Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases.

The covalent attachment of ubiquitin onto proteins can elicit a variety of downstream consequences. Attachment is mediated by a large array of E3 ubiquitin ligases, each thought be subject to regulatory control and to have a specific repertoire of substrates. Assessing the biological roles of ligases, and in particular, identifying their biologically relevant substrates has been a persistent yet challenging question. In this study, we describe tools that may help achieve both of these goals. We describe a strategy whereby the activity of a ubiquitin ligase has been enzymatically reversed, accomplished by fusing it to a catalytic domain of an exogenous deubiquitinating enzyme. We present a library of 72 "anti-ligases" that appear to work in a dominant-negative fashion to stabilize their cognate substrates against ubiquitin-dependent proteasomal and lysosomal degradation. We then used the ligase-deubiquitinating enzyme (DUb) library to screen for E3 ligases involved in post-Golgi/endosomal trafficking. We identify ligases previously implicated in these pathways (Rsp5 and Tul1), in addition to ligases previously localized to endosomes (Pib1 and Vps8). We also document an optimized workflow for isolating and analyzing the "ubiquitome" of yeast, which can be used with mass spectrometry to identify substrates perturbed by expression of particular ligase-DUb fusions.

CaMKII is essential for the proasthmatic effects of oxidation.

Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.

Biochemical and biological characterization of exosomes containing prominin-1/CD133.

Exosomes can be viewed as complex "messages" packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic-microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived.

ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting.

Mutations affecting ciliary components cause a series of related genetic disorders in humans, including nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and Bardet-Biedl syndrome (BBS), which are collectively termed "ciliopathies." Recent protein-protein interaction studies combined with genetic analyses revealed that ciliopathy-related proteins form several functional networks/modules that build and maintain the primary cilium. However, the precise function of many ciliopathy-related proteins and the mechanisms by which these proteins are targeted to primary cilia are still not well understood. Here, we describe a protein-protein interaction network of inositol polyphosphate-5-phosphatase E (INPP5E), a prenylated protein associated with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted to the primary cilium through a motif near the C terminus and prenyl-binding protein phosphodiesterase 6D (PDE6D)-dependent mechanisms. Ciliary targeting of INPP5E is facilitated by another JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by ARL2 or ARL3. ARL13B missense mutations that cause JBTS in humans disrupt the ARL13B-INPP5E interaction. We further demonstrate interactions of INPP5E with several ciliary and centrosomal proteins, including a recently identified ciliopathy protein centrosomal protein 164 (CEP164). These findings indicate that ARL13B, INPP5E, PDE6D, and CEP164 form a distinct functional network that is involved in JBTS and NPHP but independent of the ones previously defined by NPHP and MKS proteins.