Molecular Characterization of Two Arabidopsis Ire1 Homologs, Endoplasmic Reticulum-Located Transmembrane Protein Kinases

A major response of eukaryotic cells to the presence of unfolded proteins in the lumen of the endoplasmic reticulum (ER) is to activate genes that encode ER-located molecular chaperones, such as the binding protein. This response, called the unfolded protein response, requires the transduction of a signal from the ER to the nucleus. In yeast (Saccharomyces cerevisiae) and mammalian cells, an ER-located transmembrane receptor protein kinase/ribonuclease called Ire1, with a sensor domain in the lumen of the ER, is the first component of this pathway. Here, we report the cloning and derived amino acid sequences of AtIre1-1 and AtIre1-2, two Arabidopsis homologs of Ire1. The two proteins are located in the perinuclear ER (based on heterologous expression of fusions with green fluorescent protein). The expression patterns of the two genes (using beta-glucuronidase fusions) are nearly nonoverlapping. We also demonstrate functional complementation of the sensor domains of the two proteins in yeast and show that the Ire1-2 protein is capable of autotransphosphorylation. These and other findings are discussed in relation to the involvement of these genes in unfolded protein response signaling in plants.     

Do differences in plant and flower age change mating patterns and alter offspring fitness in Raphanus sativus (Brassicaceae)?

When more pollen is present on stigmas than needed to fertilize all ovules, selection among pollen grains may occur due to effects of both pollen donors and maternal plants. We asked whether increasing plant age and flower age, two changes in maternal condition, altered the pattern of seed paternity after mixed pollination. We also asked whether changes in seed paternity affected offspring success in an experimental garden. While flower age did not affect seed paternity, there was a dramatic shift in pollen donor performance as plants aged. These differences were seen in the offspring as well, where the offspring of one pollen donor, which sired more seeds on young plants, flowered earlier in the season, and the offspring of another pollen donor, which sired more seeds on old plants, flowered later in the season. Thus, change in maternal condition resulted in altered seed paternity, perhaps because the environment for pollen tube growth was different. The pattern of seed paternity and offspring performance suggests that pollen donors may show temporal specialization.     

Hydraulic properties of Pinus halepensis,Pinus pinea and Tetraclinis articulata in a dune ecosystem of Eastern Spain

The hydraulic properties of Pinus pinea, Pinus halepensis and Tetraclinis articulata were studied in a coastal dune area from Eastern Spain. The measured variables include vulnerability to xylem embolism (vulnerability curves), hydraulic conductivity and carbon isotopic discrimination in leaves. Leaf water potentials were also monitored in the three studied populations during an extremely dry period. Our results showed that roots had always wider vessels and higher hydraulic conductivity than branches. Roots were also more vulnerable to xylem embolism and operated closer to their hydraulic limit (i.e., with narrower safety margins). Although it was not quantified, extensive root mortality was observed in the two pines during the study period, in agreement with the high values of xylem embolism (> 75%) predicted from vulnerability curves and the water potentials measured in the field. T. articulata was much more resistant to embolism than P. pinea and P. halepensis. Since T. articulata experienced also lower water potentials, safety margins from hydraulic failure were only slightly wider in this species than in the pines. Combining species and tissues, high resistance to xylem embolism was associated with low hydraulic conductivity and with high wood density. Both relationships imply a cost of having a resistant xylem. The study outlined very different water-use strategies for T. articulata and the pines. Whereas T. articulata had a conservative strategy that relied on the low vulnerability of its conducting system to drought-induced xylem embolism, the two pines showed regulatory mechanisms at different levels (i.e., embolism, root demography) that constrained the absorption of water when it became scarce. This revised version was published online in August 2006 with corrections to the Cover Date.     

Lapatinib,a dual HER1/HER2 tyrosine kinase inhibitor,augments basal cleavage of HER2 extracellular domain (ECD) to inhibit HER2‐driven cancer cell growth

The ultimate biological and clinical meaning of shed HER2 extracellular domain (ECD) has remained largely unclear until recently. Oversecretion of soluble HER2 ECD has been shown to inhibit growth of HER2‐overexpressing cancer cells by promoting HER2 ECD dimerization with HER transmembrane receptors thus impairing their cross‐tyrosine phosphorylation and decreasing their activation status. HER2‐targeted drugs capable to enhance the occurrence of basal HER2 ECD shedding but simultaneously preventing formation of truncated cell membrane‐bound HER2 intracellular fragment, which exhibits an undesirable constitutive kinase activity, might be extremely efficient at managing HER2‐positive cancer disease. The dual HER1/HER2 Tyrosine Kinase inhibitor lapatinib, which works intracellularly and directly targets the TK domain of HER2, drastically augments basal shedding of HER2 ECD to inhibit HER2‐driven cancer cell growth. Lapatinib treatment significantly augments the concentration of the inactive (unphosphorylated) form of HER2 protein at the tumor cell membrane and promotes an exacerbated HER2 ECD shedding to the extracellular milieu of HER2‐overexpressing cancer cells. Exacerbated sensitivity of trastuzumab‐resistant cancer cells, which contain nearly undetectable levels of soluble HER2 ECD when compared with trastuzumab‐sensitive parental cells to lapatinib‐induced cell growth inhibition, takes place when lapatinib treatment fully restores high levels of basal HER2 ECD shedding. The dramatic augmentation of HER2 ECD shedding that occurs upon treatment of with lapatinib is fully suppressed in lapatinib‐refractory HER2‐positive cells. These findings, altogether, may provide crucial insights concerning clinical studies aimed to accurately describe HER2 ECD as a potential predictor of response or resistance to the HER2‐targeted drugs trastuzumab and lapatinib. J. Cell. Physiol. 226: 52–57, 2010. © 2010 Wiley‐Liss, Inc.     

CD40 Gene Silencing Reduces the Progression of Experimental Lupus Nephritis Modulating Local Milieu and Systemic Mechanisms

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders.     

Protein Array Profiling of Tic Patient Sera Reveals a Broad Range and Enhanced Immune Response against Group A Streptococcus Antigens

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.     

Surfome Analysis as a Fast Track to Vaccine Discovery: IDENTIFICATION OF A NOVEL PROTECTIVE ANTIGEN FOR GROUP B STREPTOCOCCUS HYPERVIRULENT STRAIN COH1

Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against infectious diseases. In the present work, we confirmed previous data from our laboratory showing that whole viable bacterial cell treatment with proteases followed by the identification of released peptides by mass spectrometry is the method of choice for the rapid and reliable identification of vaccine candidates in Gram-positive bacteria. When applied to the Group B Streptococcus COH1 strain, 43 surface-associated proteins were identified, including all the protective antigens described in the literature as well as a new protective antigen, the cell wall-anchored protein SAN_1485 belonging to the serine-rich repeat protein family. This strategy overcomes the difficulties so far encountered in the identification of novel vaccine candidates and speeds up the entire vaccine discovery process by reducing the number of recombinant proteins to be tested in the animal model.Vaccination is the safest, most attractive, and cost-effective solution to combat infectious diseases (1). Unfortunately vaccines against several pathogens are not yet available, and this is largely because of the difficulties encountered in the identification of the few pathogen components capable of eliciting protective immune responses.Recently new genomics-based approaches have been described and shown to be very powerful for the discovery of vaccine candidates (2–4). However, these methods are labor-intensive and time-consuming in that the identification of the few protective antigens requires the screening of a large number of recombinant proteins in biological assays, which usually involve animal models. Therefore, the development of new strategies capable of substantially reducing the number of proteins to be tested would be highly desirable. Looking at the list of vaccines, either licensed or in advanced phase of development, that protect by eliciting antibody-mediated immunity, it appears that they include secreted toxins and/or highly expressed, surface-exposed molecules (5, 6). Hence the development of strategies capable of singling out this relatively small group of antigens from the plethora of pathogen components would substantially accelerate the vaccine discovery process.We have recently proposed a novel proteomics-based approach, which has allowed the identification of Group A Streptococcus (GAS)¹ proteins having domains protruding out of the bacterial surface (7). The approach is based on (i) the proteolytic treatment of bacteria under conditions that preserve cell viability and (ii) the analysis of the released peptides by mass spectrometry. The approach proved to be rapid and highly selective in that the large majority (>90%) of the identified proteins fell into the categories of cell wall proteins, lipoproteins, membrane proteins, and secreted proteins. Furthermore the method also allowed a semiquantitative evaluation of protein exposition and level of expression because, in general, the number of peptides identified from a given protein nicely correlates with the extent of its recognition by specific antibodies as judged by fluorescence-activated cell sorting analysis (7). Interestingly the list of surface-associated proteins included most of the published GAS protective antigens as well as new protective components such as the cell envelope proteinase Spy0416 (7), a protein attracting the interest of several laboratories for its important role in pathogenesis (8–10). To demonstrate that the proteomics-based approach represents a reliable and generally applicable strategy for the identification of vaccine components in Gram-positive bacteria, we have applied the same protocol to the Group B Streptococcus (GBS) for which a vaccine is not yet available on the market. GBS is a multiserotype Gram-positive opportunistic human pathogen that can lead to life-threatening infections in newborns and elderly adults (11–16).Here we show that on the surface of the hypervirulent GBS COH1 strain there are 43 major proteins belonging to the families of cell wall proteins, lipoproteins, and membrane proteins. As in the case of GAS (7), the proteins identified comprise all protective antigens so far described in the literature (6, 17,–26) as well as a new antigen, SAN_1485, which appears to be highly protective in the active maternal immunization mouse model. These data confirm the effectiveness of protease digestion coupled to mass spectrometry for the identification of surface-exposed antigens in Gram-positive bacteria and demonstrate the power of this technology for the rapid discovery of new vaccines.     

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co‐express KCNQ1 with KCNE1‐5 proteins. KCNQ1 may co‐associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K⁺ channels in sphingolipid–cholesterol‐enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK‐293 cells were transiently transfected with KCNQ1 and KCNE1–5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co‐localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3–5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation. J. Cell. Physiol. 225: 692–700, 2010. © 2010 Wiley‐Liss, Inc.