Atomic structure of mutant PPARγ LBD complexed with 15d-PGJ2: Novel modulation mechanism of PPARγ/RXRα function by covalently bound ligands

15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) activates a nuclear receptor heterodimer, peroxisome proliferators-activated receptor γ (PPARγ)/ retinoid X receptor (RXRα) through covalent binding to Cys285 in PPARγ ligand-binding domain (LBD). Here, we present the 1.9 Å crystal structure of C285S mutant LBD complexed with 15d-PGJ₂, corresponding to the non-covalently bound state. The ligand lies adjacent to a hydrogen-bond network around the helix H2 and the nearby β-sheet. Comparisons with previous structures clarified the relationships between PPARγ function and conformational alterations of LBD during the process of covalently binding ligands, such as 15d-PGJ₂, and thus suggested a mechanism, by which these ligands modulate PPARγ/RXRα function through conformational changes of the loop following helix H2′ and the β-sheet.     

Sebaceous epithelial cell differentiation requires cyclic adenosine monophosphate generation

The cyclic adenosine monophosphate (cAMP) generator choleratoxin is known to promote the growth of sebaceous epithelial cells (sebocytes) in monolayer culture in classical serum-containing media. Now that sebocytes can be grown in serum-free medium, we have examined whether choleratoxin or other cAMP generators are required for differentiation of rat preputial sebocytes in response to specific ligand activators of peroxisome proliferator-activated receptors (PPARs). Unexpectedly, choleratoxin reduced sebocyte proliferation. However, sebocyte differentiation in response to specific PPARalpha and PPARgamma agonists required a cAMP generator such as choleratoxin, and this response was suppressed by a protein kinase A inhibitor. In contrast, the stable prostacyclin analog, carbaprostacyclin (cPGI2), a PPARalpha,delta agonist that also generates cAMP, stimulated differentiation independently of choleratoxin. Furthermore, unlike the selective PPARalpha and PPARgamma agonists, cPGI2 stimulated both sebocyte DNA synthesis and proliferation. These data are compatible with the evidence that prostacyclin has the additional effect of generating cAMP. In addition, we addressed the possibility that choleratoxin may act as a surrogate for beta-adrenergic catecholamines in generating cAMP. In contrast with choleratoxin, both alpha- and beta-adrenergic catecholamines stimulated sebocyte growth and interfered with the choleratoxin effect on differentiation. These data suggest ligand-dependent, complex interactions between cAMP and the other signal transduction pathways involved in sebocyte growth and development.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Temperature sensitivity of phospho-Ser(473)-PKB/AKT

The phospho-PKB/Akt status is often used as surrogate marker to measure activation of the PI3K/Akt/mTOR signal transduction pathway. Though, inconsistencies of the p-Ser⁴⁷³-PKB/Akt status have raised doubts in the validity of p-Ser⁴⁷³-PKB/Akt phosphorylation as endpoint. Here, we determined that p-Ser⁴⁷³-PKB/Akt but not p-Thr³⁰⁸-PKB/Akt phosphorylation is highly temperature sensitive. p-Ser⁴⁷³-PKB/Akt phosphorylation was rapidly reduced to levels below 50% on exposure to 20-25 °C in murine and human cell lines including cells expressing constitutively active PI3K or lacking PTEN. Down-regulation of p-Ser⁴⁷³-PKB/Akt was reversible and re-exposure to physiological temperature resulted in increased p-Ser⁴⁷³-PKB/Akt phosphorylation levels. Phosphatase activity at low temperature was sustained at 75% baseline level and phosphatase inhibition prevented p-Ser⁴⁷³-PKB/Akt dephosphorylation induced by the low temperature shift. Interestingly temperature-dependent deregulation of the p-Ser⁴⁷³-PKB/Akt status was also observed in response to irradiation. Thus our data demonstrate that minimal additional stress factors deregulate the PI3K/Akt-survival pathway and the p-Ser⁴⁷³-PKB/Akt status as experimental endpoint.     

Activation of Asparaginyl Endopeptidase Leads to Tau Hyperphosphorylation in Alzheimer Disease

Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I₂^PP2A. In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I₂^PP2A is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I_2NTF) and the C-terminal fragment (I_2CTF). Both I_2NTF and I_2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I₂^PP2A translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I₂^PP2A, except when I₂^PP2A was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I₂^PP2A, inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I₂^PP2A-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.     

Very-long-chain fatty acid metabolism in adrenoleukodystrophy protein-deficient mice

X-linked adrenoleukodystrophy (X-ALD) is characterized by progressive mental and motor deterioration, with demyelination of the central and peripheral nervous system. Its principal biochemical abnormality is the accumulation of very-long-chain fatty acids (VLCFAs) in tissues and body fluids, caused by the impairment of peroxisomal β-oxidation. The authors have generated a line of mice deficient in ALD protein (ALDP) by gene targeting. ALDP-deficient mice appeared normal clinically, at least up to 12 mo. Western blot analysis showed absence of ALDP in the brain, spinal cord, lung, and kidney. The amounts of C26∶0 increased by 240% in the spinal cord. VLCFA β-oxidation in cultured hepatocytes was reduced to 50% of normal. The authors investigated the roles of ALDP in VLCFA β-oxidation using the ALDP-deficient mice. Very-long-chain acyl-CoA synthetase (VLACS) is functionally deficient in ALD cells. The impairment of VLCFA β-oxidation in the ALDP-deficient fibroblasts was not corrected by overexpression of VLACS only, but was done by co-expression of VLACS and ALDP, suggesting that VLACS requires ALDP to function. VLACS was detected in the peroxisomal and microsomal fractions of the liver from both types of mice. Peroxisomal VLACS was clearly decreased in the ALDP-deficient mouse. Thus, ALDP is involved in the peroxisomal localization of VLACS.     

Endotoxin Conditioning Induces VCP/p97-mediated and Inducible Nitric-oxide Synthase-dependent Tyr284 Nitration in Protein Phosphatase 2A

Endotoxins activate Toll-like receptors and reprogram cells to be refractory to secondary exposure. Here we found that activation of different Toll-like receptors elicited a time- and dose-dependent increase in the levels of the protein phosphatase 2A catalytic subunit (PP2Ac) but not its partner A subunit. We purified the lipopolysaccharide-induced form of PP2A by chromatography plus immunoprecipitation and used mass spectrometry to identify VCP/p97 as a novel partner for PP2Ac. Endogenous VCP/p97 and PP2Ac were co-immunoprecipitated from primary murine macrophages and human lymphocytes. GST-VCP/p97 bound purified PP2A in pulldown assays, showing direct protein-protein interaction. Endotoxin conditioning of macrophages induced formation of 3-nitrotyrosine in the PP2Ac associated with VCP/p97, a response severely reduced in macrophages from iNOS knock-out mice. The reaction of purified PP2A with peroxynitrite dissociated the A subunit, and 3-nitro-Tyr²⁸⁴ was identified in PP2Ac by mass spectrometry. Myc-PP2Ac (Y284F) expressed in cells was resistant to peroxynitrite-induced nitration and reduction of A subunit binding. Transient expression of either VCP/p97 or PP2Ac was sufficient to elevate levels of the dual specificity phosphatase DUSP1, reduce p38 MAPK activation, and suppress tumor necrosis factor-α release. We propose that VCP/p97-mediated Tyr nitration of PP2A increases the levels of phosphatases PP2A and DUSP1 to contribute to the refractory response of conditioned cells.     

Docosahexaenoic acid and synaptic protection in Alzheimer's disease mice

Alzheimer's disease (AD) is a major public health concern due to longer life expectancy in the Western countries. Amyloid-beta (Aβ) oligomers are considered the proximate effectors in the early stages of AD. AD-related cognitive impairment, synaptic loss and neurodegeneration result from interactions of Aβ oligomers with the synaptic membrane and subsequent activation of pro-apoptotic signalling pathways. Therefore, membrane structure and lipid status appear determinant in Aβ-induced toxicity. Numerous epidemiological studies have highlighted the beneficial influence of docosahexaenoic acid (DHA, C22:6 n-3) on the preservation of synaptic function and memory capacities in aged individuals or upon Aβ exposure, whereas its deficiency is presented as a risk factor for AD. An elevated number of studies have been reporting the beneficial effects of dietary DHA supplementation on cognition and synaptic integrity in various AD models. In this review, we describe the important potential of DHA to preserve neuronal and brain functions and classified its numerous molecular and cellular effects from impact on membrane lipid content and organisation to activation of signalling pathways sustaining synaptic function and neuronal survival. DHA appears as one of the most valuable diet ingredients whose neuroprotective properties could be crucial for designing nutrition-based strategies able to prevent AD as well as other lipid- and age-related diseases whose prevalence is progressing in elderly populations.     

Dephosphorylation of Barrier-to-autointegration Factor by Protein Phosphatase 4 and Its Role in Cell Mitosis

Barrier-to-autointegration factor (BAF or BANF1) is highly conserved in multicellular eukaryotes and was first identified for its role in retroviral DNA integration. Homozygous BAF mutants are lethal and depletion of BAF results in defects in chromatin segregation during mitosis and subsequent nuclear envelope assembly. BAF exists both in phosphorylated and unphosphorylated forms with phosphorylation sites Thr-2, Thr-3, and Ser-4, near the N terminus. Vaccinia-related kinase 1 is the major kinase responsible for phosphorylation of BAF. We have identified the major phosphatase responsible for dephosphorylation of Ser-4 to be protein phosphatase 4 catalytic subunit. By examining the cellular distribution of phosphorylated BAF (pBAF) and total BAF (tBAF) through the cell cycle, we found that pBAF is associated with the core region of telophase chromosomes. Depletion of BAF or perturbing its phosphorylation state results not only in nuclear envelope defects, including mislocalization of LEM domain proteins and extensive invaginations into the nuclear interior, but also impaired cell cycle progression. This phenotype is strikingly similar to that seen in cells from patients with progeroid syndrome resulting from a point mutation in BAF.