BBA - Molecular Basis of Disease (v.1772, #10)

Annexin V is directly involved in cystic fibrosis transmembrane conductance regulator's chloride channel function by Pascal Trouvé; Marie-Anne Le Drévo; Mathieu Kerbiriou; Gaëlle Friocourt; Yann Fichou; Danièle Gillet; Claude Férec (1121-1133).
The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-activated chloride channel, which is regulated by protein–protein interactions. The extent to which CFTR is regulated by these interactions remains unknown. Annexin V is overexpressed in cystic fibrosis (CF), and given the functional properties of annexin V and CFTR we considered whether they are associated and if so whether this has implications for CFTR function. Using co-immunoprecipitation and overlay experiments, we show that annexin V is associated with nucleotide-binding domain 1 (NBD1) of CFTR. Surface plasmon resonance (SPR) indicated different K D values in the absence and presence of both calcium and ATP, suggesting that this interaction is calcium- and ATP-dependent. Using an siRNA approach and overexpression, we showed that CFTR chloride channel function and its localization in the cell membranes were dependent on annexin V expression. We concluded that annexin V is necessary for normal CFTR chloride channel activity. Furthermore, we show that CFTR and annexin V are partially co-distributed in normal epithelial cells in human bronchi. In conclusion, we show for the first time that annexin V is associated with CFTR and is involved in its function.
Keywords: Protein–protein interaction; Normal CFTR function; Protein localization;

Co-existence of high levels of the PTEN protein with enhanced Akt activation in renal cell carcinoma by Lizhi He; Catherine Fan; Aubrey Gillis; Xinchang Feng; Michael Sanatani; Sebastien Hotte; Anil Kapoor; Damu Tang (1134-1142).
Recruiting Akt to the membrane-bound phosphatidylinositol (3,4,5) trisphosphate (PIP3) is required for Akt activation. While PI3 kinase (PI3K) produces PIP3, PTEN dephosphorylates the 3-position phosphate from PIP3, thereby directly inhibiting Akt activation. PTEN is the dominant PIP3 phosphatase, as knockdown of PTEN results in increases in Akt activation in mice. The PTEN tumor suppressor gene is frequently mutated in a variety of human cancers, consistent with an inverse correlation between levels of the PTEN protein and Akt activation. We have examined PTEN expression and Akt activation in 35 primary clear cell renal cell carcinomas RCCs (ccRCCs) and 9 papillary RCCs (pRCCs) and their respective non-tumor kidney tissues. The PTEN protein was reduced in 16 ccRCCs (16/35 = 45.7%) and 8 pRCCs (8/9 = 88.9%). In these RCCs, 25.0% (4/16) of ccRCCs and 25.0% (2/8) of pRCCs expressed elevated Akt activation. 19 ccRCCc (19/35 = 54.3%) expressed comparable or higher levels of PTEN. Of these ccRCCs, 31.6% (6/19) showed increases in Akt activation. As PTEN dominantly inhibits Akt activation, the coexistence of high levels of the PTEN protein with enhanced Akt activation suggests the existence of novel mechanisms which attenuate PTEN function in ccRCC. These mechanisms may reduce PTEN function or increase PIP3 production.
Keywords: PTEN; Akt; Renal Cell Carcinoma (RCC);

Alteration in kidney glucose and amino acids are implicated in renal pathology in MRL/lpr mice by Jessy J. Alexander; Claudia Zwingmann; Alexander Jacob; Richard Quigg (1143-1149).
This study has employed high-resolution NMR spectroscopy of kidney extracts to study alterations in the concentrations of amino acids and glucose in systemic lupus erythematosus (SLE). We used the well-established mouse model of SLE, MRL/lpr, and their congenic controls, MRL/+. There was a substantial increase in the tissue concentration of branched-chain amino acids (133%), aromatic amino acids (134%) and glutathione (122%) in the lupus mice, compared to the controls. Since increased glucose can lead to fibrosis, we used [1-13C] glucose as a tracer to study its transport into the kidney. Significant increases in the levels of [1-13C] glucose (200% of controls) were observed in the MRL/lpr mice 15 min after its injection. 13C NMR spectra demonstrated that the 13C-label from [1-13C] glucose was not incorporated into glycolytic and Krebs cycle related metabolites within 15 min. Furthermore, we found that the expression of the profibrotic cytokine, TGFβ and the regulatory transcription factor Smad3 are significantly enhanced in MRL/lpr mice compared to the MRL/+ controls. The mRNA and protein expression of extracellular matrix proteins, fibronectin, laminin, and collagen IV were upregulated in the MRL/lpr mice compared to the controls. All these changes were significantly reduced by the complement (C) inhibitor, Crry. Our results suggest that C activation causes increased glucose concentration in the kidney, which can lead to the observed hyperglycemia. This may be one of the important factors that cause increased extracellular matrix (ECM) deposition through the TGFβ signaling in lupus mice and thereby lead to glomerulosclerosis that translates into increased kidney disease.
Keywords: Autoimmune; MRL/lpr; Glucose; [13C] NMR spectroscopy; Kidney;

Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation by Shanshan Lin; Min Wu; Yang Xu; Wei Xiong; Zhigang Yi; Xiaonan Zhang; Yuan Zhenghong (1150-1157).
In our previous paper, we reported that myeloid differential primary response protein (MyD88), a key adaptor in the signaling cascade of the innate immune response, inhibits hepatitis B virus (HBV) replication. The MyD88 activated nuclear factor-kappaB (NF-κB) signaling pathway and the intracellular upregulation of NF-κB signaling can induce an antiviral effect. Therefore, the association between the inhibition of HBV replication by MyD88 and NF-κB activation was investigated further. The results show that NF-κB activation was moderately increased after MyD88 expression. The strong activation of NF-κB by the IkappaB kinase complex IKKα/IKKβ dramatically suppressed HBV replication; the MyD88 dominant negative mutant that abrogated NF-κB activity did not inhibit HBV replication. Furthermore, the IκBα dominant negative mutant restored the inhibition of HBV replication by MyD88. These results support a role for NF-κB activation in the inhibition of HBV replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88 protein.
Keywords: Hepatitis B virus; Myeloid differential primary response protein (MyD88); Antiviral activity; Nuclear factor-kappaB;

Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus by Tatiana El-Bacha; Victor Midlej; Ana Paula Pereira da Silva; Leandro Silva da Costa; Marlene Benchimol; Antonio Galina; Andrea T. Da Poian (1158-1166).
Dengue virus infection affects millions of people all over the world. Although the clinical manifestations of dengue virus-induced diseases are known, the physiopathological mechanisms involved in deteriorating cellular function are not yet understood. In this study we evaluated for the first time the associations between dengue virus-induced cell death and mitochondrial function in HepG2, a human hepatoma cell line. Dengue virus infection promoted changes in mitochondrial bioenergetics, such as an increase in cellular respiration and a decrease in ΔΨ m. These alterations culminated in a 20% decrease in ATP content and a 15% decrease in the energy charge of virus-infected cells. Additionally, virus-infected cells showed several ultrastructural alterations, including mitochondria swelling and other morphological changes typical of the apoptotic process. The alterations in mitochondrial physiology and energy homeostasis preceded cell death. These results indicate that HepG2 cells infected with dengue virus are under metabolic stress and that mitochondrial dysfunction and alterations in cellular ATP balance may be related to the pathogenesis of dengue virus infection.
Keywords: Dengue virus infection; Mitochondrial dysfunction; Metabolism; Energy charge; Apoptosis; HepG2 cell;

It is controversial whether mutation in the melancortin-3 receptor (MC3R) gene is a cause for monogenic obesity in humans. Three novel mutations in the MC3R, A293T, I335S, and X361S, were identified from morbidly obese subjects. We investigated whether these mutations caused loss-of-function and the molecular defects if any. Ligand binding, signaling, and cell surface expression of the mutant MC3Rs were studied. I335S resulted in a complete loss of ligand binding and signaling due to intracellular retention. A293T and X361S MC3Rs had normal ligand binding and signaling as wild type MC3R. Co-expression studies showed that the mutants did not affect wild type MC3R signaling. Hence the I335S variant previously identified from obese patients is not expressed at the cell surface when expressed in vitro, suggesting that it might contribute to obesity in carriers of this variant. Whether A293T and X361S cause obesity remains to be investigated. Additional mutations at I335 showed that I335, part of the highly conserved N/DPxxY motif, was critical for multiple aspects of the MC3R function, including cell surface expression, ligand binding, and signaling.
Keywords: Melanocortin-3 receptor; Mutation; Obesity; G protein-coupled receptor; Trafficking;

Glutathione is a small tripeptide to maintain overall reducing environment in vivo. Reduced endogenous glutathione level has been associated with aging, obesity and diabetes. In this study, the direct impact of low endogenous glutathione level on energy homeostasis is investigated at molecular level. Depletion of endogenous glutathione in rat primary hepatocytes by BSO, an inhibitor of γ-glutamylcysteine synthase, leads to reduced mRNA levels of several key enzymes in energy homeostasis, including phosphoenolpyruvate carboxylkinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. Supplementation of various reducing reagents, including N-acetylcysteine, DTT and glutathione, reverses the inhibitory effect of BSO on PEPCK mRNA level. The suppressive effect of BSO on PEPCK mRNA level is also reversed through co-treatment with either SB210290, a specific p38 kinase inhibitor, or wortmannin and LY294002, the well-established PI-3 kinase inhibitors, suggesting the involvement of these kinases in this process. These observations correlate well with the observations that reduced endogenous glutathione level and reduced gluconeogenesis coincide with aging process, implying a causal relationship between these changes in aged population. More importantly, this study suggests that endogenous glutathione level tightly associates with energy homeostasis at molecular level, identifying reduced endogenous glutathione level as a potential contributing factor to dysregulated metabolic processes in aging, obese and diabetic populations. In addition, the different responses of PEPCK expression to the alteration of endogenous glutathione level in rat hepatoma cells from primary hepatocytes raises caution against using established cell lines in examining the dysregulated metabolic process related to altered endogenous glutathione level.
Keywords: Glutathione; PEPCK; Hepatocytes; Ageing and diabetes; PI-3 kinase;