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Hutchinson–Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder characterized by premature death from myocardial infarction or stroke. It is caused by de novo single-nucleotide mutations in the LMNA gene that activate a cryptic splice donor site, resulting in the production of a toxic form of lamin A, which is termed progerin. Here we present a potential genetic therapeutic strategy that utilizes antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to block pathogenic splicing of mutant transcripts. Of several candidates, PPMO SRP-2001 provided the most significant decrease in progerin transcripts in patient fibroblasts. Intravenous delivery of SRP-2001 to a transgenic mouse model of HGPS produced significant reduction of progerin transcripts in the aorta, a particularly critical target tissue in HGPS. Long-term continuous treatment with SRP-2001 yielded a 61.6% increase in lifespan and rescue of vascular smooth muscle cell loss in large arteries. These results provide a rationale for proceeding to human trials. A modified antisense oligonucleotide that blocks pathogenic splicing of progeria transcripts reduces the production of progerin in key target organs and extends animal lifespan in mouse model of progeria syndrome.

Protein–metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein–lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid‐binding fingerprints of 172 proteins, including 91 with predicted lipid‐binding domains. We identified 530 protein–lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live‐cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid‐binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids. Synopsis Deciphering the molecular mechanisms behind cellular processes requires the systematic charting of the multitude of interactions between all cellular components. While protein–protein and protein–DNA networks have been the subject of many systematic surveys, other critically important cellular components, such as lipids, have to date rarely been studied in large‐scale interaction screens. Growing numbers of lipids are known to operate as signaling molecules. The importance of protein–lipid interactions is evident from the variety of protein domains that have evolved to bind particular lipids (Lemmon, 2008 #392) and from the large list of disorders, such as cancer and bipolar disorder, arising from altered protein–lipid interactions. The current understanding of protein–lipid recognition comes from the study of a limited number of lipids, principally PtdInsPs (Zhu et al , 2001 #16), and lipid‐binding domains (LBDs) in isolation (Dowler et al , 2000 #81; Yu and Lemmon, 2001 #396; Yu et al , 2004 #31). For other signaling lipids, such as sphingolipids, intracellular targets and molecular mechanisms are only partially understood (Hannun and Obeid, 2008 #397). The importance of lipids in biological processes and their under‐representation in current biological networks suggest the need for systematic, unbiased biochemical screens. To systematically study protein–lipid interactions, we developed miniaturized arrays that contained sets of 56 lipids covering the main lipid classes in yeast. We used the arrays to determine the binding profiles of 172 soluble proteins. The selection included proteins that contained one or several predicted LBD that were lipid regulated or enzymes involved in lipid metabolism (Figure 1 ). We obtained 530 protein–lipid interactions (accuracy and coverage: 61 and 60%, respectively). More than half were supported by additional experimental evidences obtained from a large validation effort using a variety of biochemical and cell biology approaches, and the integration of a data set of genetic interactions (Figure 1 ). As a substantial fraction (45%) of the analyzed proteins were conserved in humans, the protein–lipid data set will have functional implications for higher eukaryotes and thus for human biology. Overall, 68% of all interactions were novel or unexpected from either protein sequences or known LBDs specificities. We discovered cryptic LBDs that were previously undetected in Ecm25 (a RhoGAP) and Ira2 (a RasGAP). We also identified a set of proteins that bound sphingolipids, a class of bioactive lipids that play important signaling functions in yeast and higher eukaryotes. The exact mode of action for these lipids remains elusive and the data set points to series of new cellular targets. We identified 63 proteins, involved in endocytosis, cell polarity and lipid metabolism that interacted with sphingoid long‐chain bases (LCBs), ceramides or phosphorylated LCBs (Figure 5 ). Despite the importance of sphingolipids in signaling processes, only a few domains, such as START or Saposins, have been reported to specifically bind these lipids in higher eukaryotes, and none of them have been found in yeast. Interestingly, almost 60% of proteins binding to phosphorylated LCBs in our assay also contained a pleckstrin homology (PH) domain and bound PtdInsPs (Figure 5 ). This suggests some PH domains might have unanticipated ligands and also have a function in sphingolipid recognition. We showed, using a variety of biochemical and cell‐based assays, that the PH domain of Slm1, a component of the TORC2 signaling pathway (Fadri et al , 2005 #429), can bind PtdIns(4,5)P 2 and sphingolipid cooperatively. The structure of Slm1‐PH, which we solved by X‐ray crystallography at 2 Å resolution, suggests the presence of two positively charged binding pockets for anionic lipids. These results indicate that the PH domain of Slm1 might work as a coincidence sensor to integrate both PtdInsP and sphingolipid signaling pathways. This reinforces the emerging notion that cooperative mechanisms have important functions in PH domains functioning (Maffucci and Falasca, 2001 #528). These mechanisms initially described between PtdInsPs and proteins can now be extended to new lipid classes, illustrating the benefit of unbiased and systematic analyses. This work shows the feasibility and benefits of large‐scale analyses combining biochemical arrays and live‐cell imaging for charting protein–lipid interactions. Accurate representations of biological processes require systematic charting of the physical and functional links between all cellular components. There is a clear need to expand molecular interaction space from proteome‐ to metabolome‐wide efforts and of systematic classifications of bioactive molecules based on their binding profiles. The data provided here represents an excellent resource to enhance the understanding of lipids function in eukaryotic systems. Lipids are important cellular metabolites, with a wide range of structural and functional diversity. Many operate as signaling molecules. Lipids though have rarely been studied in large‐scale interaction screen; they are poorly represented in current biological networks. Here, we describe the use of miniaturized lipid–arrays for the large‐scale study of protein–lipid interactions. In yeast, we show general feasibility with a systematic screen implying 172 proteins. We report 530 protein–lipid associations, the majority is novel and several were validated using other techniques. The screen uncovers numerous insights into lipid function in yeast and equivalent systems in humans. It revealed (i) previously undetected cryptic lipid‐binding domains, (ii) series of new cellular targets for sphingolipids and (iii) new ligands for some PH domains that can cooperatively bind additional lipids and work as coincidence sensor to integrate both phosphatidylinositol phosphates and sphingolipid signaling pathways. The significant number of biological insights uncovered shows that even major classes of metabolites have been insufficiently studied. This illustrates the general relevance of such systematic screens and calls for further system‐wide analyses.

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS 8–13 , the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity. The X-ray crystal structure of a rat neurotensin receptor in complex with the C-terminal portion of neurotensin is presented; this is the first structure of a member of the β group of class A G-protein-coupled receptors. GPCR–peptide agonist structure determined Neurotensin is a short peptide that can act as a neurotransmitter, a digestive hormone, and a regulator of cardiac output and blood pressure. In this manuscript, the authors solve an X-ray crystal structure of the carboxy-terminal portion of neurotensin bound to a rat neurotensin receptor. This is the first structure of a member of the beta group of class A G-protein-coupled receptors (GPCRs), and the first published structure of a GPCR bound to a peptide agonist. This structure should facilitate the development of non-peptide drugs that could be used to treat neurological disorders, cancer and obesity.

Malignant tumors develop through multiple steps of initiation and progression, and tumor initiation is of singular importance in tumor prevention, diagnosis, and treatment. However, the molecular mechanism whereby a signaling network of interacting pathways restrains proliferation in normal cells and prevents tumor initiation is still poorly understood. Here, we have reported that the Hippo, Wnt/β-catenin, and Notch pathways form an interacting network to maintain liver size and suppress hepatocellular carcinoma (HCC). Ablation of the mammalian Hippo kinases Mst1 and Mst2 in liver led to rapid HCC formation and activated Yes-associated protein/WW domain containing transcription regulator 1 (YAP/TAZ), STAT3, Wnt/β-catenin, and Notch signaling. Previous work has shown that abnormal activation of these downstream pathways can lead to HCC. Rigorous genetic experiments revealed that Notch signaling forms a positive feedback loop with the Hippo signaling effector YAP/TAZ to promote severe hepatomegaly and rapid HCC initiation and progression. Surprisingly, we found that Wnt/β-catenin signaling activation suppressed HCC formation by inhibiting the positive feedback loop between YAP/TAZ and Notch signaling. Furthermore, we found that STAT3 in hepatocytes is dispensable for HCC formation when mammalian sterile 20-like kinase 1 and 2 (Mst1 and Mst2) were removed. The molecular network we have identified provides insights into HCC molecular classifications and therapeutic developments for the treatment of liver tumors caused by distinct genetic mutations.

In a new study, Yingzi Yang and her colleagues show that a careful balance between Wnt and Hedgehog signaling is required to maintain proper differentiation of osteogenic precursor cells. Upon mutation of GNAS , this balance is disturbed and severe bone disease develops, including either heterotopic ossification or fibrous dysplasia. Heterotopic ossification, the pathologic formation of extraskeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gα s restricts bone formation to the skeleton by inhibiting Hedgehog signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia, a human disease caused by null mutations in GNAS , which encodes Gα s , Hedgehog signaling is upregulated in ectopic osteoblasts and progenitor cells. In animal models, we show that genetically-mediated ectopic Hedgehog signaling is sufficient to induce heterotopic ossification, whereas inhibition of this signaling pathway by genetic or pharmacological means strongly reduces the severity of this condition. As our previous work has shown that GNAS gain-of-function mutations upregulate WNT–β-catenin signaling in osteoblast progenitor cells, resulting in their defective differentiation and fibrous dysplasia, we identify Gα s as a key regulator of proper osteoblast differentiation through its maintenance of a balance between the Wnt–β-catenin and Hedgehog pathways. Also, given the results here of the pharmacological studies in our mouse model, we propose that Hedgehog inhibitors currently used in the clinic for other conditions, such as cancer, may possibly be repurposed for treating heterotopic ossification and other diseases caused by GNAS inactivation.

Malignant tumors develop through multiple steps of initiation and progression, and tumor initiation is of singular importance in tumor prevention, diagnosis, and treatment. However, the molecular mechanism whereby a signaling network of interacting pathways restrains proliferation in normal cells and prevents tumor initiation is still poorly understood. Here, we have reported that the Hippo, Wnt/β-catenin, and Notch pathways form an interacting network to maintain liver size and suppress hepatocellular carcinoma (HCC). Ablation of the mammalian Hippo kinases Mst1 and Mst2 in liver led to rapid HCC formation and activated Yes-associated protein/WW domain containing transcription regulator 1 (YAP/TAZ), STAT3, Wnt/β-catenin, and Notch signaling. Previous work has shown that abnormal activation of these downstream pathways can lead to HCC. Rigorous genetic experiments revealed that Notch signaling forms a positive feedback loop with the Hippo signaling effector YAP/TAZ to promote severe hepatomegaly and rapid HCC initiation and progression. Surprisingly, we found that Wnt/β- catenin signaling activation suppressed HCC formation by inhibiting the positive feedback loop between YAP/TAZ and Notch signaling. Furthermore, we found that STAT3 in hepatocytes is dispensable for HCC formation when mammalian sterile 20-like kinase 1 and 2 (Mst1 and Mst2) were removed. The molecular network we have identified provides insights into HCC molecular classifications and therapeutic developments for the treatment of liver tumors caused by distinct genetic mutations.

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS.sub.8-13, the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

Abstract Heterotopic ossification (HO), either acquired or hereditary, is featured by ectopic bone formation outside of the normal skeleton. The acquired form of HO is a debilitating and common complication of musculoskeletal trauma, central nervous system injury, burns, combat trauma, hip and elbow fractures, and total joint replacement surgeries. It can be characterized as abnormal bone formation that occurs mostly by endochondral ossification. Recent studies have implicated inflammation and dysregulation of Hedgehog (Hh) signaling as major early contributors to HO formation. Here, we demonstrate that administration of the Hh pathway inhibitor, arsenic trioxide (ATO), prevented acquired HO in a clinically-relevant trauma/burn mouse model. We further evaluated the effects of two additional Hh pathway antagonists: cholecalciferol and pravastatin on mitigating osteoblast differentiation. Finally, we assessed the effect of a combination of Hh pathway inhibitors on reducing systemic proinflammatory responses. A targeted combination approach using Hh pathway inhibitors may offer potential therapeutic benefits though targeting differential components of the Hh pathway. Taken together, our study demonstrates that the administration of single or multiple Hh pathway inhibitors may have the potential to reduce the formation of acquired HO. Competing Interest Statement JGJ and EVW are co-founders of Nostopharma LLC. All other authors declare no conflicts of interest with this work.

Skeletni sistem je kompleksan organ koji se formira tokom embriogeneze, znatno uvećava tokom detinjstva, remodeluje tokom celog života i regeneriše nakon povreda. Formiranje kostiju je striktno kontrolisan proces, vremenski i prostorno. Prostorno gledano, granice skeletnog sistema su jasno ograničene u ljudskom telu. Van skeletna formacija kostiju ili heterotopna osifikacija (HO) je patološka pojava koja se javlja sporadično i predstavlja klinički problem jer dovodi do bola, otoka i potpune imobilizacije zglobova. Heterotopna osifikacija je definisana prisustvom lamelarne koštane mase tkiva van skeletnog sistema. Heterotopna ili ektopična osifikacija je problem koji se javlja kao posledica traume na koštano ili meko tkivo, ili mnogo ređe kao posledica genetske mutacije kod retkih naslednih bolesti kao što je, na primer, progresivna koštana heteroplazija (engleski Progressive Osseous Heteroplasia ili POH). Mehanizam ove osifikacije nije dovoljno razjašnjen, ali je poznato da se glavni defekt dešava na nivou neadekvatne i ubrzane metaplastične osifikacije koja ima za rezultat formiranje lamelarne kortikosteroidne kosti. Pretpostavlja se da su multipotente mezenhimske matične ćelije (MSC), ćelije koje se diferenciraju u osteoblaste. Stimulatorni G proteinski kompleks je nedavno prepoznat kao ključni regulator mezenhimskih ćelija skeletnog sistema. Inaktivirajuća mutacija alfa subjedinice stimulatornog G proteinskog regulatornog kompleksa (Gαs) adenil ciklaze (GNAS1), koja se javlja kod retke genetske bolesti POH, je glavni uzrok fenotipa - nekontrolisanog i progresivnog formiranja patološke osifikacije van skeletnog sistema, najčešće u okolnom mekom tkivu. Kao što često biva, iz primera retkih bolesti i mutacija koje one nose, možemo naučiti dosta o osnovnim biološkim mehanizmima i biološkim procesima u ćeliji.Cilj ovog rada je bio da se rasvetli mehanizam nastanka HO kao i da se ispita efikasnost tri inhibitora Hedgehog (Hh) signalnog puta, arsen trioksida (ATO), lovastatina (Lov) i aktivne forme vitamin D3 (VitD3) u sprečavanju osteogeneze u progenitorskim mezenhimalnim ćelijama i koncipira efikasna kombinacija leka primenom niskih koncentracija navedenih agenasa.Korišćen je animalni model za POH kao i humane i progenitorske mezenhimalnee ćelije miša. Analizom genske ekspresije praćen je sadržaj iRNK Gli1 gena koji je glavni marker Hh signalnog puta.Dokazano je da je Hedgehog signalni put dovoljan i neophodan za nastanak HO. Primena molekula male molekularne mase, Hh inhibitora, je rezultirala u značajnom smanjenju osteogene aktivnosti progenitorskih mezenhimskih ćelija. Pokazana je funkcionalna veza između inhibicije Hh signalnog puta i osteogeneze u progenitorskim mezenhimskim ćelijama. Analizom ekspresije gena utvrđene su smanjene koncentracije iRNK Gli1 gena. Kombinacija koja je sadržala niske koncentracije navedena tri Hh inhibitora se pokazala podjednako efikasna u inhibiciji Hh signalnog puta i osteogeneze u testiranim humanim i ćelijama miša kao i pojedinačno dejstvo agenasa.Predložena formulacija koja sadrži niske koncentracije antagonista Hh signalnog puta može biti efikasna u profilaksi HO.