Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways.

Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.

Regulation of actin polymerization and adhesion-dependent cell edge protrusion by the Abl-related gene (Arg) tyrosine kinase and N-WASp.

Extracellular cues stimulate the Abl family nonreceptor tyrosine kinase Arg to promote actin-based cell edge protrusions. Several Arg-interacting proteins are potential links to the actin cytoskeleton, but exactly how Arg stimulates actin polymerization and cellular protrusion has not yet been fully elucidated. We used affinity purification to identify N-WASp as a novel binding partner of Arg. N-WASp activates the Arp2/3 complex and is an effector of Abl. We find that the Arg SH3 domain binds directly to N-WASp. Arg phosphorylates N-WASp on Y256, modestly increasing the affinity of Arg for N-WASp, an interaction that does not require the Arg SH2 domain. The Arg SH3 domain stimulates N-WASp-dependent actin polymerization in vitro, and Arg phosphorylation of N-WASp weakly stimulates this effect. Arg and N-WASp colocalize to adhesion-dependent cell edge protrusions in vivo. The cell edge protrusion defects of arg-/- fibroblasts can be complemented by re-expression of an Arg-yellow fluorescent protein (YFP) fusion, but not by an N-WASp binding-deficient Arg SH3 domain point mutant. These results suggest that Arg promotes actin-based protrusions in response to extracellular stimuli through phosphorylation of and physical interactions with N-WASp.

Inhibition of Rho via Arg and p190RhoGAP in the postnatal mouse hippocampus regulates dendritic spine maturation, synapse and dendrite stability, and behavior.

The RhoA (Rho) GTPase is a master regulator of dendrite morphogenesis. Rho activation in developing neurons slows dendrite branch dynamics, yielding smaller, less branched dendrite arbors. Constitutive activation of Rho in mature neurons causes dendritic spine loss and dendritic regression, indicating that Rho can affect dendritic structure and function even after dendrites have developed. However, it is unclear whether and how endogenous Rho modulates dendrite and synapse morphology after dendrite arbor development has occurred. We demonstrate that a Rho inhibitory pathway involving the Arg tyrosine kinase and p190RhoGAP is essential for synapse and dendrite stability during late postnatal development. Hippocampal CA1 pyramidal dendrites develop normally in arg-/- mice, reaching their mature size by postnatal day 21 (P21). However, dendritic spines do not undergo the normal morphological maturation in these mice, leading to a loss of hippocampal synapses and dendritic branches by P42. Coincident with this synapse and dendrite loss, arg-/- mice exhibit progressive deficits in a hippocampus-dependent object recognition behavioral task. p190RhoGAP localizes to dendritic spines, and its activity is reduced in arg-/- hippocampus, leading to increased Rho activity. Although mutations in p190rhogap enhance dendritic regression resulting from decreased Arg levels, reducing gene dosage of the Rho effector ROCKII can suppress the dendritic regression observed in arg-/- mice. Together, these data indicate that signaling through Arg and p190RhoGAP acts late during synaptic refinement to promote dendritic spine maturation and synapse/dendrite stability by attenuating synaptic Rho activity.

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase.

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.