A targeted antisense therapeutic approach for Hutchinson-Gilford progeria syndrome.

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.

Enpp1 inhibits ectopic joint calcification and maintains articular chondrocytes by repressing hedgehog signaling.

The differentiated phenotype of articular chondrocytes of synovial joints needs to be maintained throughout life. Disruption of the articular cartilage, frequently associated with chondrocyte hypertrophy and calcification, is a central feature in osteoarthritis (OA). However, the molecular mechanisms whereby phenotypes of articular chondrocytes are maintained and pathological calcification is inhibited remain poorly understood. Recently, the ecto-enzyme Enpp1, a suppressor of pathological calcification, was reported to be decreased in joint cartilage with OA in both human and mouse, and Enpp1 deficiency causes joint calcification. Here, we found that hedgehog (Hh) signaling activation contributes to ectopic joint calcification in the Enpp1-/- mice. In the Enpp1-/- joints, Hh signaling was upregulated. Further activation of Hh signaling by removing the patched 1 gene in the Enpp1-/- mice enhanced ectopic joint calcification, whereas removing Gli2 partially rescued the ectopic calcification phenotype. In addition, reduction of Gαs in the Enpp1-/- mice enhanced joint calcification, suggesting that Enpp1 inhibits Hh signaling and chondrocyte hypertrophy by activating Gαs-PKA signaling. Our findings provide new insights into the mechanisms underlying Enpp1 regulation of joint integrity.

Hippo signaling interactions with Wnt/ß-catenin and Notch signaling repress liver tumorigenesis.

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/ß-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/ß-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/ß-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.

Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification.

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 Gαs 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 Gαs, 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-ß-catenin signaling in osteoblast progenitor cells, resulting in their defective differentiation and fibrous dysplasia, we identify Gαs as a key regulator of proper osteoblast differentiation through its maintenance of a balance between the Wnt-ß-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.

Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination.

Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination.

Structure of the agonist-bound neurotensin receptor.

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.

Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid.

Membrane lipids have been implicated to influence the activity of G-protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus, insight gained from rhodopsin signaling may not be simply translated to other nonvisual GPCRs. Here, we investigated the effect of lipid head group charges on the signal transduction properties of the class A GPCR neurotensin (NT) receptor 1 (NTS1) under defined experimental conditions, using self-assembled phospholipid nanodiscs prepared with the zwitter-ionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), or a POPC/POPG mixture. A combination of dynamic light scattering and sedimentation velocity showed that NTS1 was monomeric in POPC-, POPC/POPG-, and POPG-nanodiscs. Binding of the agonist NT to NTS1 occurred with similar affinities and was essentially unaffected by the phospholipid composition. In contrast, Gq protein coupling to NTS1 in various lipid nanodiscs was significantly different, and the apparent affinity of Gαq and Gß(1)γ(1) to activated NTS1 increased with increasing POPG content. NTS1-catalyzed GDP/GTPγS nucleotide exchange at Gαq in the presence of Gß(1)γ(1) and NT was crucially affected by the lipid type, with exchange rates higher by 1 or 2 orders of magnitude in POPC/POPG- and POPG-nanodiscs, respectively, compared to POPC-nanodiscs. Our data demonstrate that negatively charged lipids in the immediate vicinity of a nonvisual GPCR modulate the G-protein-coupling step.

A systematic screen for protein-lipid interactions in Saccharomyces cerevisiae.

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.

Up-regulation of the cell integrity pathway in saccharomyces cerevisiae suppresses temperature sensitivity of the pgs1Delta mutant.

We have previously shown that mutants in the cardiolipin (CL) pathway exhibit temperature-sensitive growth defects that are not associated with mitochondrial dysfunction. The pgs1Delta mutant, lacking the first enzyme of the CL pathway, phosphatidylglycerolphosphate synthase (Pgs1p), has a defective cell wall due to decreased beta-1,3-glucan (Zhong, Q., Gvozdenovic-Jeremic, J., Webster, P., Zhou, J., and Greenberg, M. L. (2005) Mol. Biol. Cell 16, 665-675). Disruption of KRE5, a gene involved in cell wall biogenesis, restores beta-1,3-glucan synthesis and suppresses pgs1Delta temperature sensitivity. To gain insight into the mechanisms underlying the cell wall defect in pgs1Delta, we show in the current report that pgs1Delta cells have reduced glucan synthase activity and diminished levels of Fks1p, the glucan synthase catalytic subunit. In addition, activation of Slt2p, the downstream effector of the protein kinase C (PKC)-activated cell integrity pathway, was defective in pgs1Delta. The kre5W1166X suppressor restored Slt2p activation and dramatically increased (>10-fold) mRNA levels of FKS2, the alternate catalytic subunit of glucan synthase, partially restoring glucan synthase activity. Consistent with these results, up-regulation of PKC-Slt2 signaling and overexpression of FKS1 or FKS2 alleviated sensitivity of pgs1Delta to cell wall-perturbing agents and restored growth at elevated temperature. These findings demonstrate that functional Pgs1p is essential for cell wall biogenesis and activation of the PKC-Slt2 signaling pathway.

Loss of function of KRE5 suppresses temperature sensitivity of mutants lacking mitochondrial anionic lipids.

Disruption of PGS1, which encodes the enzyme that catalyzes the committed step of cardiolipin (CL) synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol (PG) and CL. The pgs1Delta mutant exhibits severe growth defects at 37 degrees C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1Delta that grew at 37 degrees C. One of the suppressors has a loss of function mutation in KRE5, which is involved in cell wall biogenesis. The cell wall of pgs1Delta contained markedly reduced beta-1,3-glucan, which was restored in the suppressor. Stabilization of the cell wall with osmotic support alleviated the cell wall defects of pgs1Delta and suppressed the temperature sensitivity of all CL-deficient mutants. Evidence is presented suggesting that the previously reported inability of pgs1Delta to grow in the presence of ethidium bromide was due to defective cell wall integrity, not from "petite lethality." These findings demonstrated that mitochondrial anionic lipids are required for cellular functions that are essential in cell wall biogenesis, the maintenance of cell integrity, and survival at elevated temperature.