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Highlights d Motins are destabilized upon serum or LPA treatment in a NF2-dependent manner d NF2 recruits RNF146 to Motins, promoting ubiquitination and degradation of Motins d Inactivation of Motins in NF2-deficient cells enhances oncogenic activity of YAP d High AMOT expression in NF2-null mesotheliomas is associated with good prognosis

Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials. gov: NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of factor VIII (FVIII) expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. In this study, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (e.g., cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking) with rAAV serotype 2, while rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed sitedirected mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy.

The Hippo pathway is a central regulator of organ size and tumorigenesis and is commonly depicted as a kinase cascade, with an increasing number of regulatory and adaptor proteins linked to its regulation over recent years. Here, we propose that two Hippo signaling modules, MST1/2-SAV1-WWC1-3 (HPO1) and MAP4K1-7-NF2 (HPO2), together regulate the activity of LATS1/2 kinases and YAP/TAZ transcriptional co-activators. In mouse livers, the genetic inactivation of either HPO1 or HPO2 module results in partial activation of YAP/TAZ, bile duct hyperplasia, and hepatocellular carcinoma (HCC). On the contrary, inactivation of both HPO1 and HPO2 modules results in full activation of YAP/TAZ, rapid development of intrahepatic cholangiocarcinoma (iCCA), and early lethality. Interestingly, HPO1 has a predominant role in regulating organ size. HPO1 inactivation causes a homogenous YAP/TAZ activation and cell proliferation across the whole liver, resulting in a proportional and rapid increase in liver size. Thus, this study has reconstructed the order of the Hippo signaling network and suggests that LATS1/ 2 and YAP/TAZ activities are finetuned by HPO1 and HPO2 modules to cause different cell fates, organ size changes, and tumorigenesis trajectories.

Type I interferon (IFN)‐induced Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling drives the expression of IFN‐stimulated genes (ISGs) to mediate antiviral response. The strength and duration of JAK‐STAT signaling are tightly regulated to ensure effective antiviral defense while avoiding pathological inflammation and autoimmunity. Here, we report that cTAZ, an isoform of the Hippo pathway effector TAZ, is transcribed by an alternative promoter. Although majority of C‐terminal sequences of TAZ is retained, cTAZ is not regulated by the Hippo signaling and does not mediate its growth‐inhibitory functions. Instead, cTAZ negatively regulates JAK‐STAT signaling by inhibiting STAT1/2 nuclear localization and ISG expression, and its expression is induced by type I IFN. Thus, cTAZ functions as a modulator of JAK‐STAT signaling and may play a role in fine‐tuning cellular antiviral response.

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WWC1 regulates episodic learning and memory, and genetic nucleotide polymorphism of WWC1 is associated with neurodegenerative diseases such as Alzheimer's disease. However, the molecular mechanism through which WWC1 regulates neuronal function remains unclear. Here, we show that WWC1 and its paralogs (WWC2/3) bind directly to angiomotin (AMOT) family proteins (Motins), and recruit USP9X to deubiquitinate and stabilize Motins. Deletion of WWC in different cell types, including neurons, leads to reduced protein levels of Motins. In mice, neuron-specific deletion of Wwc1 and Wwc2 results in lower density of the dendritic spine and impairment of cognitive functions. Interestingly, ectopic expression of AMOT partially rescues the neuronal phenotypes associated with Wwc1/2 deletion. Thus, WWC proteins modulate spinogenesis and cognition in part by regulating protein stability of Motins.

The scaffolding protein angiomotin (AMOT) is indispensable for vertebrate embryonic angiogenesis. Here, we report that AMOT undergoes cleavage in the presence of lysophosphatidic acid (LPA), a lipid growth factor also involved in angiogenesis. AMOT cleavage is mediated by aspartic protease DNA damage‐inducible 1 homolog 2 (DDI2), and the process is tightly regulated by a signaling axis including neurofibromin 2 (NF2), tankyrase 1/2 (TNKS1/2), and RING finger protein 146 (RNF146), which induce AMOT membrane localization, poly ADP ribosylation, and ubiquitination, respectively. In both zebrafish and mice, the genetic inactivation of AMOT cleavage regulators leads to defective angiogenesis, and the phenotype is rescued by the overexpression of AMOT‐CT, a C‐terminal AMOT cleavage product. In either physiological or pathological angiogenesis, AMOT‐CT is required for vascular expansion, whereas uncleavable AMOT represses this process. Thus, our work uncovers a signaling pathway that regulates angiogenesis by modulating a cleavage‐dependent activation of AMOT.