Maintaining a fluid bilayer is essential for cell signaling and survival. Lipid saturation is a key factor determining lipid packing and membrane fluidity, and it must be tightly controlled to guarantee organelle function and identity. A dedicated eukaryotic mechanism of lipid saturation sensing, however, remains elusive. Here we show that Mga2, a transcription factor conserved among fungi, acts as a lipid-packing sensor in the ER membrane to control the production of unsaturated fatty acids. Systematic mutagenesis, molecular dynamics simulations, and electron paramagnetic resonance spectroscopy identify a pivotal role of the oligomeric transmembrane helix (TMH) of Mga2 for intra-membrane sensing, and they show that the lipid environment controls the proteolytic activation of Mga2 by stabilizing alternative rotational orientations of the TMH region. This work establishes a eukaryotic strategy of lipid saturation sensing that differs significantly from the analogous bacterial mechanism relying on hydrophobic thickness.

Abstract-Recently we demonstrated that mechanical stress induces apoptosis of vascular smooth muscle cells in vitro and in vein grafts (Mayr et al. FASEB J. 2000;15:261-270). The current study was designed to investigate molecular mechanisms of mechanical stretch-induced apoptosis. Smooth muscle cells cultivated on silicone elastomer plates precoated with collagen I, elastin, laminin, or Pronectin were subjected to cyclic mechanical stretch. Interestingly, in response to mechanical stress, the number of apoptotic cells increased significantly in cells growing on collagen I-coated plates but not on other matrixes. We therefore thought that receptors mediating binding to collagen I, such as integrin ␤ 1 containing receptors, might be involved in signaling pathways leading to stretch-induced apoptosis. On collagen plates, mechanical stress rapidly activated p38 MAPK that phosphorylated p53 in smooth muscle cells. Lack of functional Rac completely abrogated p38 MAPK-p53 activation as well as apoptosis. Furthermore, mechanical stress resulted in increases of both integrin ␤ 1 protein expression and activity as identified by Western blotting and Shc immunoprecipitation assays. Treatment with a ␤ 1 -integrin-blocking antibody or integrin signaling inhibitor cytochalasin B but not growth factor receptor inhibitor suramin abrogated both stretch-induced phosphorylation of p38 MAPK and p53 expression. Akin to the inhibition of p38 MAPK-p53 signaling, pretreatment with a ␤ 1 -integrin-blocking antibody or cytochalasin B but not suramin inhibited stretch-induced apoptosis on collagen plates. These results suggest that mechanical stress-induced apoptosis in vascular smooth muscle cells is mediated by ␤ 1 -integrin-rac-p38-p53 signaling pathways.

Vascular smooth muscle cells (SMCs) are exposed to altered mechanical stress that may contribute to SMC migration in the development of atherosclerosis. Signal transduction pathways in SMCs activated by mechanical stress that instigate cell migration are undefined. Herein, we provide evidence that mechanical stress enhances SMC migration, which is mediated, at least in part, by protein kinase C (PKC)delta. When rat SMCs cultivated on a flexible membrane were subjected to cyclic strain stress (60 cycles/min, 5, 15, or 20% elongation), PKCdelta was translocated to the Triton-insoluble fraction, whereas PKCalpha was translocated to the membrane, which was confirmed by PKC kinase assays. Immunofluorescence and actin staining revealed a cytoskeleton translocation of PKCdelta in SMCs stimulated by cyclic strain. PKCdelta-deficient SMCs cultivated from PKCdelta-/- mice showed an abnormal cytoskeleton structure, which was related to a diminished phosphorylation of paxillin, focal adhesion kinase, and vinculin in response to mechanical stress. Mechanical stress enhanced SMC migration, which was diminished in PKCdelta-/- SMCs. Taken together, our data demonstrated that mechanical stress activates PKCdelta translocation to the cytoskeleton, which is related to decreased SMC migration and indicates that PKCdelta is a key signal transducer between mechanical stress and cell migration.

leads to changes in protein dynamics, followed by binding of the cognate G-protein (transducin) initiating the biological signaling. Present X-ray crystallographic structures do not reveal changes in protein dynamics which are the key for understanding the activation mechanism. Here we compare an energy landscape model (ELM) and spatial motion model (SMM) analysis of both elastic and quasielastic neutron scattering (QENS) data to explain regulation of integral membrane protein mobility by the retinal cofactor of rhodopsin. Mean-square displacements calculated from elastic incoherent neutron scattering (EINS) are consistent with a dynamical transition as observed for globular proteins. In the SMM analysis the quasielastic spectrum is dissected into an elastic peak due to quasistatic atoms, and quasielastic wings due to homogenous line broadening from the mobile atoms in the protein. By contrast, the ELM adopts a wave-mechanical approach and describes the QENS spectrum in terms of inhomogeneous lines due to the various conformational substates of the protein [2]. Application of mode-coupling theory as developed for glass-forming liquids to SMM analysis of the QENS spectra reveals a slowing down of picosecond-nanosecond dynamics in the b-relaxation region for ligand-free opsin versus dark-state rhodopsin. Alternatively, ELM analysis reveals that the ensemble of conformational substates in opsin is smaller versus the dark state. The results are consistent with increased local crowding due to a more collapsed protein structure in ligand-free opsin versus the dark-state rhodopsin. A novel powdered GPCR preparation method together with the QENS technique uncovers changes in structural fluctuations governed by retinal cofactor of rhodopsin.[1] A.V.

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Background: Graves' disease is one of the most common autoimmune conditions, but treatment remains imperfect. This study explores the first-inhuman use of antigen-specific immunotherapy with a combination of two thyrotropin receptor (TSHR) peptides (termed ATX-GD-59) in Graves' hyperthyroidism. Methods: Twelve participants (11 female) with previously untreated mild to moderate Graves' hyperthyroidism were enrolled in a Phase I open label trial to receive 10 doses of ATX-GD-59 administered intradermally over an 18-week period. Adverse events, tolerability, changes in serum free thyroid hormones, and TSHR autoantibodies were measured. Results: Ten subjects received all 10 doses of ATX-GD-59, five (50%) of whom had free triiodothyronine within the reference interval by the 18-week visit. Two further subjects had improved free thyroid hormones by the end of the study (7/10 responders), whereas three subjects showed worsening thyrotoxicosis during the study. Serum TSHR autoantibody concentrations reduced during the study and correlated with changes in free thyroid hormones (r = 0.85, p = 0.002 for TSHR autoantibody vs. free triiodothyronine). Mild injection-site swelling and pain were the most common adverse events. Conclusions: These preliminary data suggest that ATX-GD-59 is a safe and well-tolerated treatment. The improvement in free thyroid hormones in 70% of subjects receiving the medication suggests potential efficacy as a novel treatment for Graves' hyperthyroidism.

Context Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are responsible for a subset of familial isolated pituitary adenoma (FIPA) cases and sporadic pituitary neuroendocrine tumors (PitNETs). Objective To compare prospectively diagnosed AIP mutation-positive (AIPmut) PitNET patients with clinically presenting patients and to compare the clinical characteristics of AIPmut and AIPneg PitNET patients. Design 12-year prospective, observational study. Participants & Setting We studied probands and family members of FIPA kindreds and sporadic patients with disease onset ≤18 years or macroadenomas with onset ≤30 years (n = 1477). This was a collaborative study conducted at referral centers for pituitary diseases. Interventions & Outcome AIP testing and clinical screening for pituitary disease. Comparison of characteristics of prospectively diagnosed (n = 22) vs clinically presenting AIPmut PitNET patients (n = 145), and AIPmut (n = 167) vs AIPneg PitNET patients (n = 1310). Results Prospectively diagnosed AIPmut PitNET patients had smaller lesions with less suprasellar extension or cavernous sinus invasion and required fewer treatments with fewer operations and no radiotherapy compared with clinically presenting cases; there were fewer cases with active disease and hypopituitarism at last follow-up. When comparing AIPmut and AIPneg cases, AIPmut patients were more often males, younger, more often had GH excess, pituitary apoplexy, suprasellar extension, and more patients required multimodal therapy, including radiotherapy. AIPmut patients (n = 136) with GH excess were taller than AIPneg counterparts (n = 650). Conclusions Prospectively diagnosed AIPmut patients show better outcomes than clinically presenting cases, demonstrating the benefits of genetic and clinical screening. AIP-related pituitary disease has a wide spectrum ranging from aggressively growing lesions to stable or indolent disease course.

The transcription factor p53 is essentially involved in regulation of cell death and proliferation. Recently, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of p53 in the development of vein graft arteriosclerosis in p53(-/-) mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in p53(-/-) mice was increased 2-fold compared with that of wild-type controls. Cell component analysis revealed that neointimal lesions in p53(-/-) mice consisted mainly of alpha-actin positive smooth muscle cells (SMCs), whereas the majority of cells in wild-type mice were MAC-1 (CD11b/18)-positive at 4 weeks. Importantly, SMC apoptosis as determined by TUNEL assay was significantly reduced in p53(-/-) vein grafts. TUNEL positive cells in wild-type vein grafts markedly increased from 0.5% to 6.4% of total cells 4 weeks postoperatively, but remained virtually unchanged in p53(-/-) grafts (0.8%). Immunofluorescence analysis revealed that increased p53 expression in neointimal SMCs of wild-type, but not p53(-/-), mice coincided with oxidative DNA damage in vein grafts. Interestingly, SMCs of p53(-/-) mice showed increased apoptosis in response to TNFalpha and decreased apoptosis in response to sodium nitroprusside. Additionally, p53-deficient SMCs showed a higher rate of proliferation and migration and expressed higher levels of matrix metalloproteinases. Thus, p53 deficiency accelerates neointima formation by facilitating SMC proliferation as well as abrogating cell apoptosis.