18 research outputs found
Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns
Regulating levels of the neuromodulator D-serine in human brain: structural insight into pLG72 and D-amino acid oxidase interaction
The human flavoenzyme D-amino acid oxidase (hDAAO) degrades the NMDA-receptor modulator D-serine in the brain. Whereas hDAAO has been extensively characterized, little is known about its main modulator pLG72, a small protein encoded by the primate-specific gene G72 that has been associated with schizophrenia susceptibility. pLG72 interacts with neosynthesized hDAAO, promoting its inactivation and degradation. In this work we used low-resolution techniques to characterize the surface topology of the hDAAO-pLG72 complex. By using limited proteolysis coupled to mass spectrometry we could map the exposed regions in the two proteins after complex formation and highlighted an increased sensitivity to proteolysis of hDAAO in complex with pLG72. Cross-linking experiments by using bis(sulfosuccinimidyl)suberate identified the single covalent bond between T182 in hDAAO and K62 in pLG72. In order to validate the designed mode of interaction, three pLG72 variants incrementally truncated at the C-terminus, in addition to a form lacking the 71 N-terminal residues, were produced. All variants were dimeric, folded, and interacted with hDAAO. The strongest decrease in affinity for hDAAO (as well as for the hydrophobic drug chlorpromazine) was apparent for the N-terminally deleted pLG7272-153 form, which lacked K62. On the other hand, eliminating the disordered C-terminal tail yielded a more stable pLG72 protein, improved the binding to hDAAO, although giving lower enzyme inhibition. Elucidation of the mode of hDAAO-pLG72 interaction now makes it possible to design novel molecules that, by targeting the protein complex, can be therapeutically advantageous for diseases related to impairment in D-serine metabolism. This article is protected by copyright. All rights reserved
Ophiobolin A Induces Autophagy and Activates the Mitochondrial Pathway of Apoptosis in Human Melanoma Cells.
Ophiobolin A, a fungal toxin from Bipolaris species known to affect different cellular processes in plants, has recently been shown to have anti-cancer activity in mammalian cells. In the present study, we investigated the anti-proliferative effect of Ophiobolin A on human melanoma A375 and CHL-1 cell lines. This cellular model was chosen because of the incidence of melanoma malignant tumor on human population and its resistance to chemical treatments. Ophyobolin A strongly reduced cell viability of melanoma cells by affecting mitochondrial functionality. The toxin induced depolarization of mitochondrial membrane potential, reactive oxygen species production and mitochondrial network fragmentation, leading to autophagy induction and ultimately resulting in cell death by activation of the mitochondrial pathway of apoptosis. Finally, a comparative proteomic investigation on A375 cells allowed to identify several Ophiobolin A down-regulated proteins, which are involved in fundamental processes for cell homeostasis and viability
Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns.
Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP-PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense
Abnormality of Auditory Mismatch Negativity in Depression and Its Dependence on Stimulus Intensity
Mismatch negativity (MMN) is thought to reveal several abnormalities of cognitive functioning. Although depression often affects cognitive functioning, previous studies concerning MMN in depressed patients provided conflicting results. In recent reports, it has been suggested that depressed patients may show abnormal auditory response to regular auditory stimuli presented with at a relatively high intensity. We thus recorded acoustic MMN in 16 drug-free patients suffering from moderate depression and in 10 healthy subjects at 2 different stimulus intensities. Differences in MMN latency and amplitude between depressed patients and healthy subjects reached the significance level only for high intensity stimulation, and they were consistent with a dysfunction of frontal MMN subcomponents in depressed patients. This finding suggests that consistent MMN abnormalities can be observed in depressed patients by using high-intensity stimulation; moreover, it supports the hypothesis of disturbances of frontal networks in depression even in early stages of disease
Enhancing the secretion of a glyco-engineered anti-CD20 scFv-Fc antibody in hairy root cultures
Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study is to enhance the accumulation of a tumorâtargeting antibody with a humanâcompatible glycosylation profile in HR culture medium. To this aim, the authors produce Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4âdichlorophenoxyacetic acid (2,4âD) is found to induce high accumulation levels (334âmgâLâ1) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumorâtargeting, CD20âspecific 2B8âFcÎXF recombinant antibody from glycoâengineered ÎXTFT N. benthamiana HR cultures. The addition of 2,4âD determine a 28âfold increase of the accumulation of fully functional 2B8âFcÎXF in the culture medium, at levels of â16âmgâLâ1. Antibody Nâglycosylation profiling reveal the prominent occurrence of GnGn structures and low levels of xyloseâ and fucoseâcontaining counterparts. This result is the first example of the expression of an engineered antiâCD20 antibody with a scFvâFc format at high levels in HR
Corpeptins, new bioactive lipodepsipeptides from cultures of Pseudomonas corrugata
The structure of the corpeptins, bioactive lipodepsipeptides produced in culture by Pseudomonas corrugata, the causal agent of tomato pith necrosis, has been determined. The combined use of FAB-mass spectrometry, NMR spectroscopy and chemical procedures has allowed us to assign the following primary structure to the peptide moiety: Dhb-Pro-Ala-Ala-Ala-Val-V al-Dhb-Hse-Val-alle-Dhp-Ala-Ala-Ala-Val-Dhb-aThr-Al-a-Dab-Ser-lle with the terminal carboxy group closing a macrocyclic ring on the hydroxy; group of the allo-threonine residue. The N-terminus is in turn acylated by 3-hydroxydecanoate in corpeptin A and by cis-3-hydroxy-5-dodecenoate in corpeptin B, Some preliminary data on the biological activity of corpeptins are included. (C) 1998 Federation of European Biochemical Societies
Exposure to Moringa oleifera microRNAs induces proteomic changes linked to tumorigenesis and epithelial-mesenchymal transition in HeLa cells
Cervical cancer (CC) is one of the most frequent cancers in women worldwide. The epithelial-mesenchymal transition (EMT) and the extracellular release of TGF-β are phenomena typically associated with different tumorigenic processes, including tumour cell proliferation and metastatization. Specific human microRNAs (miRNAs; miRs) involved in these tumorigenic processes have been identified, becoming important diagnostic and prognostic markers, and even potential therapeutic targets. In parallel, different studies have also shown that plant miRNAs can mediate a cross-kingdom regulation (CKR) of mammalian genes and modulate host's gene expression under pathological conditions, restoring the regulatory activity of endogenous miRNAs lost in cancer. In our previous studies, the miRNome from Moringa oleifera Lam. (henceforth moringa or mol) has been sequenced, showing the presence of several conserved miRNAs in the plant kingdom, whose ability to differentially regulate proliferation and apoptosis in healthy and cancer cells has been demonstrated. Furthermore, the effects of mol-miR treatment on tumorigenesis and EMT have been proved in liver tumour cells. According to these premises, we here investigated the proteomic profile of CC-derived HeLa cells exposed to a mol-miRNA pool, demonstrating the down-representation of specific factors involved in tumorigenesis. The treatment with plant miRs was able to modulate proteins involved in several biological processes linked to EMT. Furthermore, it reduced the expression of TGF-β and significantly inhibited cell motility, as observed following Scratch test and cell viability measurements, with a significant increase of apoptotic events. In conclusion, our results suggest and pave the way for the development of new potential therapeutic approaches based on CKR mediated by plant miRNAs for contrasting human cervical cancer, even in the form of adjuvants to classic treatments for limiting their side effects
OP-A induced PINK1 accumulation, Bax and Bak translocation and cytochrome <i>c</i> release.
<p>HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 ΟM OP-A for 24 h and whole cell extracts (WCE), mitochondrial-enriched and cytosolic fractions were analysed by western blot (A) with antibodies directed against PINK1, Bak, Bax, and cytochrome <i>c</i>. Panels B, C and D, densitometric analysis of PINK1, Bak and Bax levels present in the mitochondrial fraction. β-tubulin was used as loading control and standard for densitometry. Data, expressed as %, or fold variation with respect to β-tubulin, were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p
OP-A induced autophagy and cell death by apoptosis.
<p>A, flow cytometry analysis of cell death. HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 ÎźM OP-A for 24 h and cell death analysis performed by propidium iodide staining and flow cytometry evaluation of the sub-G1 population. Where indicated, 20 ÎźM necrostatin-1 was added to evaluate the occurrence of necrotic cell death. B, western blot of PARP, caspase-9 and -3, and LC3II proteins. C, D and E. Densitometric analysis of immunorecognized protein bands of caspase-3, caspase-9 and LC3II, respectively. Data were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p