pigment epithelium (RPE) is a ciliated monolayer in the eye that borders the retina and is vital for visual function. Maturation of the RPE is absolutely critical for visual function and the role of the primary cilium in this process has been largely ignored to date. We show that primary cilia are transiently present during RPE development and that as the RPE matures, primary cilia retract, and gene expression of ciliary disassembly components decline. We observe that ciliary-associated BBS proteins protect against HDAC6-mediated ciliary disassembly via their recruitment of Inversin to the base of the primary cilium. Inhibition of ciliary disassembly components was able to rescue ciliary length defects in BBS deficient cells. This consequently affects ciliary regulation of Wnt signaling. Our results shed light onto the mechanisms by which cilia-mediated signaling facilitates tissue maturation.

Intraflagellar transport is deeply integrated in hedgehog signaling.

The vertebrate hedgehog pathway is organized in primary cilia, and hedgehog components relocate into or out of cilia during signaling. Defects in intraflagellar transport (IFT) typically disrupt ciliary assembly and attenuate hedgehog signaling. Determining whether IFT drives the movement of hedgehog components is difficult due to the requirement of IFT for building cilia. Unlike most IFT proteins, IFT27 is dispensable for cilia formation but affects hedgehog signaling similarly to other IFTs, allowing us to examine its role in the dynamics of signaling. Activating signaling at points along the pathway in Ift27 mutant cells showed that IFT is extensively involved in the pathway. Similar analysis of Bbs mutant cells showed that BBS proteins participate at many levels of signaling but are not needed to concentrate Gli transcription factors at the ciliary tip. Our analysis showed that smoothened delivery to cilia does not require IFT27, but the role of other IFTs is not known. Using a rapamycin-induced dimerization system to sequester IFT-B proteins at the mitochondria in cells with fully formed cilia did not affect the delivery of Smo to cilia, suggesting that this membrane protein may not require IFT-B for delivery.

BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

Bardet-Biedl syndrome is a model ciliopathy. Although the characterization of BBS proteins has evidenced their involvement in cilia, extraciliary functions for some of these proteins are also being recognized. Importantly, understanding both cilia and cilia-independent functions of the BBS proteins is key to fully dissect the cellular basis of the syndrome. Here we characterize a functional interaction between BBS4 and the secreted protein FSTL1, a protein linked to adipogenesis and inflammation among other functions. We show that BBS4 and cilia regulate FSTL1 mRNA levels, but BBS4 also modulates FSTL1 secretion. Moreover, we show that FSTL1 is a novel regulator of ciliogenesis thus underscoring a regulatory loop between FSTL1 and cilia. Finally, our data indicate that BBS4, cilia and FSTL1 are coordinated during the differentiation of 3T3-L1 cells and that FSTL1 plays a role in this process, at least in part, by modulating ciliogenesis. Therefore, our findings are relevant to fully understand the development of BBS-associated phenotypes such as obesity.

Bardet-Biedl syndrome: Genetics, molecular pathophysiology, and disease management.

Primary cilia play a key role in sensory perception and various signaling pathways. Any defect in them leads to group of disorders called ciliopathies, and Bardet–Biedl syndrome (BBS, OMIM 209900) is one among them. The disorder is clinically and genetically heterogeneous, with various primary and secondary clinical manifestations, and shows autosomal recessive inheritance and highly prevalent in inbred/consanguineous populations. The disease mapped to at least twenty different genes (BBS1-BBS20), follow oligogenic inheritance pattern. BBS proteins localizes to the centerosome and regulates the biogenesis and functions of the cilia. In BBS, the functioning of various systemic organs (with ciliated cells) gets deranged and results in systemic manifestations. Certain components of the disease (such as obesity, diabetes, and renal problems) when noticed earlier offer a disease management benefit to the patients. However, the awareness of the disease is comparatively low and most often noticed only after severe vision loss in patients, which is usually in the first decade of the patient's age. In the current review, we have provided the recent updates retrieved from various types of scientific literature through journals, on the genetics, its molecular relevance, and the clinical outcome in BBS. The review in nutshell would provide the basic awareness of the disease that will have an impact in disease management and counseling benefits to the patients and their families.

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms.

Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators.

Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients.

Cilia born out of shock and stress

Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins. Centrioles are barrel‐shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule‐organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle‐dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule‐dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM‐1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet‐Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: …

Bardet–Biedl syndrome proteins control the cilia length through regulation of actin polymerization

Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet–Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanisms by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4- and Bbs6-deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, fewer mutant cells were ciliated [48% ± 6 for wild-type (WT) cells versus 23% ± 7 for Bbs4 null cells; P < 0.0001] and their cilia were shorter (2.55 μm ± 0.41 for WT cells versus 2.16 μm ± 0.23 for Bbs4 null cells; P < 0.0001). While the microtubular cytoskeleton and cortical actin were intact, actin stress fibre formation was severely disrupted, forming abnormal apical stress fibre aggregates. Furthermore, we observed over-abundant focal adhesions (FAs) in Bbs4-, Bbs6- and Bbs8-deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerization, we showed that RhoA-GTP levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4-deficient cells with chemical inhibitors of RhoA, we were able to restore the cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control the cilia length through alteration of RhoA levels.

Bardet-Biedl syndrome proteins control cilia length through regulation of actin polymerisation

Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet-Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanism by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4 and Bbs6 deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, few cells were ciliated (48% ± 6 for WT cells vs 23% ± 7 for Bbs4 null cells) and bear shorter cilia (2.55 μm ± 0.41 for WT cells vs 2.16 μm ± 0.23 for Bbs4 null cells). Whilst the microtubular cytoskeleton and cortical actin was intact, the actin cytoskeleton was severely disrupted, forming abnormal apical stress fiber aggregates. Furthermore, we observed over-abundant focal adhesions in Bbs4, Bbs6 and Bbs8-deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerisation, we showed that RhoA-GTP (activated form) levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4-deficient cells with a RhoA inhibitor, Y27632, we were able to restore cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control cilia length through alteration of RhoA levels.

Alteration of nephrocystins and IFT-A proteins causes similar ciliary phenotypes leading to Nephronophthisis

Nephronophtisis (NPH) is a kidney ciliopathy often associated with extra-renal defects and for which 12 genes (NPHP1-12) have been identified. NPHP1 and NPHP4 control the ciliary access at the transition zone and the velocity of some intraflagellar transport (IFT)/BBS proteins in C.elegans. Recently, in a collaborative effort, we have identified, in families with isolated NPH, mutations in TTC21B as well as in WDR19, which encode the retrograde IFT-A proteins IFT139 and IFT144, respectively. By ciliome sequencing of 1600 candidate genes from 14 NPH patients followed by Sanger sequencing of a cohort of 52 patients, we have found respectively 8 and 7 patients carrying pathogenic missense mutations in genes coding IFT-A proteins, including WDR35, TTC21B and IFT140, which could partially affect their function. Together, these results indicate that IFT-A are involved in nephronophtisis. Moreover, alteration of cilia length was observed in patient kidney, Nphp4-/- mice kidney tubules and NPHP1 or NPHP4 knockdown IMCD3 cell lines. In these cells, primary cilia present swellings at the distal region accompanied by an accumulation of IFT-B at the base and the tip, similar to what was observed in IFT-A mutants, suggesting a possible alteration of retrograde transport. Additionally, ARL13B, a small GTPase required for proper cilium shape and IFT stability, is absent along the axoneme of NPHP4-KD-IMCD cells. By controlling the entry of ciliary components at the transition zone, NPHP1 and NPHP4 may modulate IFT-A cargos thus participating in the same pathway (i.e. Wnt/PCP), alteration of which would lead to renal lesions observed in nephronophthisis.

The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.

A Rab8 Guanine Nucleotide Exchange Factor-Effector Interaction Network Regulates Primary Ciliogenesis

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we show that a highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Using immunofluorescence microscopy, we found that Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.

Hyperactive Neuroendocrine Secretion Causes Size, Feeding, and Metabolic Defects of C. elegans Bardet-Biedl Syndrome Mutants

Bardet-Biedl syndrome, BBS, is a rare autosomal recessive disorder with clinical presentations including polydactyly, retinopathy, hyperphagia, obesity, short stature, cognitive impairment, and developmental delays. Disruptions of BBS proteins in a variety of organisms impair cilia formation and function and the multi-organ defects of BBS have been attributed to deficiencies in various cilia-associated signaling pathways. In C. elegans, bbs genes are expressed exclusively in the sixty ciliated sensory neurons of these animals and bbs mutants exhibit sensory defects as well as body size, feeding, and metabolic abnormalities. Here we show that in contrast to many other cilia-defective mutants, C. elegans bbs mutants exhibit increased release of dense-core vesicles and organism-wide phenotypes associated with enhanced activities of insulin, neuropeptide, and biogenic amine signaling pathways. We show that the altered body size, feeding, and metabolic abnormalities of bbs mutants can be corrected to wild-type levels by abrogating the enhanced secretion of dense-core vesicles without concomitant correction of ciliary defects. These findings expand the role of BBS proteins to the regulation of dense-core-vesicle exocytosis and suggest that some features of Bardet-Biedl Syndrome may be caused by excessive neuroendocrine secretion.

Abstract SY14-02: From disease genetics to diagnostics to functional biology: High-throughput proteomics and protein interaction network building guides discovery of novel disease genes in ciliopathies, cystic disease, and cancer

Abstract With the advent of deep sequencing, genome-wide association studies, and rapid advances in human genetics, extensive genomic data linking human gene variants to specific diseases, including cancer, neurological disease, and metabolic syndromes, is rapidly accumulating. These disease associations, especially mutations, provide a definitive link between protein function and disease state, and can provide critical diagnostics for the disease. However, in many cases, the disease links are unclear and fail to 1) connect individual mutations to specific biological pathways, 2) provide functional hypotheses for understanding disease mechanism, or 3) identify targets for therapeutic intervention. Bioinformatic analyses can provide key clues to specific experimental tests, but a more extensible methodology is required to build from genomic hits to functional links. We have developed and implemented automated, a recombination-driven “tandem-affinity” proteomic probes, called the g-LAP (Gateway-Location and Affinity Purification) system. This approach is amenable to high-throughput and allows us to purify proteins associated with disease-related “bait” proteins and thus identify protein complexes and networks with high confidence. We have also established similarly high-throughput tools for studying protein localization, protein-protein interaction both in vivo and in vitro, and for testing interactions among biochemical complexes and activities. To date, we have tagged over 100 new human genetic disease genes, which have lent insight into a number of important pathways, notably in ciliary diseases or ciliopathies, neurological disease, and for novel tumor suppressors. Within the ciliopathies, the identification of assembly and regulatory complexes has provided new insight into the cell biology and signaling pathways associated with primary cilia. In the case of Bardet-Biedl syndrome, patients present with retinal degeneration, olfactory and hearing loss, polydactyly, neurological problems, and obesity linked to hyperphagia. Purifying proteins associated with LAP-tagged BBS proteins, we discovered seven of fourteen known BBS disease genes were present in the highly conserved BBSome complex, important to activate Rab8 vesicles for ciliary membrane formation1. In the tubby/Tubby-like (TULP) family of proteins, we recently purified the Hedgehog regulatory TULP3 protein and found the protein forms a complex together with the complete, six-component intraflagellar transport A (IFT-A) complex, which function together at the base of the cilia to regulate entry of G-protein coupled receptors into cilia2. A link between cilia and cancer was demonstrated by the link between cilia, Hedgehog signaling, and specific receptor tyrosine kinases, including the PDGF receptor α. Among cancers and proliferative diseases, specific ciliary pathways function in the kidney to limit polycystic kidney disease and renal cell carcinoma. Notably, the Von Hippel-Lindau (VHL) tumor suppressor protein and autosomal dominant polycystic kidney disease genes PKD1 and PKD2 proteins are present in primary cilia. Among ciliopathies, several may control the polarity and function of ductal structures of the kidney, but also of the pancreas, and liver. Do ciliary genes play an important role in cancer progression in these tissues? The molecular mechanism suppressing formation of cysts is poorly understood, but may include changes in the apical-basal polarity of cells, cell cycle, or secretory behavior of cells. Specific changes in ciliary signaling mechanisms may be important for cyst suppression. Among cystic regulators, nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Nephronophthisis (NPHP) is a rare pediatric autosomal cystic kidney disease caused by mutations of nine known genes, NPHP1-9. NPHP patients also show cerebellar defects, situs inversus, and retinitis pigmentosa (RP). The pediatric Joubert (JBST) and Meckel-Gruber (MKS) syndromes also present nephronophthisis and RP, but are characterized by cerebellar malformation or severe neural tube defects. There are nine JBST and seven MKS genes, with considerable allelism among NPHP, JBST, and MKS genes. We used high-confidence proteomics to identify over 200 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules functioning at the apical cell surface and ciliary “transition zone” (NPHP1/4/8), at the basal body (NPHP5/6), or in a novel Hedgehog pathway (Mks1/Mks6/Tectonic). We employed assays for ciliogenesis and cell polarity defects using 3-D renal cultures to demonstrate that renal cystic disease links primarily to apical organization defects, whereas ciliary and Hedgehog pathway defects appear central to neural malformations. Using 38 interactors as candidates, linkage and sequencing analysis of 250 NPHP patients identified TCTN2 as a new Joubert syndrome disease gene, plus two other novel NPHP human. Our Tctn2 mouse knockout showed neural tube and Hedgehog signaling defects. Our studies demonstrate the power of linking proteomic networks and human genetics to uncover critical disease pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY14-02. doi:10.1158/1538-7445.AM2011-SY14-02