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Vobruba, Simon, Zdenek Kamenik, Stanislav Kadlcik, and Jiri Janata. "N-Deacetylation in Lincosamide Biosynthesis Is Catalyzed by a TldD/PmbA Family Protein." ACS Chemical Biology 15, no. 8 (August 6, 2020): 2048–54. http://dx.doi.org/10.1021/acschembio.0c00224.
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Bozulic, Larry, Mohammad Malik, David Powell, Adrian Nanez, Andrew Link, Kenneth Ramos, and William Dean. "Plasma membrane Ca2+-ATPase associates with CLP36, α-actinin and actin in human platelets." Thrombosis and Haemostasis 97, no. 04 (2007): 587–97. http://dx.doi.org/10.1160/th06-08-0438.
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SummaryThe plasma membrane Ca2+-ATPase (PMCA) plays an essentialrole in maintaining low cytosolic Ca2+ in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts viaits C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the lastten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pumpto the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitationassays coupled with liquid chromatography/tandemmass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing proteinassociated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extractusing a fusion protein containing the C-terminal PDZ domainbinding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized plateletsdemonstrated the existence of a 1,000-kDa complex containingPMCA and CLP36, and in addition, α -actinin and actin. Immunoflourescencemicroscopy confirmed the co-localization ofPMCA with CLP36 in resting and activated platelets. Taken togetherthese results suggest that PMCA is localized in non-filamentousactin complexes in resting platelets by means of PDZdomain interactions and then associates with the actin cytoskeletonduring cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine andtyrosine phosphorylation events previously described in humanplatelets, PMCA function may be regulated by interactions withanchoring and cytoskeletal proteins.
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Markham, Philip F., Michelle D. Glew, Glenn F. Browning, Kevin G. Whithear, and Ian D. Walker. "Expression of Two Members of the pMGA Gene Family of Mycoplasma gallisepticum Oscillates and Is Influenced by pMGA-Specific Antibodies." Infection and Immunity 66, no. 6 (June 1, 1998): 2845–53. http://dx.doi.org/10.1128/iai.66.6.2845-2853.1998.
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ABSTRACT Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the expression of pMGA and instead expressed an antigenically unrelated new polypeptide (p82). Upon the removal of antibody, the progeny of these cells regained pMGA expression and produced antigenically sectored colonies. The basis of this switch between pMGA+ and pMGA− states was shown to be transcriptional. The p82 polypeptide, the expression of which resulted from growth of cells in antibodies, was another member of the pMGA gene family and was located just downstream from the pMGA gene normally expressed by the M. gallisepticum cells used. Collectively the results of this work suggest that this organism has evolved an unusual means of altering the antigenic composition of its surface in response to antibodies or to other environmental cues.
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Drouault, Sophie, Jamila Anba, Sophie Bonneau, Alexander Bolotin, S. Dusko Ehrlich, and Pierre Renault. "The Peptidyl-Prolyl Isomerase Motif Is Lacking in PmpA, the PrsA-Like Protein Involved in the Secretion Machinery of Lactococcus lactis." Applied and Environmental Microbiology 68, no. 8 (August 2002): 3932–42. http://dx.doi.org/10.1128/aem.68.8.3932-3942.2002.
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ABSTRACT The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.
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Liu, Li, D. Michael Payne, Vicky L. van Santen, Kevin Dybvig, and Victor S. Panangala. "A Protein (M9) Associated with Monoclonal Antibody-Mediated Agglutination of Mycoplasma gallisepticum Is a Member of the pMGA Family." Infection and Immunity 66, no. 11 (November 1, 1998): 5570–75. http://dx.doi.org/10.1128/iai.66.11.5570-5575.1998.
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ABSTRACT A 62-kDa cell surface antigen (M9) of Mycoplasma gallisepticum PG31 that mediates antibody-induced agglutination of the organism was purified and subjected to N-terminal amino-acid sequencing. A 999-bp region of the cDNA encoding the M9 protein was generated by reverse transcription-PCR, and its nucleotide sequence was determined. PCR primers based on this sequence were used to screen a genomic DNA library of PG31. A full-length M9 protein-encoding gene was isolated and sequenced, revealing 96% nucleotide identity with thepMGA1.1 gene of M. gallisepticum S6. Sequence analyses of the M9 gene and flanking open reading frames that encode other pMGA family members suggest that a tandemly repeated GAA sequence may influencepMGA gene expression.
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Markham, P. F., M. D. Glew, J. E. Sykes, T. R. Bowden, T. D. Pollocks, G. F. Browning, K. G. Whithear, and I. D. Walker. "The organisation of the multigene family which encodes the major cell surface protein, pMGA, ofMycoplasma gallisepticum." FEBS Letters 352, no. 3 (October 3, 1994): 347–52. http://dx.doi.org/10.1016/0014-5793(94)00991-0.
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Yano, Koichi, Takashi Imaeda, and Tomoaki Niimi. "Transcriptional activation of the human claudin-18 gene promoter through two AP-1 motifs in PMA-stimulated MKN45 gastric cancer cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 294, no. 1 (January 2008): G336—G343. http://dx.doi.org/10.1152/ajpgi.00328.2007.
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Claudin-18 ( CLDN18), a member of the claudin family of proteins that are structural components of tight junctions, has two alternatively spliced variants, claudin-18a1 and claudin-18a2, which are highly expressed in lung and stomach, respectively. Downregulation of claudin-18a2 is associated with gastric cancers of an intestinal phenotype; however, the mechanisms regulating its expression have not been defined. Here, we found that phorbol 12-myristate 13-acetate (PMA) treatment of MKN45 human gastric cancer cell line increased claudin-18a2 expression. In addition, this study aimed to characterize the human CLDN18a2 promoter. Using reporter gene assays and deletion analysis, we mapped the critical promoter region of the PMA-stimulated claudin-18a2 expression to the −923/−286 region. Electrophoretic mobility shift assays and mutational analyses revealed that two activator protein (AP)-1 binding sites played an important role in the expression of claudin-18a2 in PMA-stimulated MKN45 cells. Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors suppressed the upregulation of claudin-18a2. These results indicate that the PKC/MAPK/AP-1 dependent pathway regulates claudin-18a2 expression in gastric cells.
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Nurden, Paquita, Najet Debili, William Vainchenker, Regis Bobe, Raymonde Bredoux, Elisabeth Corvazier, Robert Combrie, et al. "Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia." Blood 108, no. 8 (October 15, 2006): 2587–95. http://dx.doi.org/10.1182/blood-2006-03-009449.
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AbstractIn type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ibα (GPIbα). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbα N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbα, αIIb, and β3 were normally present. Proteins involved in Ca2+ homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca2+ ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP3-R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal megakaryocytes cultured from peripheral blood CD34+ cells with TPO. Significantly, megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production.
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Waxman, Ian, Dongxu Xie, Carmella van de Ven, Janet Ayello, Nancy Day, Megan S. Lim, Sherrie L. Perkins, and Mitchell S. Cairo. "Correlation Between Increases in Apoptosis in Primary Mediastinal B-Cell Lymphoma (PMBL) and NF-κB Pathway Blockade: Bortezomib (BTZ), Small Molecule IKK Inhibitor ML120B and Combination Therapy Significantly Decrease NF-κB Family Member Protein Activation." Blood 112, no. 11 (November 16, 2008): 4983. http://dx.doi.org/10.1182/blood.v112.11.4983.4983.
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Abstract Primary mediastinal B-cell lymphoma (PMBL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) with a significantly lower event-free survival in children than other identically treated DLBCLs (Lones/Cairo, JCO, 2000). It has a unique gene expression profile with upregulation of a subset of NF-κB signal pathway genes (Rosenwald, J Exp Med, 2003). We previously reported significant increases in apoptosis in a PMBL cell line with NF-κB pathway blocking agents bortezomib (BTZ) and small molecule IKK inhibitor ML120B (supplied by Millennium Pharmaceuticals, MA) used alone (BTZ alone: 5.25%±1.43 increase in apoptosis vs. control; ML120B alone: 4.0%±1.13 increase in apoptosis vs. control, p<.0005) and in combination (ML120B+BTZ vs. ML120B single agent therapy; 17-fold increase in apoptosis, p<.01; ML120B+BTZ vs. BTZ single agent therapy; 4-fold increase in apoptosis, p<.01) (Waxman, Ann Onc, 2008a). We now report the effect of single and combination treatment in PMBL on expression of NF-κB family member protein expression and its correlation with apoptosis. PMBL line Karpas-1106P was incubated with BTZ (5 ng/ml), ML120B (10 μg/ml), and ML120B+BTZ for 24h. Activation of NF-κB transcription factors was measured by ELISA (TransAM Kit, Active Motif) and results were correlated with changes in apoptosis, determined by flow cytometry with Annexin V-FITC detection kit (BD Pharmingen) using identical drug doses and incubation times. In untreated PMBL, p50 had the highest activation (0.093±0.005OD/μg protein), followed in order of decreasing activation by P52 (0.045±0.0006), P65 (RelA) (0.040±0.0019), RelB (0.039±0.0016) and c-Rel (0.028±0.0036). ML120B monotherapy was associated with inhibition of the activation of P50 (32% decrease vs. untreated control, 0.064±0.0046; p<0.01), c-Rel (31% decrease vs. untreated control, 0.019±0.0004; p<0.05), P52 (16% decrease vs. untreated control, 0.038±0.0019; p<0.01) and P65 (RelA) (55% decrease vs. untreated control, 0.018±0.0006; p<0.001). BTZ monotherapy inhibited the activation of P52 (9% decrease vs. untreated control, 0.041±0.0006; p<0.01) and RelB (14% decrease vs. untreated control, 0.034±0.0009; p<0.05). ML120B+BTZ combination therapy strongly inhibited the activation of P50 (45% decrease vs. untreated control, 0.051±0.0015; p<0.001), P52 (50% decrease vs. untreated control, 0.022±0.0002; p<0.001) and RelB (43% decrease vs. untreated control, 0.023±0.0002; p<0.001). These results indicate that increases in apoptosis in PMBL previously reported for ML120B monotherapy, BTZ monotherapy, and ML120B+BTZ combination therapy are associated with inhibition of members of the NF-κB family of transcription factors. These results suggest that ML120B and BTZ increase apoptosis in PMBL by preventing activation of NF-κB transcription factors and subsequent transcription of anti-apoptotic genes that would otherwise promote cell survival. As ML120B+BTZ combination therapy leads to greater inhibition of P50 and P52 than single-agent therapy, there appears to be an additive effect when both agents are used together. Furthermore, as only ML120B inhibited c-Rel and RelA activation and only BTZ inhibited RelB activation, it is clear that each NF-κB blocker does not affect all 5 NF-κB transcription factor family members and gene expression profiling may therefore play a role in choosing optimal therapy. Finally, our findings suggest that ML120B and BTZ inhibit will also increase apoptosis in tumors other than PMBL with constitutive activation of the NF-κB pathway and these drugs should therefore be studied in other tumor types with upregulation of NF-κB genes.
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Bandyopadhyay, Sumita, Tapas K. Sengupta, and Eleanor K. Spicer. "PMA induces stabilization of oncostatin M mRNA in human lymphoma U937 cells." Biochemical Journal 410, no. 1 (January 29, 2008): 177–86. http://dx.doi.org/10.1042/bj20070311.
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OSM (oncostatin M) is a pleiotropic cytokine belonging to the IL (interleukin) 6 family that modulates the growth of some cancer cell lines. We have found that PMA treatment of human U937 lymphoma cells increased the steady-state levels of OSM mRNA. Furthermore, the half-life of OSM mRNA was increased from 2.3 to 6.2 h. Measurement of mRNA/hnRNA (heterogeneous nuclear RNA) ratios in PMA-treated cells suggests further that the increase in OSM mRNA is due to enhanced mRNA stability. Consistent with this, synthetic OSM mRNA transcripts decayed faster in extracts of untreated U937 cells than in extracts of PMA-treated cells. The 3′-untranslated region of OSM mRNA contains a putative ARE (AU-rich element) that may play a role in mRNA stabilization. Addition of the OSM ARE motif to the 3′-end of β-globin mRNA increased its decay rate in vitro. Decay assays with β-globin–AREOSM and β-globin transcripts indicate that PMA induces mRNA stabilization in an ARE-dependent manner. PMA also induces at least five OSM ARE-binding proteins. Supershift assays indicated that HuR is present in PMA-induced OSM mRNA–protein complexes. PMA treatment appears to induce translocation of HuR from the nucleus to the cytoplasm. RNA-decay assays indicated that HuR stabilizes OSM RNA in vitro. Additionally, immunodepletion of HuR from U937 cell extracts led to more rapid decay of OSM transcripts. Collectively, these findings suggest that the ARE plays a role in PMA-induced stabilization of OSM mRNA and that this process involves multiple ARE-binding proteins, including HuR.
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Lescasse, Rachel, Jeanine Grisvard, Ghislaine Fryd, Anne Fleury-Aubusson, and Anne Baroin-Tourancheau. "Proposed Function of the Accumulation of Plasma Membrane-Type Ca2+-ATPase mRNA in Resting Cysts of the Ciliate Sterkiella histriomuscorum." Eukaryotic Cell 4, no. 1 (January 2005): 103–10. http://dx.doi.org/10.1128/ec.4.1.103-110.2005.
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ABSTRACT From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca2+-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca2+-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca2+-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.
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Strehler, Emanuel E., and David A. Zacharias. "Role of Alternative Splicing in Generating Isoform Diversity Among Plasma Membrane Calcium Pumps." Physiological Reviews 81, no. 1 (January 1, 2001): 21–50. http://dx.doi.org/10.1152/physrev.2001.81.1.21.
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Calcium pumps of the plasma membrane (also known as plasma membrane Ca2+-ATPases or PMCAs) are responsible for the expulsion of Ca2+ from the cytosol of all eukaryotic cells. Together with Na+/Ca2+ exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca2+concentration. Like the Ca2+ pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca2+ from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca2+ pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca2+ signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA “knockout” mice further support the concept of specific, nonredundant roles for each Ca2+ pump isoform in cellular Ca2+ regulation.
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Papazisi, Leka, Timothy S. Gorton, Gerald Kutish, Philip F. Markham, Glenn F. Browning, Di Kim Nguyen, Steven Swartzell, Anup Madan, Greg Mahairas, and Steven J. Geary. "The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain Rlow." Microbiology 149, no. 9 (September 1, 2003): 2307–16. http://dx.doi.org/10.1099/mic.0.26427-0.
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The complete genome of Mycoplasma gallisepticum strain Rlow has been sequenced. The genome is composed of 996 422 bp with an overall G+C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91 % coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10·4 % (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum.
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Geraghty, Kathryn M., Shuai Chen, Jean E. Harthill, Adel F. Ibrahim, Rachel Toth, Nick A. Morrice, Franck Vandermoere, Greg B. Moorhead, D. Grahame Hardie, and Carol MacKintosh. "Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR." Biochemical Journal 407, no. 2 (September 25, 2007): 231–41. http://dx.doi.org/10.1042/bj20070649.
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AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA–AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.
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Mata, Ana M., María Berrocal, and M. Rosario Sepúlveda. "Impairment of the activity of the plasma membrane Ca2+-ATPase in Alzheimer's disease." Biochemical Society Transactions 39, no. 3 (May 20, 2011): 819–22. http://dx.doi.org/10.1042/bst0390819.
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AD (Alzheimer's disease) is an age-associated neurodegenerative disorder where the accumulation of neurotoxic Aβ (amyloid β-peptide) in senile plaques is a typical feature. Recent studies point out a relationship between Aβ neurotoxicity and Ca2+ dyshomoeostasis, but the molecular mechanisms involved are still under discussion. The PMCAs (plasma membrane Ca2+-ATPases) are a multi-isoform family of proteins highly expressed in brain that is implicated in the maintenance of low intraneural Ca2+ concentration. Therefore the malfunction of this pump may also be responsible for Ca2+ homoeostasis failure in AD. We have found that the Ca2+-dependence of PMCA activity is affected in human brains diagnosed with AD, being related to the enrichment of Aβ. The peptide produces an inhibitory effect on the activity of PMCA which is isoform-specific, with the greatest inhibition of PMCA4. Besides, cholesterol blocked the inhibitory effect of Aβ, which is consistent with the lack of any Aβ effect on PMCA4 found in cholesterol-enriched lipid rafts isolated from pig brain. These observations suggest that PMCAs are a functional component of the machinery that leads to Ca2+ dysregulation in AD and propose cholesterol enrichment in rafts as a protector of the Aβ-mediated inhibition on PMCA.
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Boczek, Tomasz, Marta Sobolczyk, Joanna Mackiewicz, Malwina Lisek, Bozena Ferenc, Feng Guo, and Ludmila Zylinska. "Crosstalk among Calcium ATPases: PMCA, SERCA and SPCA in Mental Diseases." International Journal of Molecular Sciences 22, no. 6 (March 10, 2021): 2785. http://dx.doi.org/10.3390/ijms22062785.
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Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca2+ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca2+, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are considered efficient line of defense against abnormal Ca2+ rises. However, their role is not limited only to Ca2+ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein—calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca2+-ATPases to neuropathology, with a special emphasis on mental diseases.
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Nurden, Paquita, Najet Debili, William Vainchenker, Jean-Max Pasquet, Regis Bobe, Raymonde Bredoux, Alan T. Nurden, and Jocelyne Enouf. "Severe Thrombocytopenia with Impaired Megakaryocytopoiesis in a Family with von Willebrand Disease Type 2B." Blood 106, no. 11 (November 16, 2005): 733. http://dx.doi.org/10.1182/blood.v106.11.733.733.
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Abstract Thrombocytopenia associated with type 2B von Willebrand disease (VWD) is said to result from an increased elimination of platelets due to an abnormal binding of the higher molecular weight VWF multimers. Here we report a family (brother and sister) with severe thrombocytopenia, presence of agglutinated platelets and a very severe spontaneous bleeding syndrome since childhood. Their mutation concerns the VWF A1 domain, with a R1308P substitution which corresponds to an interactive site for GPIb (French VWD network). Electron microscopy showed agglutinates composed of adjacent discoid platelets with plasma membrane domains either in very close contact or showing signs of being fusioned. By flow cytometry, increased amounts of VWF and sometimes also fibrinogen were found at the platelet surface, but not P-selectin. Binding of antibodies to the GP Ibalpha N-terminal domain was decreased whereas GPIX and GPV were normally detected. In Western Blotting (WB), GPIbalpha was normally present but there were signs of filamin degradation. Basal Ca2+ levels were elevated. Platelet proteins involved in Ca2+ regulation were analysed by quantitating mRNA or by WB. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-type 3b was found to be increased but not the isoforms 2b or 3a. Furthermore, plasma membrane Ca2+ ATPase (PMCA)-4b as well as Inositol triphosphate receptor (InsP3-R3) were also increased in concentration. Interestingly, this platelet profile, mimics that observed in normal immature MK before proplatelet formation. As PMCA4b is a target of caspase-3 in MK, another substrate the polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) was evaluated. For both patients, platelets contained increased PARP and a degradation product indicating an abnormal persistance of caspase-3 activity. In vitro, megakaryocyte (MK) culture from peripheral blood CD34+ cells in the presence of SCF and TPO showed impaired MK maturation and the production of self-associated proplatelets. All these results are in favour of a role for abnormal platelet production with abnormal apoptosis that can explain at least in part the marked thrombocytopenia in this family with VWD type 2B.
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Ritz, Olga, Jochen K. Lennerz, Karolin Rommel, Karola Dorsch, Elena Kelsch, Julia Melzner, Michaela Buck, Karen Leroy, Silke Brüderlein, and Peter Moeller. "Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma." Blood 120, no. 21 (November 16, 2012): 2417. http://dx.doi.org/10.1182/blood.v120.21.2417.2417.
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Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.
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Toufaily, Chirine, Cyndia Charfi, Bayader Annabi, and Borhane Annabi. "A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype." Cancer Growth and Metastasis 7 (January 2014): CGM.S18581. http://dx.doi.org/10.4137/cgm.s18581.
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Caveolae are specialized cell membrane invaginations known to regulate several cancer cell functions and oncogenic signaling pathways. Among other caveolar proteins, they are characterized by the presence of proteins of the cavin family. In this study, we assessed the impact of cavin-1, cavin-2, and cavin-3 on cell migration in a human HT-1080 fibrosarcoma model. We found that all cavin-1, -2 and -3 transcripts were expressed and that treatment with phorbol 12-myristate 13-acetate (PMA), which is known to prime cell migration and proliferation, specifically upregulated cavin-3 gene and protein expression. PMA also triggered matrix metalloproteinase (MMP)-9 secretion, but reduced the global cell migration index. Overexpression of recombinant forms of the three cavins demonstrated that only cavin-3 was able to reduce basal cell migration, and this anti-migratory effect was potentiated by PMA. Interestingly, cavin-3 overexpression inhibited PMA-induced MMP-9, while cavin-3 gene silencing led to an increase in MMP-9 gene expression and secretion. Furthermore, recombinant cavin-3 significantly prevented PMA-mediated dephosphorylation of AKT, a crucial regulator in MMP-9 transcription. In conclusion, our results demonstrate that cellular cavin-3 expression may repress MMP-9 transcriptional regulation in part through AKT. We suggest that the balance in cavin-3-to-MMP-9 expression regulates the extent of extracellular matrix degradation, confirming the tumor-suppressive role of cavin-3 in controlling the invasive potential of human fibrosarcoma cells.
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Grasseschi, Helen A., and Michael F. Minnick. "Transformation of Bartonella bacilliformis by electroporation." Canadian Journal of Microbiology 40, no. 9 (September 1, 1994): 782–86. http://dx.doi.org/10.1139/m94-123.
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Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae. The bacterium is an intracellular parasite of human erythrocytes. To date, members of the family Bartonellaceae have not been transformed by standard chemical methods. We report the first successful transformation of a member of the Bartonellaceae family, B. bacilliformis, by the method of electroporation. The optimal conditions for electroporation of B. bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 × 105 was obtained. Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 μg/mL. Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA. Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B. bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization. Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful.Key words: transformation, electroporation, Rickettsiales order.
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Vahling, Cheryl M., and Kevin S. McIver. "Domains Required for Transcriptional Activation Show Conservation in the Mga Family of Virulence Gene Regulators." Journal of Bacteriology 188, no. 3 (February 1, 2006): 863–73. http://dx.doi.org/10.1128/jb.188.3.863-873.2006.
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ABSTRACT Mga, or the multigene regulator of the group A streptococcus (GAS) (Streptococcus pyogenes), is a transcriptional regulator of virulence genes important for colonization and immune evasion. All serotypes of the GAS possess one of two divergent mga alleles (mga-1 or mga-2), and orthologues of Mga have also been identified in other pathogenic streptococci. To date, the only functional motifs established within Mga are two amino-terminal DNA-binding domains (HTH-3 and HTH-4). To uncover novel domains, a random mutagenesis screen using an M6 Mga (mga-1) was undertaken to find mutations leading to a defect in transcriptional activation of the Mga-regulated emm gene. In addition to mutations in the established DNA-binding domains, the screen also revealed mutations in a region conserved among several Mga orthologues. Alanine scanning helped resolve the boundaries of this conserved Mga domain (CMD-1) spanning from residues 10 to 15 of the protein, with the two flanking amino acid residues likely involved in protein stability. Transcriptional reporter analyses demonstrated the importance of CMD-1 for activation of Pemm and autoactivation of Pmga in the serotype M6 Mga. Mutational analyses showed that both CMD-1 and HTH-4 are also necessary for activation of the promoter target Pmrp in a divergent serotype M4 Mga (mga-2), suggesting a conserved functionality. However, in contrast to M6, the M4 Mga mutants did not show a defect in autoregulation. Mutation of similar conserved residues in the Mga-like regulator DmgB from S. dysgalactiae subsp. dysgalactiae showed that CMD-1 and HTH-4 are critical for transcriptional activation in this orthologue, implying that a common mechanism of virulence gene activation may exist for members of the Mga family of regulators.
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SUDBECK, Barry D., Petra BAUMANN, Gavin J. RYAN, Katja BREITKOPF, Roswitha NISCHT, Thomas KRIEG, and Cornelia MAUCH. "Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases." Biochemical Journal 339, no. 1 (March 25, 1999): 167–75. http://dx.doi.org/10.1042/bj3390167.
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Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation.
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SUDBECK, Barry D., Petra BAUMANN, Gavin J. RYAN, Katja BREITKOPF, Roswitha NISCHT, Thomas KRIEG, and Cornelia MAUCH. "Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases." Biochemical Journal 339, no. 1 (April 1, 1999): 167. http://dx.doi.org/10.1042/0264-6021:3390167.
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Torka, Pallawi, Francisco J. Hernandez-Ilizaliturri, Sarah Belliotti, Cory Mavis, Juan Gu, and Myron S. Czuczman. "Inhibition of Nuclear Factor Kappa B (NFkB)/Interferon Regulatory Factor 4 (IRF4) Signaling Pathway in Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) By Combining MLN4924, a Novel NEDD8 Activating Enzyme Inhibitor (NAE) and Ibrutinib." Blood 124, no. 21 (December 6, 2014): 1768. http://dx.doi.org/10.1182/blood.v124.21.1768.1768.
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Abstract Identification of critical signaling pathways required for the development, maintenance and progression of specific subtypes of DLBCL is essential in order to develop novel therapeutic agents, especially for patients with relapsed/refractory disease or those not eligible for high-dose chemotherapy and autologous stem cell support (HDC-ASCT). Gene expression profiling (GEP) studies have identified three distinct subtypes of DLBCL- ABC-DLBCL, germinal center B-cell (GCB) DLBCL and primary mediastinal B-cell lymphoma (PMBL). Pre-clinical and clinical studies suggest that ABC-DLBCL is driven by an abnormally high NFκB activity that deregulates expression of Bcl-2 family proteins, and is associated with resistance to chemotherapy agents resulting in inferior clinical outcomes when compared to GCB-DLBCL or PMBL. In ABC-DLBCL, activation and maintenance of NFκB is mediated by the B-cell receptor (BCR) signaling pathway, the ubiquitin-proteasome system (UPS), oncogenic CARD11 or MYD88 mutations, and/or the effect of IRF4/SPIB on CARD11 or MYD88. Optimal NFκB targeting is an attractive therapeutic strategy in ABC-DLBCL and ongoing clinical trials are incorporating specific inhibitors (bortezomib, lenalidomide, or ibrutinib) in combination with chemo-immunotherapy in the front-line setting. On the other hand, multi-step targeting of the NFκB signaling pathway in ABC-DLBCL using investigational and currently available small molecule inhibitors could result in novel, active, and potentially less toxic regimens for ABC-DLBCL patients. To this end we studied the biological activity of MLN4924, a NAE inhibitor that selectively blocks the UPS up-stream by preventing activation of a subset of ubiquitin ligases known as cullin-RING ligases in combination with ibrutinib in lymphoma pre-clinical models. A panel of rituximab sensitive or resistant lymphoma cell lines representing ABC- and GCB-DLBCL were exposed to MLN4924. Changes in cell viability, cell cycle/NFκB activity, and expression of key regulatory proteins of the cell cycle, Bcl-2 family members, and the UPS were evaluated using the cell titer glo assay, flow cytometry and western blotting respectively. Subsequently, ABC- or GCB-DLBCL cell lines and tumor cells isolated from previously untreated or relapsed/refractory B-cell lymphoma were exposed to MLN4924 in combination with various chemotherapy agents or other available NFkB inhibitors (i.e. ibrutinib) for 24 or 48 hrs. Changes in viability were determined and coefficient of synergy was calculated using the CalcuSyn software. MLN4924 induced cell death in ABC-DLBCL cell lines and to a lesser degree in GCB-DLBCL cell lines. Anti-tumor activity plateau was seen after 48 hrs of drug exposure. In MLN4924 sensitive cells we consistently observed cell cycle arrest in G1 phase, down-regulation of Bcl-XL and PARP cleavage. MLN4924 exposure in vitro resulted in a decrease in Bcl-XL mRNA as determined by quantitative polymerase chain reaction (qPCR),due to the inhibition of NFkB activity as demonstrated in MLN4924-exposed cells by p65 co-localization studies using the imagestream technology. MLN4924 enhanced activity of cytarabine, cisplatin, doxorubicin and etoposide in ABC-, but not in the GCB-DLBCL cell lines. MLN4924 also exhibited synergistic effects when combined with ibrutinib in ABC-DLBCL cell lines at the doses tested. Our data suggests that multi-step targeting of NFκB signaling pathway in ABC-DLBCL is a viable therapeutic strategy and supports further in vivo pre-clinical studies and clinical studies in relapsed/refractory or HDC-ASCT ineligible ABC-DLBCL patients. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.
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Ranuncolo, Stella M., Wenming Xiao, George Wright, Mones S. Abu-Asab, Stefania Pittaluga, Elaine S. Jaffe, and Brian A. Lewis. "A Constitutivly Active NFkB Non Canonical Pathway Is Maintained by RelB Repression of TRAF2 to Prevent Autophagy-Mediated Cell Death in Hodgkin Lymphomas." Blood 118, no. 21 (November 18, 2011): 428. http://dx.doi.org/10.1182/blood.v118.21.428.428.
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Abstract Abstract 428 NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell processes such as proliferation, apoptosis and inflammation. The REL or NFkB family is comprised of hetero or homo dimeric combinations of five members: RELA (p65), RELB, c-REL, NFKB1 (p50) and NFkB2 (p52). Despite considerable evidence supporting the role of the REL members in the immune system and lymphomagenesis, it has never been accurately established whether specific NFkB dimers control a particular set of target genes that account for the biological functions known to be mediated by these TFs. Furthermore, it is not clear whether NFkB controls different targets in distinct subsets of germinal center (GC) B cell derived lymphomas. We performed loss of function experiments delivering specific shRNAs to knock down each REL member in lymphoma cell lines modeling sequential stages of the GC B cell maturation: Mantle Cell Lymphoma (MCL), Diffuse Large B Cell Lymphoma [DLBCL including ABC-Like (Activated B Cell), GCB-Like (Germinal Center B Cell) and PMBL (Primary Mediastinal B cell Lymphoma)], Burkitt Lymphoma (BL) and Hodgkin Lymphoma (HL). All the NHL subsets showed a dependency on RelA and c-Rel to survive but were largely unaffected by RelB depletion. Only HL cells were dramatically sensitive to RelB knock-down. ChIP-seq analysis uncovered an extensive NFkB network in BJAB (GCB), HBL-1 (ABC), U-H01 (HL) and human primary centroblasts. This data shaped two network architectures: a RelB hierarchical network in HL and a c-rel hierarchy in NHL. Out of 4,581 genes that had relB binding (+/− 2kB TSS) in HL cells, more than 70% were only relB targets in HL as compared to NHL cells. Microarray analysis of these cell lines following RelA, RelB or c-Rel knockdown was merged with our ChIP-seq data. The overlap of RelB peaks and RelB shRNA downregulated targets showed that RelB directly controlled cell cycle regulators and apoptosis inhibitors including CCND3, CDK6, BCL2 and BCLxL. Interestingly, RelB maintained HL cell viability through BCL2 induction since expression of a BCL2 cDNA rescued HL cell lines from the shRNA RelB lethality. Strikingly, the RelB induced BCL2 and BCLxL protein down regulation in HL cells does not kill these cells by predicted apoptotic mechanisms. Morphological and molecular evidence indicated that RelB inhibition lead to cell death by autophagy. Electron microscopy revealed intact nucleus with lack of condensed chromatin and citosolic vacuoles. After RelB inhibition, the well known autophagy marker L3C, is completely degraded meanwhile ATG7 protein expression, a key player in the autophagosomes development, is enhanced. RelB suppressed BCL2/BCL-xL dependent autophagic cell death by maintaining high levels of these two pro-survival molecules interacting with the autophagy executing protein Beclin-1. The interaction is disrupted by the RelB shRNA. We found that RelB is engaged in a positive regulatory loop that keeps the non canonical arm of the NFkB pathway on. Activation of the alternative pathway centers on the modulation of NIK (NFkB inducing kinase). In physiological conditions, NIK undergoes constitutive ubiquitin-dependent degradation through the TRAF-2/TRAF-3/c-IAP-1/c-IAP-2 complex, which keeps its abundance below the threshold required for its function. We found that HL cell lines lack TRAF-2 or TRAF-3 protein expression which leads to stabilization and accumulation of NIK. RelB knock down is followed by up-regulation and/or re-expression of TRAF members and depletion of NIK protein. Remarkably, lymph nodes of HL patient biopsies showed NIK protein citosolic expression by IHC in the Reed-Sternberg Cells, indicating stabilization and accumulation of NIK that accounts for constitutive NFkB-2/p100b processing to release active p52-RelB dimers. In summary, we found that HL is the only GC B cell derived malignancy that relies on RelB for viability, and thus differentiates Hodgkin Lymphomas from non-Hodgkin lymphomas. These data argue that each REL member has specific and unique functions and targets, as we predicted from the lack of similarities in their transactivation domains. Of clinical relevance, HL patient samples reproduced the alternative NFkB pathway molecular alterations that we initially observed in HL cell lines. Furthermore, the unique Hodgkin Lymphoma RelB dependency makes the non canonical NF-kB signaling pathway an attractive therapeutic target. Disclosures: No relevant conflicts of interest to declare.
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Davenport, Kristen A., Clare E. Hoover, Nathaniel D. Denkers, Candace K. Mathiason, and Edward A. Hoover. "Modified Protein Misfolding Cyclic Amplification Overcomes Real-Time Quaking-Induced Conversion Assay Inhibitors in Deer Saliva To Detect Chronic Wasting Disease Prions." Journal of Clinical Microbiology 56, no. 9 (June 27, 2018). http://dx.doi.org/10.1128/jcm.00947-18.
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ABSTRACT Chronic wasting disease (CWD), a fatal neurodegenerative prion disease of cervids, has spread across North America and has been detected in The Republic of Korea, Finland, and Norway. CWD appears to spread by horizontal transmission, and prions shed in saliva, feces, and urine are thought to contribute. However, studies investigating the rapid spread of CWD have been hampered by assay inhibitors and a lack of consistent and sensitive means to detect the relatively low levels of prions in these samples. Here we show that saliva frequently contains an inhibitor of the real-time quaking-induced conversion assay (RT-QuIC) and that the inhibitor is a member of the mucin family. To circumvent the inhibitor, we developed a modified protein misfolding cyclic amplification (PMCA) method to amplify CWD prions in saliva that were undetectable or ambiguous by RT-QuIC. Our results reinforce the impact of saliva in horizontal CWD transmission and highlight the importance of detection optimization.
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Palusińska-Szysz, Marta, Agnieszka Szuster-Ciesielska, Monika Janczarek, Sylwia Wdowiak-Wróbel, Jürgen Schiller, Emilia Reszczyńska, Wiesław I. Gruszecki, and Beate Fuchs. "Genetic diversity of Legionella pcs and pmtA genes and the effect of utilization of choline by Legionella spp. on induction of proinflammatory cytokines." Pathogens and Disease 77, no. 7 (October 1, 2019). http://dx.doi.org/10.1093/femspd/ftz065.
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ABSTRACT Legionella species synthesize phosphatidylcholine (PC) in two independent pathways: the three-step methylation of phosphatidylethanolamine PMT pathway and the one-step PCS pathway, in which the Pcs enzyme catalyzes the reaction between choline and CDP-diacylglycerol to form PC. Legionella pcs genes encode highly hydrophobic proteins with phosphatidylcholine synthase activity, which contain up to eight transmembrane helices with N- and C-termini located inside the bacterial cell. The comparative analysis of nucleotide sequences of pcs showed that these genes share high sequence identity among members of the Legionellaceae family. Legionella pmtA genes involved in the PMT pathway encoded small cytosolic proteins with putative phosphatidylethanolamine N-methyltransferase activity. The pmtA genes identified in Legionella species had lower sequence identity to each other than the pcs genes. The phylogenetic tree constructed based on the pcs and pmtA gene sequences showed phylogenetic relatedness between Legionella spp. and other bacteria. The utilization of extracellular choline by the four Legionella species leads to changes not only in the lipid components but also in proteins, and the interactions between these components lead to changes in cell surface properties, which result in a decline in induction of proinflammatory cytokines (TNF-α and IL-6).
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Yano, Junji, Russell Wells, Ying-Wai Lam, and Judith L. Van Houten. "Ciliary Ca2+ pumps regulate intraciliary Ca2+ from the action potential and may co-localize with ciliary voltage-gated Ca2+ channels." Journal of Experimental Biology 224, no. 9 (May 1, 2021). http://dx.doi.org/10.1242/jeb.232074.
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ABSTRACT Calcium ions (Ca2+) entering cilia through the ciliary voltage-gated calcium channels (CaV) during the action potential causes reversal of the ciliary power stroke and backward swimming in Paramecium tetraurelia. How calcium is returned to the resting level is not yet clear. Our focus is on calcium pumps as a possible mechanism. There are 23 P. tetraurelia genes for calcium pumps that are members of the family of plasma membrane Ca2+ ATPases (PMCAs). They have domains homologous to those found in mammalian PMCAs. Of the 13 pump proteins previously identified in cilia, ptPMCA2a and ptPMCA2b are most abundant in the cilia. We used RNAi to examine which PMCA might be involved in regulating intraciliary Ca2+ after the action potential. RNAi for only ptPMCA2a and ptPMCA2b causes cells to significantly prolong their backward swimming, which indicates that Ca2+ extrusion in the cilia is impaired when these PMCAs are depleted. We used immunoprecipitations (IP) to find that ptPMCA2a and ptPMCA2b are co-immunoprecipitated with the CaV channel α1 subunits that are found only in the cilia. We used iodixanol (OptiPrep) density gradients to show that ptPMCA2a and ptPMCA2b and CaV1c are found in the same density fractions. These results suggest that ptPMCA2a and ptPMCA2b are located in the proximity of ciliary CaV channels.