Transmembrane BAX Inhibitor Motif Research Articles

The characteristics of the ancient cell death suppressor, TMBIM6, and its related signaling pathways after endoplasmic reticulum stress

Activation of the unfolded protein response in combination with generation of reactive oxygen species, from cytochrome P450 members and NADPH-P450 reductases, are two major consequences of Endoplasmic Reticulum (ER) stress that cause oxidative damage and cell death. Herein, we reviewed the role of Bax Inhibitor-1 (BI-1), an evolutionarily conserved protein encoded by the Transmembrane Bax inhibitor Motif Containing 6 gene, in protection from ER stress. As BI-1 has multimodal properties that can target a wide array of pathophysiological consequences after injury, our main objective was to explore BI-1's protective role in ER stress and its potential signaling pathways.

Ion and pH Sensitivity of a TMBIM Ca2+ Channel

The anti-apoptotic transmembrane Bax inhibitor motif (TMBIM) containing protein family regulates Ca2+ homeostasis, cell death, and the progression of diseases including cancers. The recent crystal structures of the TMBIM homolog BsYetJ reveal a conserved Asp171-Asp195 dyad that is proposed in regulating a pH-dependent Ca2+ translocation. Here we show that BsYetJ mediates Ca2+ fluxes in permeabilized mammalian cells, and its interaction with Ca2+ is sensitive to protons and other cations. We report crystal structures of BsYetJ in additional states, revealing the flexibility of the dyad in a closed state and a pore-opening mechanism. Functional studies show that the dyad is responsible for both Ca2+ affinity and pH dependence. Computational simulations suggest that protonation of Asp171 weakens its interaction with Arg60, leading to an open state. Our integrated analysis provides insights into the regulation of the BsYetJ Ca2+ channel that may inform understanding of human TMBIM proteins regarding their roles in cell death and diseases.

Abstract TP322: Viral Mediated Gene Delivery of TMBIM6 Protects the Neonatal Brain

Introduction: A wide variety of conditions may contribute to oxidative protein folding processes in the endoplasmic reticulum (ER) which cause ER stress and play a major role in the pathogenesis of neonatal hypoxic ischemic (HI) injury. ER stress triggers the unfolded protein response (UPR) resulting in the activation of pro-apoptotic signaling causing cells to undergo apoptosis. Here were explored the role of Bax Inhibitor-1 (BI-1) protein, an evolutionary protein encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, as a novel modulator of ER stress induced apoptosis after HI brain injury in a neonatal rat pup. BI-1 may modulate the UPR response via a direct physical interaction with the stress sensor receptor, IRE1α, and thus inhibiting it’s pro-apoptotic signaling. Hypothesis: Overexpression of BI-1, using viral-mediated gene delivery of human adenoviral-TMBIM6 (Ad-TMBIM6) vector, will protect the neonatal brain from HI by inhibiting ER stress induced apoptosis. Methods: Ten-day old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). To determine BI-1 confers protection via inhibition of IRE1α, we administered BI-1 siRNA, to silence BI-1, and an IRE1α CRISPR activation plasmid (AP), to counteract BI-1’s effects, at 48h pre-HI. Percent infarct area, immunofluorescent staining and western blot were performed at 72h post HI. Results: Overexpression of BI-1 showed to significantly reduce percent infarcted area, attenuated neuronal degeneration, reduced ER stress induced neuronal apoptosis and improved long-term neurological outcomes. Either silencing BI-1 or over activating IRE1α with a CRISPR AP significantly reversed BI-1’s protective effects. Conclusions: Overexpression of BI-1 attenuated the morphological and neurological consequences post neonatal HI via inhibition of the ER stress sensor, IRE1α. This study showed a novel role for BI-1 and ER stress in the pathophysiology of HI and provided a basis for BI-1 as a potential therapeutic target.

Apoptosis in Hydra: function of HyBcl-2 like 4 and proteins of the transmembrane BAX inhibitor motif (TMBIM) containing family.

Mechanisms of programmed cell death differ between animals, plants and fungi. In animals, apoptotic cell death depends on caspases and Bcl-2 family proteins. These protein families are only found in multicellular animals, including cnidarians, insects and mammals. In contrast, members of the TMBIM-family of transmembrane proteins are conserved across all eukaryotes. Sequence comparisons of cell death related proteins between phyla indicate strong conservation of the genes involved. However, often it is not known whether this is paralleled by conservation of function. Here we present the first study to support an anti-apoptotic function of Bcl-2 like proteins in the cnidarian Hydra within a physiological context. We used transgenic Hydra expressing GFP-tagged HyBcl-2-like 4 protein in epithelial cells. The protein was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis induced by either the PI(3) kinase inhibitor wortmannin or by starvation. Moreover, we identified members of the TMBIM-family in Hydra including HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4. Expressing these TMBIM-family members in Hydra and human HEK cells, we found HyBax-inhibitor-1 protein localised to ER-membranes and HyLifeguard-family members localised to the plasma membrane and Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from camptothecin induced apoptosis. This work illustrates that the investigated Bcl-2- and TMBIM-family members represent evolutionarily conserved mitochondrial, ER, Golgi and plasma membrane proteins with anti-apoptotic functions. The participation of ER and Golgi proteins in the regulation of programmed cell death might be a very ancient feature.

Gene Co-expression Analysis Indicates Potential Pathways and Regulators of Beef Tenderness in Nellore Cattle.

Beef tenderness, a complex trait affected by many factors, is economically important to beef quality, industry, and consumer’s palatability. In this study, RNA-Seq was used in network analysis to better understand the biological processes that lead to differences in beef tenderness. Skeletal muscle transcriptional profiles from 24 Nellore steers, selected by extreme estimated breeding values (EBVs) for shear force after 14 days of aging, were analyzed and 22 differentially expressed transcripts were identified. Among these were genes encoding ribosomal proteins, glutathione transporter ATP-binding cassette, sub-family C (CFTR/MRP), member 4 (ABCC4), and synaptotagmin IV (SYT4). Complementary co-expression analyses using Partial Correlation with Information Theory (PCIT), Phenotypic Impact Factor (PIF) and the Regulatory Impact Factor (RIF) methods identified candidate regulators and related pathways. The PCIT analysis identified ubiquitin specific peptidase 2 (USP2), growth factor receptor-bound protein 10 (GBR10), anoctamin 1 (ANO1), and transmembrane BAX inhibitor motif containing 4 (TMBIM4) as the most differentially hubbed (DH) transcripts. The transcripts that had a significant correlation with USP2, GBR10, ANO1, and TMBIM4 enriched for proteasome KEGG pathway. RIF analysis identified microRNAs as candidate regulators of variation in tenderness, including bta-mir-133a-2 and bta-mir-22. Both microRNAs have target genes present in the calcium signaling pathway and apoptosis. PIF analysis identified myoglobin (MB), enolase 3 (ENO3), and carbonic anhydrase 3 (CA3) as potentially having fundamental roles in tenderness. Pathways identified in our study impacted in beef tenderness included: calcium signaling, apoptosis, and proteolysis. These findings underscore some of the complex molecular mechanisms that control beef tenderness in Nellore cattle.

Abstract 308: Yeast Bax Inhibitor, BXI1, is involved in calcium homeostasis of the ER in Saccharomyces cerevisiae

Abstract Yeast Bax inhibitor-1 (BXI1/YBH3) encodes a protein that belongs to the Bax Inhibitor (TMBIM) family of proteins that all contain a transmembrane BAX inhibitor motif. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks. In mammals, the Bax inhibitor family of proteins has cytoprotective properties that are most evident in paradigms of endoplasmic reticulum (ER) stress. Our published studies have shown that yeast Bxi1p is localized to the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. BXI1 is thought to act via a mechanism involving altered calcium dynamics. We now show that cells lacking BXI1 accumulate higher levels of calcium in their ER as compared to their wildtype counterparts. We have also over expressed Bxi1p in E.coli and have used a fura-2 based calcium assay to show that the protein facilitates the influx of extracellular calcium into the cell. Our preliminary data with Δbxi1 Δpmr1 double mutants suggest that Bxi1p and Pmr1p, the Golgi-ER calcium pump, function in an antagonistic manner. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.] Citation Format: Nicholas Andrews, Nicholas Mello, Liam McDonough, Joseph Alisch, David Eagan, James Mullin, Alfredo Gonzalez, Nicanor Austriaco. Yeast Bax Inhibitor, BXI1, is involved in calcium homeostasis of the ER in Saccharomyces cerevisiae [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 308.

New insights into the phylogeny of the TMBIM superfamily across the tree of life: Comparative genomics and synteny networks reveal independent evolution of the BI and LFG families in plants

The Transmembrane BAX Inhibitor Motif containing (TMBIM) superfamily, divided into BAX Inhibitor (BI) and Lifeguard (LFG) families, comprises a group of cytoprotective cell death regulators conserved in prokaryotes and eukaryotes. However, no research has focused on the evolution of this superfamily in plants. We identified 685 TMBIM proteins in 171 organisms from Archaea, Bacteria, and Eukarya, and provided a phylogenetic overview of the whole TMBIM superfamily. Then, we used orthology and synteny network analyses to further investigate the evolution and expansion of the BI and LFG families in 48 plants from diverse taxa. Plant BI family forms a single monophyletic group; however, monocot BI sequences transposed to another genomic context during evolution. Plant LFG family, which expanded trough whole genome and tandem duplications, is subdivided in LFG I, LFG IIA, and LFG IIB major phylogenetic groups, and retains synteny in angiosperms. Moreover, two orthologous groups (OGs) are shared between bryophytes and seed plants. Other several lineage-specific OGs are present in plants. This work clarifies the phylogenetic classification of the TMBIM superfamily across the three domains of life. Furthermore, it sheds new light on the evolution of the BI and LFG families in plants providing a benchmark for future research.

Targeting Transmembrane BAX Inhibitor Motif Containing 1 Alleviates Pathological Cardiac Hypertrophy.

Cardiac hypertrophy and its resultant heart failure are among the most common causes of mortality worldwide. Abnormal protein degradation, especially the impaired lysosomal degradation of large organelles and membrane proteins, is involved in the progression of cardiac hypertrophy. However, the underlying mechanisms have not been fully elucidated. We investigated cardiac transmembrane BAX inhibitor motif containing 1 (TMBIM1) mRNA and protein expression levels in samples from patients with heart failure and mice with aortic banding (AB)-induced cardiac hypertrophy. We generated cardiac-specific Tmbim1 knockout mice and cardiac-specific Tmbim1-overexpressing transgenic mice and then challenged them with AB surgery. We used microarray, confocal image, and coimmunoprecipitation analyses to identify the downstream targets of TMBIM1 in cardiac hypertrophy. Tmbim1/Tlr4 double-knockout mice were generated to investigate whether the effects of TMBIM1 on cardiac hypertrophy were Toll-like receptor 4 (TLR4) dependent. Finally, lentivirus-mediated TMBIM1 overexpression in a monkey AB model was performed to evaluate the therapeutic potential of TMBIM1. TMBIM1 expression was significantly downregulated on hypertrophic stimuli in both human and mice heart samples. Silencing cardiac Tmbim1 aggravated AB-induced cardiac hypertrophy. This effect was blunted by Tmbim1 overexpression. Transcriptome profiling revealed that the TLR4 signaling pathway was disrupted dramatically by manipulation of Tmbim1. The effects of TMBIM1 on cardiac hypertrophy were shown to be dependent on TLR4 in double-knockout mice. Fluorescent staining indicated that TMBIM1 promoted the lysosome-mediated degradation of activated TLR4. Coimmunoprecipitation assays confirmed that TMBIM1 directly interacted with tumor susceptibility gene 101 via a PTAP motif and accelerated the formation of multivesicular bodies that delivered TLR4 to the lysosomes. Finally, lentivirus-mediated TMBIM1 overexpression reversed AB-induced cardiac hypertrophy in monkeys. TMBIM1 protects against pathological cardiac hypertrophy through promoting the lysosomal degradation of activated TLR4. Our findings reveal the central role of TMBIM1 as a multivesicular body regulator in the progression of pathological cardiac hypertrophy, as well as the role of vesicle trafficking in signaling regulation during cardiac hypertrophy. Moreover, targeting TMBIM1 could be a novel therapeutic strategy for treating cardiac hypertrophy and heart failure.

Shrimp miR-12 Suppresses White Spot Syndrome Virus Infection by Synchronously Triggering Antiviral Phagocytosis and Apoptosis Pathways.

Growing evidence has indicated that the innate immune system can be regulated by microRNAs (miRNAs). However, the mechanism underlying miRNA-mediated simultaneous activation of multiple immune pathways remains unknown. To address this issue, the role of host miR-12 in shrimp (Marsupenaeus japonicus) antiviral immune responses was characterized in the present study. The results indicated that miR-12 participated in virus infection, host phagocytosis, and apoptosis in defense against white spot syndrome virus invasion. miR-12 could simultaneously trigger phagocytosis, apoptosis, and antiviral immunity through the synchronous downregulation of the expression of shrimp genes [PTEN (phosphatase and tensin homolog) and BI-1(transmembrane BAX inhibitor motif containing 6)] and the viral gene (wsv024). Further analysis showed that miR-12 could synchronously mediate the 5′–3′ exonucleolytic degradation of its target mRNAs, and this degradation terminated in the vicinity of the 3′ untranslated region sequence complementary to the seed sequence of miR-12. Therefore, the present study showed novel aspects of the miRNA-mediated simultaneous regulation of multiple immune pathways.

Variation at 2q35 (PNKD and TMBIM1) influences colorectal cancer risk and identifies a pleiotropic effect with inflammatory bowel disease.

To identify new risk loci for colorectal cancer (CRC), we conducted a meta-analysis of seven genome-wide association studies (GWAS) with independent replication, totalling 13 656 CRC cases and 21 667 controls of European ancestry. The combined analysis identified a new risk association for CRC at 2q35 marked by rs992157 (P = 3.15 × 10−8, odds ratio = 1.10, 95% confidence interval = 1.06–1.13), which is intronic to PNKD (paroxysmal non-kinesigenic dyskinesia) and TMBIM1 (transmembrane BAX inhibitor motif containing 1). Intriguingly this susceptibility single-nucleotide polymorphism (SNP) is in strong linkage disequilibrium (r2 = 0.90, D′ = 0.96) with the previously discovered GWAS SNP rs2382817 for inflammatory bowel disease (IBD). Following on from this observation we examined for pleiotropy, or shared genetic susceptibility, between CRC and the 200 established IBD risk loci, identifying an additional 11 significant associations (false discovery rate [FDR]) < 0.05). Our findings provide further insight into the biological basis of inherited genetic susceptibility to CRC, and identify risk factors that may influence the development of both CRC and IBD.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

Depletion of three combined THOC5 mRNA export protein target genes synergistically induces human hepatocellular carcinoma cell death

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy.

TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy and reduces renal dysfunction in a cyclosporine A-induced nephrotoxicity model

Cyclosporine A (CsA) is widely used as an immunosuppressor in transplantation. Previous studies reported that CsA induces autophagy and that chronic treatment with CsA results in accumulation of autophagosomes and reduced autophagic clearance. Autophagy is a prosurvival process that promotes recovery from acute kidney injury by degrading misfolded proteins produced in the kidney. In the present study, we used TMBIM6-expressing HK-2, human kidney tubular cells (TMBIM6 cells) and Tmbim6 knockout (tmbim6−/−) mice. When exposed to CsA, the TMBIM6 cells maintained autophagy activity by preventing autophagosome accumulation. With regard to signaling, PRKKA/AMPK phosphorylation and mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) expression and its downstream target TFEB (transcription factor EB), a lysosome biogenesis factor, were regulated in the TMBIM6 cells. Lysosomal activity was highly increased or stably maintained in the presence of TMBIM6. In addition, treatment of tmbim6−/− mice with CsA resulted in increased autophagosome formation and decreased lysosome formation and activity. We also found that tmbim6−/− mice were susceptible to CsA-induced kidney injury. Taken together, these results indicate that TMBIM6 protects against CsA-induced nephrotoxicity both in vitro and in vivo by inducing autophagy and activating lysosomes.

The transmembrane Bax inhibitor motif (TMBIM) containing protein family: Tissue expression, intracellular localization and effects on the ER CA2+-filling state

Bax inhibitor-1 (BI-1) is an evolutionarily conserved pH-dependent Ca2+ leak channel in the endoplasmic reticulum and the founding member of a family of six highly hydrophobic mammalian proteins named transmembrane BAX inhibitor motif containing (TMBIM) 1-6 with BI-1 being TMBIM6. Here we compared the structure, subcellular localization, tissue expression and the effect on the cellular Ca2+ homeostasis of all family members side by side. We found that all TMBIM proteins possess the di-aspartyl pH sensor responsible for pH sensing identified in TMBIM6 and its bacterial homologue BsYetJ. TMBIM1-3 and TMBIM4-6 represent two phylogenetically distinct groups that are localized in the Golgi apparatus (TMBIM1-3), endoplasmic reticulum (TMBIM4-6) or mitochondria (TMBIM5) but share a common structure of at least seven transmembrane domains with the last domain being semi-hydrophobic. TMBIM1 is mainly expressed in muscle, TMBIM2 and 3 in the nervous system, TMBIM4 and 5 are ubiquitously expressed and TMBIM6 in skeletal muscle, kidney, liver and spleen. All TMBIM proteins reduce the Ca2+ content of the endoplasmic reticulum, and all but TMBIM5 also reduce the cytosolic resting Ca2+ concentration. These results suggest that the TMBIM family has comparable functions in the maintenance of intracellular Ca2+ homeostasis in a wide variety of tissues. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. Guest Editors: Jacques Haiech, Claus Heizmann and Joachim Krebs.

Structural basis for a pH-sensitive calcium leak across membranes.

Calcium homeostasis balances passive calcium leak and active calcium uptake. Human Bax inhibitor-1 (hBI-1) is an antiapoptotic protein that mediates a calcium leak and is representative of a highly conserved and widely distributed family, the transmembrane Bax inhibitor motif (TMBIM) proteins. Here, we present crystal structures of a bacterial homolog and characterize its calcium leak activity. The structure has a seven-transmembrane-helix fold that features two triple-helix sandwiches wrapped around a central C-terminal helix. Structures obtained in closed and open conformations are reversibly interconvertible by change of pH. A hydrogen-bonded, pKa (where Ka is the acid dissociation constant)-perturbed pair of conserved aspartate residues explains the pH dependence of this transition, and biochemical studies show that pH regulates calcium influx in proteoliposomes. Homology models for hBI-1 provide insights into TMBIM-mediated calcium leak and cytoprotective activity.

Allowing calcium to leak across a membrane

Calcium Channels Cells maintain a balance between calcium in the cytosol and calcium stored in organelles—too much stored calcium kills cells. Transmembrane Bax inhibitor motif (TMBIM) proteins form channels in organelle membranes that allow calcium to leak out. Chang et al. show that this calcium

DNA and RNA isolation from canine oncologic formalin-fixed, paraffin-embedded tissues for downstream “-omic” analyses

Formalin-fixed, paraffin-embedded (FFPE) tissues represent a unique source of archived biological material, but obtaining suitable DNA and RNA for retrospective "-omic" investigations is still challenging. In the current study, canine tumor FFPE blocks were used to 1) compare common commercial DNA and RNA extraction kits; 2) compare target gene expression measured in FFPE blocks and biopsies stored in a commercial storage reagent; 3) assess the impact of fixation time; and 4) perform biomolecular investigations on archival samples chosen according to formalin fixation times. Nucleic acids yield and quality were determined by spectrophotometer and capillary electrophoresis, respectively. Quantitative real-time polymerase chain reaction assays for the following genes: BCL-2-associated X protein, B-cell lymphoma extra large, antigen identified by monoclonal antibody Ki-67, proto-oncogene c-KIT (c-kit). Two internal control genes (Golgin A1 and canine transmembrane BAX inhibitor motif containing 4), together with direct sequencing of c-kit exons 8, 9, 11, and 17, were used as end points. Differences in DNA/RNA yield and purity were noticed among the commercial kits. Nucleic acids (particularly RNA) extracted from paraffin blocks were degraded, even at lower fixation times. Compared to samples held in the commercial storage reagent, archived tissues showed a poorer amplification. Therefore, a gold standard protocol for DNA/RNA isolation from canine tumor FFPE blocks for molecular investigations is still troublesome. More standardized storage conditions, including time between sample acquisition and fixation, fixation time, and sample thickness, are needed to guarantee the preservation of nucleic acids and, then, their possible use in retrospective transcriptomic analysis.

Transcription activity of MMP-2 and MMP-9 metalloproteinase genes and their tissue inhibitor (TIMP-2) in acute coronary syndrome patients

Acute coronary syndromes (ACS) are a consequence of coronary vessel atherosclerosis and they are a leading cause of death in industrialized countries. One of the ACS causative factors is the deranged ratio equilibrium of the matrix metalloproteinase/tissue inhibitor of metalloproteinases (MMPs/TIMPs). Assessment of transcriptional activity of metalloproteinase genes using Human Genome-U133A oligonucleotide microarrays and selection of candidate genes differentiating ACS patients from healthy subjects and finally, QRT-PCR (quantitative real time polymerase chain reaction) confirmation of the results. The study involved 67 ACS patients, admitted on a consecutive basis, to the Cardiology Clinic as well as 24 healthy subjects (control). Ribonucleic acid isolated from peripheral blood mononuclear cells was analyzed by QRT-PCR. Transcriptional activity of the analyzed gene was assessed with TaqMan gene expression assays. U Mann-Whitney test was used to compare the results. Homogeneity of the investigated group was assessed through hierarchical clusterization whereas the nine genes differentiating ACS patients from healthy persons were selected using the Bland-Altman technique. Among these genes three (platelet derived growth factor D, NUAK family SNF1-like kinase 1 and peroxisomal biogenesis factor 1) showed decreased transcriptional activity whereas the remaining six genes (MMP-2 and MMP-9, CDK5RAP3, transmembrane BAX inhibitor motif containing 1, adenylate cyclase-associated protein 1 and TIMP-2) were increased. MMP-2, MMP-9 and TIMP-2 were further characterized by QRT-PCR. The obtained results permit to conclude that the increased expression of MMP-2 and MMP-9 metalloproteinases and their tissue inhibitor (TIMP-2) is responsible for disturbed equilibrium of the metalloproteinase/tissue inhibitors system and as a consequence, for destabilization of atherosclerotic plaque and occurrence of the acute coronary syndrome in the investigated group of patients.

Mouse transmembrane BAX inhibitor Motif 3 (Tmbim3) encodes a 38 kDa transmembrane protein expressed in the central nervous system

Cell survival proteins play an important role throughout nervous system development, normal physiological processes, and pathological conditions. Transmembrane BAX inhibitor motif 3 (TMBIM3, also known as GRINA), is a member of a family of proteins that contain a conserved BAX inhibitor-1 motif. This family of proteins includes several members that have been shown to protect cells from apoptosis. In this study, the authors show that TMBIM3 is expressed in the brain including high levels in the hippocampus. Biochemical and sequence analysis of TMBIM3 demonstrates that the rat, murine, and human genes encode an approximately 38kDa protein with a predicted seven transmembrane domain topology. A Tmbim3 knockout mouse line did not have an obvious phenotype, but may prove useful in future studies of this family of proteins.

Transmembrane BAX Inhibitor Motif Containing (TMBIM) Family Proteins Perturbs a trans-Golgi Network Enzyme, Gb3 Synthase, and Reduces Gb3 Biosynthesis

Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-D-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.