Showing papers by "Fundación Instituto Leloir published in 2007"
TL;DR: It is suggested that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.
294 citations
TL;DR: In this article, the authors have manipulated separately the circadian photoentrainment pathway within E- and M-cells and show that E-cells process light information and function as master clocks in the presence of light.
231 citations
TL;DR: Different roles for the two glycans present at the E protein during d Dengue virus infection are defined, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.
Abstract: Dengue virus envelope protein (E) contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses, while the site at position 67 is thought to be unique for dengue viruses. N-linked oligosaccharide side chains on flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here, we examined the relevance of each N-linked glycan on dengue virus E protein by removing each site in the context of infectious viral particles. Dengue viruses lacking Asn-67 were able to infect mammalian cells and translate and replicate the viral genome, but production of new infectious particles was abolished. In addition, dengue viruses lacking Asn-153 in the E showed reduced infectivity. In contrast, ablation of one or both glycosylation sites yielded viruses that replicate and propagate in mosquito cells. Furthermore, we found a differential requirement of N-linked glycans for E secretion in mammalian and mosquito cells. While secretion of E lacking Asn-67 was efficient in mosquito cells, secretion of the same protein expressed in mammalian cells was dramatically impaired. Finally, we found that viruses lacking the carbohydrate at position 67 showed reduced infection of immature dendritic cells, suggesting interaction between this glycan and the lectin DC-SIGN. Overall, our data defined different roles for the two glycans present at the E protein during dengue virus infection, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.
185 citations
TL;DR: This study compares mature neurons born in the embryonic and adult hippocampus, with a focus on intrinsic membrane properties and γ‐aminobutyric acid (GABA)ergic synaptic inputs, and demonstrates that granule cells of different age, location and degree of excitability receive GABAergic inputs of equivalent functional characteristics.
Abstract: Neurogenesis in the dentate gyrus of the hippocampus follows a unique temporal pattern that begins during embryonic development, peaks during the early postnatal stages and persists through adult life. We have recently shown that dentate granule cells born in early postnatal and adult mice acquire a remarkably similar afferent connectivity and firing behavior, suggesting that they constitute a homogeneous functional population [Laplagne et al. (2006)PLoS Biol., 4, e409]. Here we extend our previous study by comparing mature neurons born in the embryonic and adult hippocampus, with a focus on intrinsic membrane properties and gamma-aminobutyric acid (GABA)ergic synaptic inputs. For this purpose, dividing neuroblasts of the ventricular wall were retrovirally labeled with green fluorescent protein at embryonic day 15 (E15), and progenitor cells of the subgranular zone were labeled with red fluorescent protein in the same mice at postnatal day 42 (P42, adulthood). Electrophysiological properties of mature neurons born at either stage were then compared in the same brain slices. Evoked and spontaneous GABAergic postsynaptic responses of perisomatic and dendritic origin displayed similar characteristics in both neuronal populations. Miniature GABAergic inputs also showed similar functional properties and pharmacological profile. A comparative analysis of the present data with our previous observations rendered no significant differences among GABAergic inputs recorded from neurons born in the embryonic, early postnatal and adult mice. Yet, embryo-born neurons showed a reduced membrane excitability, suggesting a lower engagement in network activity. Our results demonstrate that granule cells of different age, location and degree of excitability receive GABAergic inputs of equivalent functional characteristics.
127 citations
TL;DR: It is concluded that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.
Abstract: Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-gamma, but not IL-4, by Ag-specific CD4(+) T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.
104 citations
TL;DR: The mineralocorticoid receptor (MR) forms oligomers with the heat-shock protein 90 (Hsp90) -based heterocomplex, which contains tetratricopeptide repeat (TPR) domain immunophilins (IMM) and the unknown biological role of IMMs was investigated.
Abstract: The mineralocorticoid receptor (MR) forms oligomers with the heat-shock protein 90 (Hsp90) -based heterocomplex, which contains tetratricopeptide repeat (TPR) domain immunophilins (IMMs). Here we investigated the unknown biological role of IMMs in the MR.Hsp90 complex. Upon hormone binding, FKBP52 was greatly recruited to MR.Hsp90 complexes along with dynein motors, whereas FKBP51 was dissociated. Importantly, the Hsp90 inhibitor geldanamycin impaired the retrograde transport of MR, suggesting that the Hsp90.IMM.dynein molecular machinery is required for MR movement. To elucidate the mechanism of action of MR, the synthetic ligand 11,19-oxidoprogesterone was used as a tool. This steroid showed equivalent agonistic potency to natural agonists and was able to potentiate their mineralocorticoid action. Importantly, aldosterone binding recruited greater amounts of FKBP52 and dynein than 11,19-oxidoprogesterone binding to MR. Interestingly, 11,19-oxidoprogesterone binding also favored the selective recruitment of the IMM-like Ser/Thr phosphatase PP5. Each hormone/MR complex yielded different proteolytic peptide patterns, suggesting that MR acquires different conformations upon steroid binding. Also, hormone/MR complexes showed different nuclear translocation rates and subnuclear redistribution. All these observations may be related to the selective swapping of associated factors. We conclude that (a) the Hsp90.FKBP52.dyenin complex may be responsible for the retrotransport of MR; (b) a differential recruitment of TPR proteins such as FKBP51, FKBP52, and PP5 takes place during the early steps of hormone-dependent activation of the receptor; (c) importantly, this swapping of TPR proteins depends on the nature of the ligand; and (d) inasmuch as FKBP51 also showed an inhibitory effect on MR-dependent transcription, it should be dissociated from the MR.Hsp90 complex to positively regulate the mineralocorticoid effect.
91 citations
TL;DR: The results suggest that xanthan does not play an essential role in citrus canker at the initial stages of infection or in the incompatible interactions between X. axonopodis pv.
Abstract: Xanthan-deficient mutants of Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, were generated by deletion and marker exchange of the region encoding the carboxy-terminal end of the first glycosyltransferase, GumD. Mutants of gumD did not produce xanthan and remained pathogenic in citrus plants to the same extent as wild-type bacteria. The kinetics of appearance of initial symptoms, areas of plant material affected, and growth of bacteria inside plant tissue throughout the disease process were similar for both wild-type and mutant inoculations. Moreover, exopolysaccharide deficiency did not impair the ability of the bacteria to induce hypersensitive response on non-host plants. Apart from variations in phenotypic aspects, no differences in growth or survival under different stress conditions were observed between the xanthan-deficient mutant and wild-type bacteria. However, gumD mutants displayed impaired survival under oxidative stress during stationary phase as well as impaired epiphytic survival on citrus leaves. Our results suggest that xanthan does not play an essential role in citrus canker at the initial stages of infection or in the incompatible interactions between X. axonopodis pv. citri and non-host plants, but facilitates the maintenance of bacteria on the host plant, possibly improving the efficiency of colonization of distant tissue.
84 citations
TL;DR: It is proposed that specific complexes formation between an HSP90 and ROF isoforms depends on their spatial and temporal expression, and that ROF/HSP90 complexes assemble in vivo.
Abstract: The plant co-chaperones FK506-binding proteins (FKBPs) are peptidyl prolyl cis-trans isomerases that function in protein folding, signal transduction and chaperone activity. We report the characterization of the Arabidopsis large FKBPs ROF1 (AtFKBP62) and ROF2 (AtFKBP65) expression and protein accumulation patterns. Transgenic plants expressing ROF1 promoter fused to GUS reporter gene reveal that ROF1 expression is organ specific. High expression was observed in the vascular elements of roots, in hydathodes and trichomes of leaves and in stigma, sepals, and anthers. The tissue specificity and temporal expression of ROF1 and ROF2 show that they are developmentally regulated. Although ROF1 and ROF2 share 85% identity, their expression in response to heat stress is differentially regulated. Both genes are induced in plants exposed to 37 degrees C, but only ROF2 is a bonafide heat-stress protein, undetected when plants are grown at 22 degrees C. ROF1/ROF2 proteins accumulate at 37 degrees C, remain stable for at least 4 h upon recovery at 22 degrees C, whereas, their mRNA level is reduced after 1 h at 22 degrees C. By protein interaction assays, it was demonstrated, that ROF1 is a novel partner of HSP90. The five amino acids identified as essential for recognition and interaction between the mammalian chaperones and HSP90 are conserved in the plant ROF1-HSP90. We suggest that ROF/HSP90 complexes assemble in vivo. We propose that specific complexes formation between an HSP90 and ROF isoforms depends on their spatial and temporal expression. Such complexes might be regulated by environmental conditions such as heat stress or internal cues such as different hormones.
81 citations
TL;DR: The purpose of the review is to describe the most significant recent findings on the mechanism of glycoprotein folding and assembly quality control and to discuss the main still unanswered questions.
77 citations
TL;DR: The results indicate that BLSOmp31 could be a useful candidate for the development of subunit vaccines against brucellosis since it elicits humoral, T helper and cytotoxic immune responses and protection against smooth and rough species of Brucella.
71 citations
TL;DR: The HPV16 E7 oncoprotein is an extended dimer, with a stable and cooperative fold, but that displays properties of "natively unfolded" proteins, which allow adaptation to a variety of protein targets and expose the PEST degradation sequence that regulates its turnover in the cell.
Abstract: The HPV16 E7 oncoprotein is an extended dimer, with a stable and cooperative fold, but that displays properties of “natively unfolded” proteins. Two regions of conserved sequence are found in E7 proteins, where the N-terminus (1−40) includes the retinoblastoma tumor suppressor binding and casein kinase II phosphorylation sites. A fragment containing the highly acidic N-terminal half shows an apparently disordered conformation by far-UV−circular dichroism (CD) at neutral pH, and its hydrodynamic radius is much larger than a neutral peptide of the same length. Trifluoroethanol and micellar concentrations of sodium dodecyl sulfate stabilize a much more helical structure at pH 4.0 than at pH 7.5, while submicellar concentrations of the detergent yield a β-strand. The shape, pH, and temperature dependence of the CD spectrum at pH 7.5 are indicative of a poly proline type II structure. This structure is stabilized by phosphorylation, which would translate into increased transforming activity in the cell. Thus, ...
TL;DR: It is shown that subtle structural changes within the circadian network are responsible for behavioral arrhythmicity, and disrupted PDF signaling by slo dysfunction directly affects the structure of the underlying circuit.
Abstract: Substantial progress has been made in elucidating the molecular processes that impart a temporal control to physiology and behavior in most eukaryotes. In Drosophila, dorsal and ventral neuronal networks act in concert to convey rhythmicity. Recently, the hierarchical organization among the different circadian clusters has been addressed, but how molecular oscillations translate into rhythmic behavior remains unclear. The small ventral lateral neurons can synchronize certain dorsal oscillators likely through the release of pigment dispersing factor (PDF), a neuropeptide central to the control of rhythmic rest-activity cycles. In the present study, we have taken advantage of flies exhibiting a distinctive arrhythmic phenotype due to mutation of the potassium channel slowpoke (slo) to examine the relevance of specific neuronal populations involved in the circadian control of behavior. We show that altered neuronal function associated with the null mutation specifically impaired PDF accumulation in the dorsal protocerebrum and, in turn, desynchronized molecular oscillations in the dorsal clusters. However, molecular oscillations in the small ventral lateral neurons are properly running in the null mutant, indicating that slo is acting downstream of these core pacemaker cells, most likely in the output pathway. Surprisingly, disrupted PDF signaling by slo dysfunction directly affects the structure of the underlying circuit. Our observations demonstrate that subtle structural changes within the circadian network are responsible for behavioral arrhythmicity.
TL;DR: GH-regulated gene transcription mediated by C/EBPbeta reflects the integration of diverse mechanisms including post-translational modifications, modulation of protein complexes associated with DNA and re-localization of gene regulatory proteins.
TL;DR: Investigation of the role of Lex in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Lex mAbs (FC-2.15 and MCS-1) on this adhesion indicates that MCf-7 cells can bind to HUVEC, and that M CS-1 but not FC- 2.15 mAb inhibit this interaction.
Abstract: Lewis x (Le x , CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galb(1-4)Fuca(1-3)GlcNAc, which is expressed on glycoconjugates in human polymor- phonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Le x in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti- Le x mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Le x+ -cells induced by anti-Le x mAbs and complement when cells were ad- hered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF- 7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Le x -dependent PMN interaction with HUVEC. Although both anti-Le x mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blot- ting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Le x -tri- saccharide, interacts with specific glycoproteins of Mr ~ 28 kD and 10 kD from MCF-7 cells. The inter- action between Le x+ -cancer cells and vascular endo- thelium is a potential target for cancer treatment.
TL;DR: The observed correlation between gal4 dosage and behavioural defects associated with apoptotic neuronal loss in the specific GAL4‐expressing neurons is observed and suggests that an excess of GAL 4 might enhance neuronal deficits observed in models of neurodegeneration.
Abstract: The GAL4/UAS system has been extensively employed in Drosophila to control gene expression in defined spatial patterns. More recently this system has been successfully applied to express genes involved in neurodegeneration to model various diseases in the fruit fly. We used transgenic lines expressing different levels of GAL4 in a particular subset of neurons involved in the control of rhythmic behaviour, so that its impact on neuronal physiology would result in altered locomotor activity, which could be readily assessed. We observed a striking correlation between gal4 dosage and behavioural defects associated with apoptotic neuronal loss in the specific GAL4-expressing neurons. Increased gal4 dosage correlated with accumulation of insoluble GAL4, suggesting that the cascade of events leading to apoptosis might be triggered by protein deposits of either GAL4 or protein intermediates. Behavioural defects were rescued by expression of hsp70, a classic chaperone that also interferes with cell death pathways. In agreement with the latter, the viral caspase inhibitor p35 also rescued GAL4-induced behavioural defects. Our observations demonstrate the intrinsic effects of GAL4 deregulation on neuronal viability and suggest that an excess of GAL4 might enhance neuronal deficits observed in models of neurodegeneration.
TL;DR: This is the first indication that a recombinant subunit vaccine elicits greater protection than whole Brucella, and the insertion of this peptide on BLS induced stronger T helper 1 responses specific for the carrier (BLS), thus the authors' results represent a case of synergic strengthening between two Bru cell antigens.
TL;DR: The results indicate that only the virulent (Bvg+) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm.
Abstract: Fil: Serra, Diego. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - La Plata. Centro de Investigacion y Desarrollo en Fermentaciones Industriales. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigacion y Desarrollo en Fermentaciones Industriales; Argentina
TL;DR: It is concluded that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3β and allows Ag cross-presentation.
Abstract: In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by γ irradiation (Apo-Nec cells). We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100. Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 ± 10.5%) at 12 hs of coculture. By 12–24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC – Dextran uptake (iDC: 81 ± 5%; DC/Apo-Nec 33 ± 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3β. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-γ secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change. We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3β and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.
TL;DR: The first purification of 2-Cys Prx from higher plants is described and it is shown that the native and the recombinant forms of rapeseed leaves stimulate the activity of chloroplast fructose-1,6-bisphosphatase (CFBPase), a key enzyme of the photosynthetic CO2 assimilation.
TL;DR: It is reported that MT-ND4, a mitochondrial gene encoding a subunit of the mitochondrial Complex I (mtCx-I), is also a CFTR-dependent gene, and a reduced expression was observed in CFDE cells (derived from a CF patient) when compared toCFDE cells ectopically expressing wild-type CFTR.
TL;DR: Studies in cell culture and in vivo have revealed that Sima is activated by the insulin receptor (InR) and target-of-rapamycin (TOR) pathways, which suggests thatSima is engaged in a negative feedback loop that limits growth upon stimulation of InR/TOR pathways.
Abstract: The fruit fly Drosophila melanogaster, a widely utilized genetic model, is highly resistant to oxygen starvation and is beginning to be used for studying physiological, developmental, and cellular adaptations to hypoxia. The Drosophila respiratory (tracheal) system has features in common with the mammalian circulatory system so that an angiogenesis-like response occurs upon exposure of Drosophila larvae to hypoxia. A hypoxia-responsive system homologous to mammalian hypoxia-inducible factor (HIF) has been described in the fruit fly, where Fatiga is a Drosophila oxygen-dependent HIF prolyl hydroxylase, and the basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins Sima and Tango are, respectively, the Drosophila homologues of mammalian HIF-alpha (alpha) and HIF-beta (beta). Tango is constitutively expressed regardless of oxygen tension and, like in mammalian cells, Sima is controlled at the level of protein degradation and subcellular localization. Sima is critically required for development in hypoxia, but, unlike mammalian model systems, it is dispensable for development in normoxia. In contrast, fatiga mutant alleles are all lethal; however, strikingly, viability to adulthood is restored in fatiga sima double mutants, although these double mutants are not entirely normal, suggesting that Fatiga has Sima-independent functions in fly development. Studies in cell culture and in vivo have revealed that Sima is activated by the insulin receptor (InR) and target-of-rapamycin (TOR) pathways. Paradoxically, Sima is a negative regulator of growth. This suggests that Sima is engaged in a negative feedback loop that limits growth upon stimulation of InR/TOR pathways.
TL;DR: The results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.
TL;DR: Downregulation of SPARC levels in melanoma cells using either an antisense RNA or a shRNA against SPARC sensitized them to hMel‐SPARC addition in proliferation and migration assays, suggesting that malignant cells developed a SPARC‐resistance mechanism.
Abstract: Cell interaction with the extracellular matrix (ECM) has profound influence in cancer progression. The secreted protein, acidic and rich in cysteine (SPARC) a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. This apparent paradox might result either from the biochemical properties of the different SPARC sources or from differential responses of malignant and stromal cells to SPARC. To test these hypotheses, we purified SPARC secreted by melanoma cells (hMel-SPARC) and compared its activity with different recombinant SPARC preparations, including a new one produced in insect cells. All 5 SPARC species were effective in inhibiting bovine aortic endothelial cell proliferation, adhesion and migration. We then used the melanoma-derived protein to assess SPARC effect on additional cell types. hMel-SPARC greatly impaired the proliferation of both normal and transformed human endothelial cells and exerted a moderate biphasic effect on human fetal fibroblasts proliferation, irrespective of their endogenous SPARC levels. However, SPARC had no effect on the proliferation of several human cancer cell lines regardless of their endogenous levels of SPARC expression. Importantly, downregulation of SPARC levels in melanoma cells using either an antisense RNA or a shRNA against SPARC sensitized them to hMel-SPARC addition in proliferation and migration assays, suggesting that malignant cells developed a SPARC-resistance mechanism. This was not a general resistance to growth suppressing agents, as melanoma cells with restricted SPARC expression were more resistant to chemotherapeutic agents. Thus, malignant cells expressing or not expressing SPARC developed alternative mechanisms that, in contrary to stromal cells, rendered them SPARC-insensitive.
TL;DR: The results indicate that the cytoplasmic species contain conformational epitopes that may arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets, those involving PDZ binding domains, which requires further investigation.
Abstract: The E6 oncoproteins of high-risk HPV types 16 and 18 are involved in the development of cervical cancer. Besides its determinant role in carcinogenic progression, HPV E6 oncoprotein has also been instrumental in elucidating fundamental aspects of p53 function and its ubiquitin-proteasome degradation, with counterpart activities in various DNA tumor viruses. Establishing the conformational state and cellular distribution unequivocally for the endogenous protein in HPV-transformed cell lines derived from carcinomas is essential for understanding the underlying mechanism. Recombinant E6 from high-risk strains 16 and 18 folds into soluble oligomers of approximately 1.2 MDa, which are thermostable and display cooperative loss of tertiary and secondary structure upon chemical denaturation. Antibodies raised against these assemblies locate E6 evenly distributed in the cells. By depleting the polyclonal serum by immunoblocking with monomeric E6, the nuclei of Hela and CaSki cells become completely devoid of label, indicating that monomeric species are mainly localized in the nucleus and that both monomers and oligomers share epitopes. The monomeric species promote degradation of p53 by the proteasome, which correlates with the nuclear localization we describe. In contrast, the oligomeric E6 does not promote p53 degradation, in agreement with its cytoplasmic localization inferred from the immunoneutralization experiments. Our results indicate that the cytoplasmic species contain conformational epitopes that may arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets, those involving PDZ binding domains, which requires further investigation.
TL;DR: It is determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the V(H)-V(L) interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen.
TL;DR: A model for the regulation of the RGP activity and its binding to golgi membranes based on the glycosylation of the protein is proposed where the sugars linked to oligomeric form of RGP in the golgi may be transferred to acceptors involved in polysaccharide biosynthesis.
Abstract: Reversible glycosylated polypeptides (RGPs) are highly conserved plant-specific proteins, which can perform self-glycosylation. These proteins have been shown essential in plants yet its precise function remains unknown. In order to understand the function of this self-glycosylating polypeptide, it is important to establish what factors are involved in the regulation of the RGP activity. Here we show that incubation at high ionic strength produced a high self-glycosylation level and a high glycosylation reversibility of RGP from Solanum tuberosum L. In contrast, incubation at low ionic strength led to a low level of glycosylation and a low glycosylation reversibility of RGP. The incubation at low ionic strength favored the formation of high molecular weight RGP-containing forms, whereas incubation at high ionic strength produced active RGP with a molecular weight similar to the one expected for the monomer. Our data also showed that glycosylation of RGP, in its monomeric form, was highly reversible, whereas, a low reversibility of the protein glycosylation was observed when RGP was part of high molecular weight structures. In addition, glycosylation of RGP increased the occurrence of non-monomeric RGP-containing forms, suggesting that glycosylation may favor multimer formation. Finally, our results indicated that RGP from Arabidopsis thaliana and Pisum sativum are associated to golgi membranes, as part of protein complexes. A model for the regulation of the RGP activity and its binding to golgi membranes based on the glycosylation of the protein is proposed where the sugars linked to oligomeric form of RGP in the golgi may be transferred to acceptors involved in polysaccharide biosynthesis.
TL;DR: The interplay between calcium and the lectin and chaperone activities of CRT stabilized the CRT structure in an apparent additive, independent manner when the protein was subjected to thermal or urea-induced denaturation.
Abstract: The ER resident protein calreticulin fulfills at least two important roles. It works as a chaperone preventing Golgi exit of non-native protein species and enhancing protein folding efficiency in either N-glycan-dependent, lectin chaperone, or classical chaperone, N-glycan-independent, modes and is one of the main calcium buffers in the cell. This last feature is independent from the lectin chaperone properties of the protein as this last activity is also observed in a CRT fragment lacking calcium buffer capacity. Here we study the interplay between calcium and the lectin and chaperone activities of CRT. The affinity of CRT for monoglucosylated glycans measured in solution by equilibrium dialysis and fluorescence anisotropy was not affected by the absence of calcium. Binding of CRT to monoglucosylated neoglycoproteins displaying either native or molten globule-like conformations was also independent of calcium concentration. Moreover, calcium and monoglucosylated glycans stabilized the CRT structure in an apparent additive, independent manner when the protein was subjected to thermal or urea-induced denaturation. In addition, the ability of CRT to decrease the level of aggregation of a chemically denatured monoglucosylated and nonglycosylated protein was also independent of calcium level.
TL;DR: The DNA binding domain of papillomavirus E2 proteins is at the center of the regulation of gene transcription and replication of the virus, and the mild perturbation required for entering an amyloid route is indicative of a preexisting equilibrium.
Abstract: The DNA binding domain of papillomavirus E2 proteins is at the center of the regulation of gene transcription and replication of the virus. Its unique fold consists of a β-barrel domain that combines an eight-stranded dimeric β-barrel core interface with two symmetrical DNA binding α-helices and other two helices, packed against the central barrel. Treatment with low amounts of trifluoroethanol readily leads to a mostly β-sheet oligomeric species, with a loss of near-UV circular dichroism signal and increase in its ANS binding capacity, indicating that buried hydrophobic surfaces become accessible to the solvent. This species subsequently undergoes a slow transition into amyloid aggregates as determined by light scattering and Congo red and thioflavin T binding. Electron microscopy shows short amyloid fibers with a curly aspect as the end product. The amyloid route is completely prevented by addition of stoichiometrical amounts of specific DNA, strongly suggesting that unfolding of the DNA binding α-helix is required for the formation of the intermediate. The slow nature of this expanded β-oligomeric species and the availability of several different conformational probes make it an excellent model for investigating amyloid mechanisms. The mild perturbation required for entering an amyloid route is indicative of a preexisting equilibrium. Oligomerization processes are required for the assembly of transcription initiation and DNA replication machineries, where proteins from different viruses must come together with host cell proteins. The E2 protein is a virus-encoded multifunctional master regulator that may exert one of its multiple functions through its ability to oligomerize.
TL;DR: This structure represents the first bona fide antibody DNA immunogen complex described in atomic detail and is five orders of magnitude higher than other reported complexes, possibly because of having gone through affinity maturation.