Auswahl der wissenschaftlichen Literatur zum Thema „PmbA protein family“

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.

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.

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.

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.

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.

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.

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.

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.

Lincosamides form a small but important group of specialized microbial metabolites with antibiotic activity. The most important members of this group are celesticetin and clinically used lincomycin. Structurally, lincosamides are composed of an amino sugar and an amino acid connected by an amide bond. The amino acid precursors of both lincosamides remarkably differ. Proteinogenic L-proline is the precursor of celesticetin, while an unusual amino acid (2S,4R)-4-propyl- L-proline (PPL) is incorporated in the more efficient compound lincomycin. Surprisingly, both these precursors are recognized and activated for further biosynthetic steps by homologous adenylation domains CcbC and LmbC, respectively. The detailed description of this amino acid recognition and activation step, which is critical for the biological activity of the resulting compound, was the aim of the first part of this thesis. The site-directed mutagenesis of the LmbC substrate binding pocket and biochemical characterization of resulting mutants were employed to identify the residues crucial for the activation of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket (like in CcbC) to the PPL-specific enzyme (LmbC). The substitution of only three amino acid...