Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCF^Fbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP, whose ubiquitination results in proteasome-mediated degradation, and c-IAP1, TRAF2 and NRAGE which are not destabilized as a result of ubiquitination. None of these substrates has been linked directly to PD, nor has it not been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCF^Fbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated Gsk3β, which can phosphorylate α-synuclein, and Tomm20, a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCF^Fbxo7substrates both in vitro and in vivo . Ubiquitin chain restriction (UbiCRest) analyses revealed Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localisation. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCF^Fbxo7 impacts directly on two proteins implicated in pathological processes leading to PD.

Ticks cause massive damage to livestock and vaccines are one sustainable substitute for the acaricides currently heavily used to control infestations. To guide antigen discovery for a vaccine that targets the gamut of parasitic strategies mediated by tick saliva and enables immunological memory, we exploited a transcriptome constructed from salivary glands from all stages of Rhipicephalus microplus ticks feeding on genetically tick-resistant and susceptible bovines. Different levels of host anti-tick immunity affected gene expression in tick salivary glands; we thus selected four proteins encoded by genes weakly expressed in ticks attempting to feed on resistant hosts or otherwise abundantly expressed in ticks fed on susceptible hosts; these sialoproteins mediate four functions of parasitism deployed by male ticks and that do not induce antibodies in naturally infected, susceptible bovines. We then evaluated in tick-susceptible heifers an alum-adjuvanted vaccine formulated with recombinant proteins. Parasite performance (i.e. weight and numbers of females finishing their parasitic cycle) and titres of antigen-specific antibodies were significantly reduced or increased, respectively, in vaccinated versus control heifers, conferring an efficacy of 73.2%; two of the antigens were strong immunogens, rich in predicted T-cell epitopes and challenge infestations boosted antibody responses against them. Mining sialotranscriptomes guided by the immunity of tick-resistant hosts selected important targets and infestations boosted immune memory against salivary antigens.

G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin biased agonists, such as [Sar1,Ile4,Ile8]-AngII (SII), and [Sar1,D-Ala8]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between “G protein-dependent” and “β-arrestin-dependent” signaling. We observed clusters of activation profiles common to AngII, SII and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization “G protein-dependent” vs. “β-arrestin-dependent” signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

In cancer, many genes are mutated by genome rearrangement, but our understanding of the functional consequences of this remains rudimentary. Here we report the F-box protein encoded byFBXL17is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased ubiquitination activity. Loss of the LRRs also differentially affected Fbxl17 binding to its targets. Thus, genomic rearrangements inFBXL17are likely to disrupt SCF^Fbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved inO-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevatedO-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway.