Mitochondrial dysfunction in the spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the neurometabolic alterations during early stages of the disease remain unknown. Here, we ...investigated the bioenergetic and proteomic changes in ALS mouse motor neurons and patients' skin fibroblasts. We first observed that SODG93A mice presymptomatic motor neurons display alterations in the coupling efficiency of oxidative phosphorylation, along with fragmentation of the mitochondrial network. The proteome of presymptomatic ALS mice motor neurons also revealed a peculiar metabolic signature with upregulation of most energy-transducing enzymes, including the fatty acid oxidation (FAO) and the ketogenic components HADHA and ACAT2, respectively. Accordingly, FAO inhibition altered cell viability specifically in ALS mice motor neurons, while uncoupling protein 2 (UCP2) inhibition recovered cellular ATP levels and mitochondrial network morphology. These findings suggest a novel hypothesis of ALS bioenergetics linking FAO and UCP2. Lastly, we provide a unique set of data comparing the molecular alterations found in human ALS patients' skin fibroblasts and SODG93A mouse motor neurons, revealing conserved changes in protein translation, folding and assembly, tRNA aminoacylation and cell adhesion processes.
The oil palm fruit mesocarp contains high lipase activity that increases free fatty acids and necessitates post-harvest inactivation by heat treatment of fruit bunches. Even before heat treatment the ...mesocarp lipase activity causes consequential oil losses and requires costly measures to limit free fatty acids quantities. Here we demonstrate that elite low-lipase lines yield oil with substantially less free fatty acids than standard genotypes, allowing more flexibility for post-harvest fruit processing and extended ripening for increased yields. We identify the lipase and its gene cosegregates with the low-/high-lipase trait, providing breeders a marker to rapidly identify potent elite genitors and introgress the trait into major cultivars. Overall, economic gains brought by wide adoption of this material could represent up to one billion dollars per year. Expected benefits concern all planters but are likely to be highest for African smallholders who would be more able to produce oil that meets international quality standards.
A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional
microdomains that play important roles in protein sorting, signal ...transduction, or infection by pathogens. Recent reports
demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic
composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho- and glycerolipids
as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant
fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used
mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with
Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed
in silico . Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity
were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications,
such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to
the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated
that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction,
others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic
stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal
cells, signaling platforms involved in these physiological functions.
GCN2 is a serine/threonine kinase involved in cellular stress response related to amino acid starvation. Previously, we showed that GCN2 interacts with HIV-1 integrase and is activated during HIV-1 ...infection. Herein, we identified HIV-1 integrase as a previously unknown substrate of GCN2 in vitro with a major site of phosphorylation at residue S255 located in the C-terminal domain of HIV-1 integrase. The underlying mechanism was investigated and it appeared that the integrase active site was required in order for GCN2 to target the integrase residue S255. Moreover, various integrases from other retroviruses (e.g. MLV, ASV) were also recognized as a substrate by GCN2. In cells, HIV-1 lentiviral particles harboring mutation at integrase position 255 were affected in their replication. Preventing phosphorylation resulted in an increase in infectivity that correlated with an increase in viral DNA integration. Infectivity of MLV was also higher in cells knocked-out for GCN2 suggesting a conserved mechanism to control viral replication. Altogether, our data suggest that GCN2 may constitute a general guardian of genome stability by regulating foreign DNA integration and as such be part of the antiviral armamentarium of the cell.
lacks a cell wall, and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of
enriched with lipoproteins was ...shown to stimulate the production of interleukin-23 (IL-23) by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon
infection. We studied here the interaction between
PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell. IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two nondenaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and enzyme-linked immunosorbent assay (ELISA). We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular mass between 20 and 35 kDa and 19 with a molecular mass between 40 and 100 kDa. Among them, lipoprotein MHO_4720 was identified as potentially bioactive, and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1β was observed. After 24 h of coincubation of hDCs with
s, downregulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction of
and hDCs.
is a human urogenital pathogen involved in gynecologic and opportunistic infections.
lacks a cell wall, and its membrane contains many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on the interaction between
and human dendritic cells and examined both sides of the interaction, the mycoplasmal lipoproteins involved in the activation of the host cell and the immune response of the cell. On the mycoplasmal side, we showed for the first time that
lipoproteins with high molecular mass were potentially bioactive. On the cell side, we reported an activation of the inflammasome, which is involved in the innate immune response.
The immunoproteasome (IP) is usually viewed as favoring the production of antigenic peptides presented by MHC class I molecules, mainly because of its higher cleavage activity after hydrophobic ...residues, referred to as the chymotrypsin-like activity. However, some peptides have been found to be better produced by the standard proteasome. The mechanism of this differential processing has not been described. By studying the processing of three tumor antigenic peptides of clinical interest, we demonstrate that their differential processing mainly results from differences in the efficiency of internal cleavages by the two proteasome types. Peptide gp100(209-217) (ITDQVPSFV) and peptide tyrosinase369-377 (YMDGTMSQV) are destroyed by the IP, which cleaves after an internal hydrophobic residue. Conversely, peptide MAGE-C2(336-344) (ALKDVEERV) is destroyed by the standard proteasome by internal cleavage after an acidic residue, in line with its higher postacidic activity. These results indicate that the IP may destroy some antigenic peptides due to its higher chymotrypsin-like activity, rather than favor their production. They also suggest that the sets of peptides produced by the two proteasome types differ more than expected. Considering that mature dendritic cells mainly contain IPs, our results have implications for the design of immunotherapy strategies.
Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we ...studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.
Aims: The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in ...a possible vaccine.
Methods and Results: The expression product of ompA is a 465‐amino‐acid protein precursor that contains a 21‐amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy‐terminal region contains the ‘OmpA/MotB’ domain/signature and five putative ‘Thrombospondin type 3 repeats’ domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti‐OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund’s adjuvant developed a high antibody titter.
Conclusions: Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine.
Significance and Impact of the Study: This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine.
Proteins in bile may have important physiological functions and serve as disease biomarkers. Here, the protein composition of human gallbladder bile was analyzed using a recently described ...chromatography-like technology capable to enhance the signal of low-abundance species. First, proteins present in bile fluid were treated with immobilized peptide ligand libraries to concentrate dilute and very dilute species while concomitantly diluting the high-abundance proteins. The analysis of resulting protein mixture was then performed using LC–MS/MS after having classically separated proteins by a mini preparative gel electrophoresis. Overall 222 gene products were found; 143 of them were not reported before in proteomics studies. Ligand libraries by themselves contributed to find 81 new gene products distributed throughout different categories. The described chromatographic approach provides a significant contribution to the bile protein repertoire and opens new perspectives for the discovery of markers for specific biliary tract diseases.
The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine ...phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.
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