BBA - Molecular Basis of Disease (v.1772, #11-12)

DM1 CTG expansions affect insulin receptor isoforms expression in various tissues of transgenic mice by Céline Guiraud-Dogan; Aline Huguet; Mário Gomes-Pereira; Edith Brisson; Guillaume Bassez; Claudine Junien; Geneviève Gourdon (1183-1191).
Myotonic dystrophy (DM1) is a dominant autosomal multisystemic disorder caused by the expansion of an unstable CTG trinucleotide repeat in the 3′ untranslated region of the DMPK gene. Nuclear accumulation of the enlarged CUG-containing DMPK transcripts has a deleterious effect on the regulation of alternative splicing of some RNAs and has a central role in causing the symptoms of DM1. In particular, Insulin Receptor (IR) mRNA splicing defects have been observed in the muscle of DM1 patients. In this study, we have investigated IR splicing in insulin-responsive tissues (i.e. skeletal muscles, adipose tissue, liver) and pancreas and we have studied glucose metabolism in mice carrying the human genomic DM1 region with expanded (> 350 CTG) or normal (20 CTG) repeats and in wild-type mice. Mice carrying DM1 expansions displayed a tissue- and age-dependent abnormal regulation of IR mRNA splicing in all the tissues that we investigated. Furthermore, these mice showed a basal hyperglycemia and glucose intolerance which disappeared with age. Our findings show that deregulation of IR splicing due to the DM1 mutation can occur in different mouse tissues, suggesting that CTG repeat expansions might also result in IR misplicing not only in muscles but also in other tissues in DM1 patients.
Keywords: Myotonic dystrophy; Trinucleotide repeat; Insulin receptor; Splicing; Transgenic mice;

The ligand activity of AGE-proteins to scavenger receptors is dependent on their rate of modification by AGEs by Ryoji Nagai; Katsumi Mera; Keisuke Nakajou; Yukio Fujiwara; Yasunori Iwao; Hiroki Imai; Toshinori Murata; Masaki Otagiri (1192-1198).
The cellular interaction of proteins modified with advanced glycation end-products (AGEs) is believed to induce several different biological responses, which are involved in the development of diabetic vascular complications. We report here that the ratio of protein glycation is implicated in its ligand activity to scavenger receptors. Although highly-modified AGE-bovine serum albumin (high-AGE-BSA) was significantly recognized by human monocyte-derived macrophages and Chinese hamster ovary cells which overexpress such scavenger receptors as CD36, SR-BI (scavenger receptor class B type-I), and LOX-1 (Lectin-like Ox-LDL receptor-1), the mildly-modified-AGE-BSA (mild-AGE-BSA) did not show any ligand activity to these cells. Furthermore, when 111In-labeled high- or mild-AGE-BSA were injected into the tail vein of mice, the high-AGE-BSA was rapidly cleared from the circulation whereas the clearance rate of the mild-AGE-BSA was very slow, similar to the native BSA. These results demonstrate the first evidence that the ligand activity of the AGE-proteins to the scavenger receptors and its pharmacokinetic properties depend on their rate of modification by AGEs, and we should carefully prepare the AGE-proteins in vitro to clarify the physiological significance of the interaction between the AGE-receptors and AGE-proteins.
Keywords: Advanced glycation end products (AGEs); N ε-(carboxymethyl)lysine (CML); Scavenger receptor; Macrophage; Atherosclerosis; Diabetes;

This study was designed to investigate possible prevention of apoptotic cell death by selenium, an antioxidant, using cultured brain-derived neural progenitor cells (NPCs) and an experimental mouse brain trauma (BT) model. We tested some of the neuroprotective effects of sodium selenite in NPC cells by monitoring thioredoxin reductase (TR) expression, optimum H2O2 removal, and consequent inhibition of pro-apoptotic events including cytochrome c release and caspase 3 and 9 activation. Analysis of key apoptotic regulators during H2O2-induced apoptosis of NPCs showed that selenite blocks the activation of c-jun N-terminal protein kinase (JNK)/P38 mitogen-activated protein kinase (MAPK), and Akt survival protein. Moreover, selenite activates p44/42 MAPK and inhibits the downregulation of Bcl2 in selenite-treated NPC cells. For in vivo experiments, the effects of selenite on H2O2 neurotoxicity were tested using several biochemical and morphologic markers. Here we show that selenite potentially inhibits H2O2-induced apoptosis of NPCs and in traumatic brain injury. This in vivo protective function was also associated with inhibition of H2O2-induced reactive oxygen species (ROS) generation, cytochrome c release and caspase 3 and 9 activation. Our data show that the protective function of selenite through attenuation of secondary pathological events most likely results from its comprehensive effects that block apoptotic cell death, resulting in the maintenance of functional neurons and in inhibition of astrogliosis. The finding that selenite administration prevents secondary pathological events in an animal model of traumatic brain injury, as well as its efficacy, may provide novel drug targets for treating brain trauma.
Keywords: Brain trauma; Hydrogen peroxide; Neuroprotection; Oxidation; Sodium selenite;

The interaction of ETV6 (TEL) and TIP60 requires a functional histone acetyltransferase domain in TIP60 by Jasmina Putnik; Chang-Dong Zhang; Leticia Fröhlich Archangelo; Belay Tizazu; Sigrun Bartels; Michael Kickstein; Philipp A. Greif; Stefan K. Bohlander (1211-1224).
The ets-family transcription factor ETV6 (TEL) has been shown to be the target of a large number of balanced chromosomal translocations in various hematological malignancies and in some soft tissue tumors. Furthermore, ETV6 is essential for hematopoietic stem cell function. We identified ETV6 interacting proteins using the yeast two hybrid system. One of these proteins is the HIV Tat interacting protein (TIP60), a histone acetyltransferase (HAT) containing the highly conserved MYST domain. TIP60 functions as a corepressor of ETV6 in reporter gene assays. Fluorescently tagged ETV6 and TIP60 colocalize in the nucleus and an increase in nuclear localization of ETV6 was seen when TIP60 was cotransfected. ETV6 interacts with TIP60 through a 63 amino acids region located in the central domain of ETV6 between the pointed and the ets domain. The ETV6 interacting region of TIP60 mapped to the C2HC zinc finger of the TIP60 MYST domain. The interaction of TIP60 with full length ETV6 required an intact acetyltransferase domain of TIP60. Interestingly, the MYST domains of MOZ and MORF were also able to interact with portions of ETV6. These observations suggest that MYST domain HATs regulate ETV6 transcriptional activity and may therefore play critical roles in leukemogenesis and possibly in normal hematopoietic development.
Keywords: ETV6; TIP60; MOZ; MORF;

The yeast mitochondrial DNA (mtDNA) replicase Mip1 has been used as a model to generate five mutations equivalent to POLG mutations associated with a broad spectrum of diseases in human. All mip1 mutations, alone or in combination in cis or in trans, increase mtDNA instability as measured by petite frequency and EryR mutant accumulation. This phenotype is associated with decreased Mip1 levels in mitochondrial extracts and/or decreased polymerase activity. We have demonstrated that (1) in the mip1G651S (hG848S) mutant the high mtDNA instability and increased frequency of point EryR mutations is associated with low Mip1 levels and polymerase activity; (2) in the mip1A692T–E900G (hA889T–hE1143G) mutant, A692T is the major contributor to mtDNA instability by decreasing polymerase activity, and E900G acts synergistically by decreasing Mip1 levels; (3) in the mip1H734Y/mip1G807R (hH932Y/hG1051R) mutant, H734Y is the most deleterious mutation and acts synergistically with G807R as a result of its dominant character; (4) the mip1E900G (h1143G) mutation is not neutral but results in a temperature-sensitive phenotype associated with decreased Mip1 levels, a property explaining its synergistic effect with mutations impairing the polymerase activity. Thus, the human E1143G mutation is not a true polymorphism.
Keywords: POLG; Mutation; Disease; Yeast; mip1 mutant; mtDNA;

Coupling cystic fibrosis to endoplasmic reticulum stress: Differential role of Grp78 and ATF6 by Mathieu Kerbiriou; Marie-Anne Le Drévo; Claude Férec; Pascal Trouvé (1236-1249).
Cystic fibrosis (CF) is the most common Caucasian autosomal recessive disease. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, which is a chloride (Cl) channel. The most common mutation leads to a missing phenylalanine at position 508 (ΔF508). The ΔF508-CFTR protein is misfolded and retained in the endoplasmic reticulum and may trigger the unfolded protein response (UPR). Furthermore, CF is accompanied by inflammation and infection, which are also involved in the UPR. To date, the UPR transducer ATF6 and ER stress sensor Grp78 have been used as UPR markers. Therefore, our aim was to study the activation of ATF6 and Grp78 in transfected human epithelial cells expressing the ΔF508-CFTR protein, and we showed that they are activated in these cells. We investigated the effect of exogenous UPR inducers thapsigargin (Tg) and tunicamycin (Tu) on Grp78 and ATF6 expression. Whereas the cells reacted to the UPR induction, we show a difference in the electrophoretic pattern of ATF6. The Grp78/ATF6 complex was previously described, but its stability during UPR is controversial. Using co-immunoprecipitation we show that it is stable in ΔF508-CFTR-expressing cells and is maintained under UPR conditions. Finally, using siRNA, we show that decreased ATF6 expression induces increased cAMP-dependent halide flux through ΔF508-CFTR due to its increased membrane localization. Therefore, our results suggest that UPR may be triggered in CF and that ATF6 may be a therapeutic target.
Keywords: Cystic fibrosis; Unfolded protein response; ATF6; Grp78; Endoplasmic reticulum;

TGF-β up-regulates serum response factor in activated hepatic stellate cells by Jens Herrmann; Ute Haas; Axel M. Gressner; Ralf Weiskirchen (1250-1257).
In differentiated smooth muscle cells (SMC) the regulation of SMC marker genes (e.g. α-smooth muscle actin) is mainly conducted by the serum response factor (SRF) and accessory co-factors like myocardin. A number of SMC markers are also expressed in activated hepatic stellate cells which are the main cellular effectors in liver fibrogenesis. In the present study we found that during cellular activation and transdifferentiation the SRF transcription factor is up-regulated by transforming growth factor-β, accumulated in the nucleus, and exhibited increased DNA-binding activity. These observations were accompanied by a forced expression of the SRF co-activator myocardin. Specific targeting of SRF by small interference RNA resulted in diminished contents of α-smooth muscle actin. Therefore, we conclude that hepatic stellate cells retain differentiation capacity to evolve characteristics that are typical for cells of the cardiac and smooth muscle lineages.
Keywords: Serum response factor; Hepatic stellate cell; Transdifferentiation; Smooth muscle cell; Transforming growth factor-β; Myocardin; Gene regulation; siRNA; α-Smooth muscle actin; Liver fibrogenesis;

Refolding of the heavy chain of the Class I HLA molecule, HLA-B27, in the absence of β2m, yields soluble high molecular weight (HMW) oligomers reminiscent of the oligomeric forms of β2m-free heavy chains (FHCs) of class I HLA antigens observed on cell surfaces. Here we examine the structural characteristics of HMW B27 in respect of features potentially relevant to autoimmunity, such as: (a) retention of native-like structure, since this could facilitate non-canonical interactions with T-cell receptors even in the absence of bound β2m and peptide, or (b) presence of non-native structure, since this could yield novel (non-self) antigenic conformational epitopes that could elicit immune attack. We report that HMW B27 is characterized by high secondary structural content, structural stability, stability to proteolysis by trypsin, and structural features that are both partly native-like, and partly non-native-like, as assessed through the binding of conformationally-distinguishing and cross-reacting scFv antibodies specifically selected against HMW B27. We also present cell ELISA data with conformation-specific scFv antibodies that distinguish between lymphocytes from individuals who are healthy and B27 positive, and those who are B27 positive but suffering from ankylosing spondylitis.
Keywords: HLA-B27; MHC dysfunction; Protein misfolding; Cross-display; Secondary structure; Thermal stability; Autoimmunity;