Amelioration of hepatic steatosis by dietary essential amino acid-induced ubiquitination.

Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.

Stuxnet fine-tunes Notch dose during development using a functional Polycomb response element.

Evolutionarily conserved Notch signaling is highly sensitive to changes in Notch receptor dose caused by intrinsic and environmental fluctuations. It is well known that epigenetic regulation responds dynamically to genetic, cellular and environmental stresses. However, it is unclear whether the Notch receptor dose is directly regulated at the epigenetic level. Here, by studying the role of the upstream epigenetic regulator Stuxnet (Stx) in Drosophila developmental signaling, we find that Stx promotes Notch receptor mRNA expression by counteracting the activity of Polycomb repressive complex 1 (PRC1). In addition, we provide evidence that Notch is a direct PRC1 target by identifying and validating in vivo the only bona fide Polycomb response element (PRE) among the seven Polycomb group (PcG)-binding sites revealed by DamID-seq and ChIP-seq analysis. Importantly, in situ deletion of this PRE results in increased Notch expression and phenotypes resembling Notch hyperactivation in cell fate specification. These results not only underscore the importance of epigenetic regulation in fine-tuning the Notch activity dose, but also the need to assess the physiological significance of omics-based PcG binding in development.

Therapeutic siRNA targeting PLIN2 ameliorates steatosis, inflammation, and fibrosis in steatotic liver disease models.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease worldwide. If left untreated, MASLD can progress from simple hepatic steatosis to metabolic dysfunction-associated steatohepatitis, which is characterized by inflammation and fibrosis. Current treatment options for MASLD remain limited, leaving substantial unmet medical needs for innovative therapeutic approaches. Here, we show that PLIN2, a lipid droplet protein inhibiting hepatic lipolysis, serves as a promising therapeutic target for MASLD. Hepatic PLIN2 levels were markedly elevated in multiple MASLD mouse models induced by diverse nutritional and genetic factors. The liver-specific deletion of Plin2 exhibited significant anti-MASLD effects in these models. To translate this discovery into a therapeutic application, we developed a GalNAc-siRNA conjugate with enhanced stabilization chemistry and validated its potent and sustained efficacy in suppressing Plin2 expression in mouse livers. This siRNA therapeutic, named GalNAc-siPlin2, was shown to be biosafe in mice. Treatment with GalNAc-siPlin2 for 6-8 weeks led to a decrease in hepatic triglyceride levels by approximately 60% in high-fat diet- and obesity-induced MASLD mouse models, accompanied with increased hepatic secretion of VLDL-triglyceride and enhanced thermogenesis in brown adipose tissues. Eight-week treatment with GalNAc-siPlin2 significantly improved hepatic steatosis, inflammation, and fibrosis in high-fat/high fructose-induced metabolic dysfunction-associated steatohepatitis models compared to control group. As a proof of concept, we developed a GalNAc-siRNA therapeutic targeting human PLIN2, which effectively suppressed hepatic PLIN2 expression and ameliorated MASLD in humanized PLIN2 knockin mice. Together, our results highlight the potential of GalNAc-siPLIN2 as a candidate MASLD therapeutic for clinical trials.

Protein modifications in Hedgehog signaling: Cross talk and feedback regulation confer divergent Hedgehog signaling activity.

The complexity of the Hedgehog (Hh) signaling cascade has increased over the course of evolution; however, it does not suffice to accommodate the dynamic yet robust requirements of differential Hh signaling activity needed for embryonic development and adult homeostatic maintenance. One solution to solve this dilemma is to apply multiple forms of post-translational modifications (PTMs) to the core Hh signaling components, modulating their abundance, localization, and signaling activity. This review summarizes various forms of protein modifications utilized to regulate Hh signaling, with a special emphasis on crosstalk between different forms of PTMs and their feedback regulation by Hh signaling.

Ubpy controls the stability of the ESCRT-0 subunit Hrs in development.

Ubiquitylated developmental membrane signaling proteins are often internalized for endocytic trafficking, through which endosomal sorting complexes required for transport (ESCRT) act sequentially to deliver internalized cargos to lysosomes. The ESCRT function in endocytic sorting is well established; however, it is not fully understood how the sorting machinery itself is regulated. Here, we show that Ubiquitin isopeptidase Y (Ubpy) plays a conserved role in vivo in the homeostasis of an essential ESCRT-0 complex component Hrs. We find that, in the absence of Drosophila Ubpy, multiple membrane proteins that are essential components of important signaling pathways accumulate in enlarged, aberrant endosomes. We further demonstrate that this phenotype results from endocytic pathway defects. We provide evidence that Ubpy interacts with and deubiquitylates Hrs. In Ubpy-null cells, Hrs becomes ubiquitylated and degraded in lysosomes, thus disrupting the integrity of ESCRT sorting machinery. Lastly, we find that signaling proteins are enriched in enlarged endosomes when Hrs activity is abolished. Together, our data support a model in which Ubpy plays a dual role in both cargo deubiquitylation and the ESCRT-0 stability during development.

Genetic interaction screens identify a role for hedgehog signaling in Drosophila border cell migration.

BACKGROUND: Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, our understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration. RESULTS: Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par-1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E-Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh-Rac and Hh-Par-1 signaling was illustrated in the wing, in which Hh-dependent phenotypes were enhanced by loss of Rac or par-1. CONCLUSIONS: We identified a pathway by which Hh signaling connects to Rac and Par-1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways.

Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle.

BACKGROUND: Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. METHODS: Plin2, a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, we used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, we purified proteins from HEK293 cells and reconstituted in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. RESULTS: As a lipolysis inhibitor, Plin2 was significantly elevated in liver (P < 0.05) and muscle (P < 0.05) in patients with steatosis. Consistently, we found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. We further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. CONCLUSIONS: We identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. Our work may provide new strategies for the prevention and treatment of steatosis.

Dissection of the microRNA Network Regulating Hedgehog Signaling in Drosophila.

The evolutionarily conserved Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. Aberrant Hh signaling often leads to various forms of developmental anomalies and cancer. Since altered microRNA (miRNA) expression is associated with developmental defects and tumorigenesis, it is not surprising that several miRNAs have been found to regulate Hh signaling. However, these miRNAs are mainly identified through small-scale in vivo screening or in vitro assays. As miRNAs preferentially reduce target gene expression via the 3' untranslated region, we analyzed the effect of reduced expression of core components of the Hh signaling cascade on downstream signaling activity, and generated a transgenic Drosophila toolbox of in vivo miRNA sensors for core components of Hh signaling, including hh, patched (ptc), smoothened (smo), costal 2 (cos2), fused (fu), Suppressor of fused (Su(fu)), and cubitus interruptus (ci). With these tools in hand, we performed a genome-wide in vivo miRNA overexpression screen in the developing Drosophila wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the in vivo Hh regulatory network. Moreover, these miRNAs may act as general regulators of Hh signaling, as their overexpression disrupts Hh signaling-mediated cyst stem cell maintenance during spermatogenesis. To identify direct targets of these newly discovered miRNAs, we used the miRNA sensor toolbox to show that miR-10 and miR-958 directly target fu and smo, respectively, while the other five miRNAs act through yet-to-be-identified targets other than the seven core components of Hh signaling described above. Importantly, through loss-of-function analysis, we found that endogenous miR-10 and miR-958 target fu and smo, respectively, whereas deletion of the other five miRNAs leads to altered expression of Hh signaling components, suggesting that these seven newly discovered miRNAs regulate Hh signaling in vivo. Given the powerful effects of these miRNAs on Hh signaling, we believe that identifying their bona fide targets of the other five miRNAs will help reveal important new players in the Hh regulatory network.

Competition between two phosphatases fine-tunes Hedgehog signaling.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. How its signaling activity is fine-tuned in response to fluctuated Hh gradient is less known. Here, we identify protein phosphatase V (PpV), the catalytic subunit of protein phosphatase 6, as a homeostatic regulator of Hh signaling. PpV is genetically upstream of widerborst (wdb), which encodes a regulatory subunit of PP2A that modulates high-level Hh signaling. We show that PpV negatively regulates Wdb stability independent of phosphatase activity of PpV, by competing with the catalytic subunit of PP2A for Wdb association, leading to Wdb ubiquitination and subsequent proteasomal degradation. Thus, regulated Wdb stability, maintained through competition between two closely related phosphatases, ensures graded Hh signaling. Interestingly, PpV expression is regulated by Hh signaling. Therefore, PpV functions as a Hh activity sensor that regulates Wdb-mediated PP2A activity through feedback mechanisms to maintain Hh signaling homeostasis.

A widely diverged locus involved in locomotor adaptation in Heliconius butterflies.

Heliconius butterflies have undergone adaptive radiation and therefore serve as an excellent system for exploring the continuum of speciation and adaptive evolution. However, there is a long-lasting paradox between their convergent mimetic wing patterns and rapid divergence in speciation. Here, we characterize a locus that consistently displays high divergence among Heliconius butterflies and acts as an introgression hotspot. We further show that this locus contains multiple genes related to locomotion and conserved in Lepidoptera. In light of these findings, we consider that locomotion traits may be under selection, and if these are heritable traits that are selected for, then they might act as species barriers.

The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development.

Wingless (Wg)/Wnt signaling is conserved in all metazoan animals and plays critical roles in development. The Wg/Wnt morphogen reception is essential for signal activation, whose activity is mediated through the receptor complex and a scaffold protein Dishevelled (Dsh). We report here that the exon junction complex (EJC) activity is indispensable for Wg signaling by maintaining an appropriate level of Dsh protein for Wg ligand reception in Drosophila. Transcriptome analyses in Drosophila wing imaginal discs indicate that the EJC controls the splicing of the cell polarity gene discs large 1 (dlg1), whose coding protein directly interacts with Dsh. Genetic and biochemical experiments demonstrate that Dlg1 protein acts independently from its role in cell polarity to protect Dsh protein from lysosomal degradation. More importantly, human orthologous Dlg protein is sufficient to promote Dvl protein stabilization and Wnt signaling activity, thus revealing a conserved regulatory mechanism of Wg/Wnt signaling by Dlg and EJC.

Stuxnet Facilitates the Degradation of Polycomb Protein during Development.

Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene, stuxnet (stx), that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Our biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity.

A targeted in vivo RNAi screen reveals deubiquitinases as new regulators of Notch signaling.

Notch signaling is highly conserved in all metazoan animals and plays critical roles in cell fate specification, cell proliferation, apoptosis, and stem cell maintenance. Although core components of the Notch signaling cascade have been identified, many gaps in the understanding of the Notch signaling pathway remain to be filled. One form of posttranslational regulation, which is controlled by the ubiquitin-proteasome system, is known to modulate Notch signaling. The ubiquitination pathway is a highly coordinated process in which the ubiquitin moiety is either conjugated to or removed from target proteins by opposing E3 ubiquitin ligases and deubiquitinases (DUBs). Several E3 ubiquitin ligases have been implicated in ubiquitin conjugation to the receptors and the ligands of the Notch signaling cascade. In contrast, little is known about a direct role of DUBs in Notch signaling in vivo. Here, we report an in vivo RNA interference screen in Drosophila melanogaster targeting all 45 DUBs that we annotated in the fly genome. We show that at least four DUBs function specifically in the formation of the fly wing margin and/or the specification of the scutellar sensory organ precursors, two processes that are strictly dependent on the balanced Notch signaling activity. Furthermore, we provide genetic evidence suggesting that these DUBs are necessary to positively modulate Notch signaling activity. Our study reveals a conserved molecular mechanism by which protein deubiquitination process contributes to the complex posttranslational regulation of Notch signaling in vivo.

In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling.

Hedgehog (Hh) signaling is highly conserved in all metazoan animals and plays critical roles in many developmental processes. Dysregulation of the Hh signaling cascade has been implicated in many diseases, including cancer. Although key components of the Hh pathway have been identified, significant gaps remain in our understanding of the regulation of individual Hh signaling molecules. Here, we report the identification of novel regulators of the Hh pathway, obtained from an in vivo RNA interference (RNAi) screen in Drosophila. By selectively targeting critical genes functioning in post-translational modification systems utilizing ubiquitin (Ub) and Ub-like proteins, we identify two novel genes (dUba3 and dUbc12) that negatively regulate Hh signaling activity. We provide in vivo and in vitro evidence illustrating that dUba3 and dUbc12 are essential components of the neddylation pathway; they function in an enzyme cascade to conjugate the ubiquitin-like NEDD8 modifier to Cullin proteins. Neddylation activates the Cullin-containing ubiquitin ligase complex, which in turn promotes the degradation of Cubitus interruptus (Ci), the downstream transcription factor of the Hh pathway. Our study reveals a conserved molecular mechanism of the neddylation pathway in Drosophila and sheds light on the complex post-translational regulations in Hh signaling.

Sequential phosphorylation of smoothened transduces graded hedgehog signaling.

The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. We show that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) and further enhanced by casein kinase I (CKI). We found that protein phosphatase 1 (PP1) directly dephosphorylated PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylated PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. We also established a functional link between sequentially phosphorylated Smo species and graded Hh activity. Thus, we propose a sequential phosphorylation model in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling.

Incredible journey: how do developmental signals travel through tissue?

How developmental signaling proteins traverse tissue during animal development, through or around tightly packed cells, remains an incompletely resolved mystery. Signaling protein movement is regulated to create gradients, control amounts, impose barriers, or provide direction. Signaling can be controlled by the rate of signal production, modification, active transport, trapping along the path, or by the properties of the receptor apparatus. Signals may move by diffusion outside cells, attached to migrating cells, attached to carrier molecules, through cells by transcytosis, along cell extensions, or in released membrane packets. Recent findings about the movement of Hedgehog, Wingless (Wnt), and TGF-beta signaling proteins have helped to clarify the molecular mechanisms used to ensure that developmental signals carry only good news.

Altered localization of Drosophila Smoothened protein activates Hedgehog signal transduction.

Hedgehog (Hh) signaling is critical for many developmental events and must be restrained to prevent cancer. A transmembrane protein, Smoothened (Smo), is necessary to transcriptionally activate Hh target genes. Smo activity is blocked by the Hh transmembrane receptor Patched (Ptc). The reception of a Hh signal overcomes Ptc inhibition of Smo, activating transcription of target genes. Using Drosophila salivary gland cells in vivo and in vitro as a new assay for Hh signal transduction, we investigated the regulation of Hh-triggered Smo stabilization and relocalization. Hh causes Smo to move from internal membranes to the cell surface. Relocalization is protein synthesis-independent and occurs within 30 min of Hh treatment. Ptc and the kinesin-related protein Costal2 (Cos2) cause internalization of Smo, a process that is dependent on both actin and microtubules. Disruption of endocytosis by dominant negative dynamin or Rab5 prevents Smo internalization. Fly versions of Smo mutants associated with human tumors are constitutively present at the cell surface. Forced localization of Smo at the plasma membrane activates Hh target gene transcription. Conversely, trapping of activated Smo mutants in the ER prevents Hh target gene activation. Control of Smo localization appears to be a crucial step in Hh signaling in Drosophila.