Alexander V. Loktev

A Core Complex of BBS Proteins Cooperates with the GTPase Rab8 to Promote Ciliary Membrane Biogenesis

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.

Prophase Destruction of Emi1 by the SCFβTrCP/Slimb Ubiquitin Ligase Activates the Anaphase Promoting Complex to Allow Progression beyond Prometaphase

Progression through mitosis occurs because cyclin B/Cdc2 activation induces the anaphase promoting complex (APC) to cause cyclin B destruction and mitotic exit. To ensure that cyclin B/Cdc2 does not prematurely activate the APC in early mitosis, there must be a mechanism delaying APC activation. Emi1 is a protein capable of inhibiting the APC in S and G2. We show here that Emi1 is phosphorylated by Cdc2, and on a DSGxxS consensus site, is subsequently recognized by the SCF(betaTrCP/Slimb) ubiquitin ligase and destroyed, thus providing a delay for APC activation. Failure of betaTrCP-dependent Emi1 destruction stabilizes APC substrates and results in mitotic catastrophe including centrosome overduplication, potentially explaining mitotic deficiencies in Drosophila Slimb/betaTrCP mutants. We hypothesize that Emi1 destruction relieves a late prophase checkpoint for APC activation.

The Ciliary G-Protein-Coupled Receptor Gpr161 Negatively Regulates the Sonic Hedgehog Pathway via cAMP Signaling

The primary cilium is required for Sonic hedgehog (Shh) signaling in vertebrates. In contrast to mutants affecting ciliary assembly, mutations in the intraflagellar transport complex A (IFT-A) paradoxically cause increased Shh signaling. We previously showed that the IFT-A complex, in addition to its canonical role in retrograde IFT, binds to the tubby-like protein, Tulp3, and recruits it to cilia. Here, we describe a conserved vertebrate G-protein-coupled receptor, Gpr161, which localizes to primary cilia in a Tulp3/IFT-A-dependent manner. Complete loss of Gpr161 in mouse causes midgestation lethality and increased Shh signaling in the neural tube, phenocopying Tulp3/IFT-A mutants. Constitutive Gpr161 activity increases cAMP levels and represses Shh signaling by determining the processing of Gli3 to its repressor form. Conversely, Shh signaling directs Gpr161 to be internalized from cilia, preventing its activity. Thus, Gpr161 defines a morphogenetic pathway coupling protein kinase A activation to Shh signaling during neural tube development.

A BBSome Subunit Links Ciliogenesis, Microtubule Stability, and Acetylation

Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth.

The Evi5 Oncogene Regulates Cyclin Accumulation by Stabilizing the Anaphase-Promoting Complex Inhibitor Emi1

The anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 controls progression to S phase and mitosis by stabilizing key APC/C ubiquitination substrates, including cyclin A. Examining Emi1 binding proteins, we identified the Evi5 oncogene as a regulator of Emi1 accumulation. Evi5 antagonizes SCF(betaTrCP)-dependent Emi1 ubiquitination and destruction by binding to a site adjacent to Emi1s DSGxxS degron and blocking both degron phosphorylation by Polo-like kinases and subsequent betaTrCP binding. Thus, Evi5 functions as a stabilizing factor maintaining Emi1 levels in S/G2 phase. Evi5 protein accumulates in early G1 following Plk1 destruction and is degraded in a Plk1- and ubiquitin-dependent manner in early mitosis. Ablation of Evi5 induces precocious degradation of Emi1 by the Plk/SCF(betaTrCP) pathway, causing premature APC/C activation; cyclin destruction; cell-cycle arrest; centrosome overduplication; and, finally, mitotic catastrophe. We propose that the balance of Evi5 and Polo-like kinase activities determines the timely accumulation of Emi1 and cyclin, ensuring mitotic fidelity.

Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia.

Human monogenic obesity syndromes, including Bardet-Biedl syndrome (BBS), implicate neuronal primary cilia in regulation of energy homeostasis. Cilia in hypothalamic neurons have been hypothesized to sense and regulate systemic energy status, but the molecular mechanism of this signaling remains unknown. Here, we report a comprehensive localization screen of 42 G-protein-coupled receptors (GPCR) revealing seven ciliary GPCRs, including the neuropeptide Y (NPY) receptors NPY2R and NPY5R. We show that mice modeling BBS disease or obese tubby mice fail to localize NPY2R to cilia in the hypothalamus and that BBS mutant mice fail to activate c-fos or decrease food intake in response to the NPY2R ligand PYY3-36. We find that cells with ciliary NPY2R show augmented PYY3-36-dependent cAMP signaling. Our data demonstrate that ciliary targeting of NPY receptors is important for controlling energy balance in mammals, revealing a physiologically defined ligand-receptor pathway signaling within neuronal cilia.

Comparative Proteomics Reveals Strain-Specific β-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex

Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and β-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated β-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets β-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote β-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.