Range Of Functional Assays Research Articles

GTP binding to translation factor eIF2B stimulates its guanine nucleotide exchange activity

SummaryeIF2B is the guanine nucleotide exchange factor (GEF) required for cytoplasmic protein synthesis initiation in eukaryotes and its regulation within the integrated stress response (ISR). It activates its partner factor eIF2, thereby promoting translation initiation. Here we provide evidence through biochemical and genetic approaches that eIF2B can bind directly to GTP and this can enhance its rate of GEF activity toward eIF2–GDP in vitro. GTP binds to a subcomplex of the eIF2Bγ and ε subunits. The eIF2Bγ amino-terminal domain shares structural homology with hexose sugar phosphate pyrophosphorylase enzymes that bind specific nucleotides. A K66R mutation in eIF2Bγ is especially sensitive to guanine or GTP in a range of functional assays. Taken together, our data suggest eIF2Bγ may act as a sensor of purine nucleotide availability and thus modulate eIF2B activity and protein synthesis in response to fluctuations in cellular nucleotide levels.

Helicobacter pylori PqqE is a new virulence factor that cleaves junctional adhesion molecule A and disrupts gastric epithelial integrity

ABSTRACT Helicobacter pylori infects approximately half of the world’s population and is the strongest risk factor for peptic ulcer disease and gastric cancer, representing a major global health concern. H. pylori persistently colonizes the gastric epithelium, where it subverts the highly organized structures that maintain epithelial integrity. Here, a unique strategy used by H. pylori to disrupt the gastric epithelial junctional adhesion molecule-A (JAM-A) is disclosed, using various experimental models that include gastric cell lines, primary human gastric cells, and biopsy specimens of infected and non-infected individuals. H. pylori preferentially cleaves the cytoplasmic domain of JAM-A at Alanine 285. Cells stably transfected with full-length JAM-A or JAM-A lacking the cleaved sequence are used in a range of functional assays, which demonstrate that the H. pylori cleaved region is critical to the maintenance of the epithelial barrier and of cell-cell adhesion. Notably, by combining chromatography techniques and mass spectrometry, PqqE (HP1012) is purified and identified as the H. pylori virulence factor that cleaves JAM-A, uncovering a previously unreported function for this bacterial protease. These findings propose a novel mechanism for H. pylori to disrupt epithelial integrity and functions, breaking new ground in the understanding of the pathogenesis of this highly prevalent and clinically relevant infection.

GTP Binding to Translation Factor eIF2B Stimulates Its Guanine Nucleotide Exchange Activity

eIF2B is the guanine nucleotide exchange factor (GEF) required for cytoplasmic protein synthesis initiation in eukaryotes and its regulation within the integrated stress response (ISR). It activates its partner factor eIF2 thereby promoting translation initiation. Here we provide evidence through biochemical and genetic approaches that eIF2B can bind directly to GTP and this can enhance its rate of GEF activity towards eIF2–GDP in vitro. GTP binds to a subcomplex of the eIF2Bγ and e subunits. The eIF2Bγ amino-terminal domain shares structural homology with hexose sugar phosphate pyrophosphorylase enzymes that bind specific nucleotides. A K66R mutation in eIF2Bγ is especially sensitive to guanine or GTP in a range of functional assays. Taken together, our data suggest eIF2Bγ may act as a sensor of purine nucleotide availability and thus modulate eIF2B activity

Abstract 10829: Biological and Pharmacological Properties of the Protease Proprotein Convertase Subtilisin-Like Kexin Type 9 Antagonists (Best of Basic Science Abstract)

Protease proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a new important target for the treatment of hypercholesterolemia. Currently, several pharmaceutical companies are racing to develop new drugs against PCSK9, which could be the newest weapon against heart disease since the introduction of statins in the 80’s. However, those companies have focused primarily on the development of mAbs, not small molecules, as drugs. PCSK9 is a serine protease that is synthesized as a zymogen that undergoes autocatalytic processing and secretion. Secreted PCSK9 binds to the LDL-receptor (LDLR) and chaperones it to the degradation pathway. This study focused on the identification of antagonists of the PCSK9/LDLR interaction and explored their mechanism of action using a range of functional assays, as well as determined pharmacokinetic (PK) and pharmacodynamic (PD) properties of the lead compounds. Potential inhibitors of the PCSK9/LDLR interaction were identified through a virtual screen at the PCSK9 surface where the LDLR-EGF-A binds. The top hits exhibited inhibition of PCSK9/LDLR binding in the low micromolar (μM) range, a 5-fold upregulation of the LDLR at an inhibitor concentration of 1.6 μM in recombinant cell-based assays, and an increase in the fluorescently labeled DiI-LDL uptake in the nanomolar (nM) range in situ . Pharmacological studies revealed that subcutaneous injection of 4 mg/kg of one of these compounds resulted in a 32% reduction of the total cholesterol and 20% reduction in LDL-C levels in mice fed a high fat/high cholesterol diet. Moreover, administration of 40mg/kg of atorvastatin resulted in a 27% total cholesterol reduction and 20% reduction in LDL-C level. Additional studies were performed to evaluate the effect of the combination of our PCSK9 inhibitor with atorvastatin. Administration of the PCSK9 inhibitor (4 mg/ kg) in combination with atorvastatin (40 mg/kg) resulted in more than a 40% reduction in LDL-C in mice fed a high fat/high cholesterol diet. In vivo PK and PD evaluation of our lead PCSK9 inhibitor demonstrated that it was efficacious and exhibited 13% oral bioavailability. To our knowledge, no small molecule orally bioavailable antagonist against PCSK9 has been reported, only mAbs.

Altered metabolic and stemness capacity of adipose tissue-derived stem cells from obese mouse and human.

Adipose stem cells (ASCs) are an appealing source of cells for therapeutic intervention; however, the environment from which ASCs are isolated may impact their usefulness. Using a range of functional assays, we have evaluated whether ASCs isolated from an obese environment are comparable to cells from non-obese adipose tissue. Results showed that ASCs isolated from obese tissue have a reduced proliferative ability and a loss of viability together with changes in telomerase activity and DNA telomere length, suggesting a decreased self-renewal capacity. Metabolic analysis demonstrated that mitochondrial content and function was impaired in obese-derived ASCs resulting in changes in favored oxidative substrates. These findings highlight the impact of obesity on adult stem properties. Hence, caution should be exercised when considering the source of ASCs for cellular therapies since their therapeutic potential may be impaired.

New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris.

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called "tumor antagonizing genes," whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.

Comparative Effects of the Endogenous Agonist Glucagon-Like Peptide-1 (GLP-1)-(7-36) Amide and the Small-Molecule Ago-Allosteric Agent “Compound 2” at the GLP-1 Receptor

Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca(2+) signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Galpha(s) in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca(2+)] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.

Abstract A51: Development and initial characterization of isogenic cell lines stably overexpressing prostate cancer candidate genes

Abstract Microarray analyses of global changes in gene expression patterns have recovered a large number of genes, which are deregulated in prostate cancer, when compared to normal prostate tissue. In order to successfully translate these data into clinical intervention strategies, it is crucial to determine genes that truly contribute to tumor development (which may be referred to as ‘driver genes’) and to separate them from the majority of secondary anomalies in expression levels (‘passenger genes’). To obtain the missing functional information, we construct isogenic cell line libraries derived from commonly used prostate (RWPE-1) and prostate cancer (LNCaP, PC-3) cell lines and subject them to a range of functional assays. Using a system for rapid generation of stably-transfected isogenic cell lines, that was developed in our group, we create a prostate cancer library in a two-step procedure. At first, acceptor clones are generated from cancer and normal prostate cell lines by stable integration of a plasmid carrying a recombinase target sequence. Incorporation of a second expression plasmid with the gene of interest is then mediated by site-specific recombination. This procedure allows a facilitated transgene integration into a predefined, transcriptionally active locus. It also minimizes the influence of genetic background, as clones generated within such a library differ among each other only by the presence of the gene of interest in the same chromosomal location. Therefore, in contrast to traditional knock-in strategies, our system ensures that readily interpretable phenotypes are obtained and thereby provides a highly standardized resource for functional gene analysis. The technology is well suited for medium throughput and was designed to link data from high-throughput genomic analyses with functional gene analysis in animal models. LNCaP and PC-3 acceptor cell lines have been generated and characterized. Recombination of PC-3 with several well-described, cancer-relevant genes under the control of an inducible promoter indicated the feasibility of the method. The next step will include initial screening of the library of roughly 100 recombinants for changes in the cell viability and proliferation. The most promising effectors from the primary screen will be followed with an expanded range of functional assays, addressing their impact on cell cycle, apoptosis and cell migration. This can then serve as a starting point for an individual characterization of the novel prostate cancer genes. Citation Information: Cancer Res 2009;69(23 Suppl):A51.

NF-κB Promotes Survival during Mitotic Cell Cycle Arrest

By activating the mitotic checkpoint, anti-microtubule drugs such as nocodazole cause mammalian cells to arrest in mitosis and then undergo apoptosis. Microtubule depolymerization is rapid and results in the activation of the transcription factor NF-kappaB and induction of NF-kappaB-dependent gene expression. However, the functional consequence of NF-kappaB activation has remained unclear. Evidence has accumulated to suggest that NF-kappaB transcriptional activity is required to suppress apoptosis. In the present study, we confirm and extend previous findings that microtubule depolymerization leads to the rapid activation of NF-kappaB and test the hypothesis that the induction of NF-kappaB regulates cell survival during mitotic cell cycle arrest in order to define its role. Using a range of functional assays, we have shown that microtubule depolymerization correlates with the activation of IKKalpha and IKKbeta; the phosphorylation, ubiquitination, and degradation of IkappaBalpha; the translocation of native p65 (RelA) into the nucleus; and increased NF-kappaB transcriptional activity. By inhibiting either the activation of the IKKs or the degradation of IkappaBalpha, we find that the level of apoptosis is significantly increased in the mitotically arrested cells. Inhibition of NF-kappaB signaling in the nonmitotic cells did not affect their survival. We establish that although NF-kappaB is activated rapidly in response to microtubule depolymerization, its cell survival function is not required until mitotic cell cycle arrest, when the mitotic checkpoint is activated and apoptosis is triggered. We conclude that NF-kappaB may regulate the transcription of one or more antiapoptotic proteins that may regulate cell survival during mitotic cell cycle arrest.