Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.
Tubulin is one of the major targets in cancer chemotherapy and the target of more than twenty percent of the cancer chemotherapic agents. The modulation of isoform content has been hypothesized as being a cause of resistance to treatment. Isoform differences lie mostly in the C-terminus part of the protein. Extensive characterization of this polypeptide region is therefore of critical importance. MALDI-TOF fragmentation of tubulin C-terminal domains was tested using synthetic peptides. Then, isotypes from HeLa cells were successfully characterized for the first time by in-source decay (ISD) fragmentation of their C-terminus coupled to a pseudo MS3 technique named T3-sequencing. The fragmentation occurred in-source, preferentially generating yn-series ions. This approach required guanidination for the characterization of the βIII-tubulin C-terminus peptide. This study is, to our knowledge, the first example of reflectron in-source decay (reISD) of the C-terminus of a 50 kDa protein. This potentially occurs via a CID-like mechanism occurring in the MALDI plume. There are now new avenues for top-down characterization of important clinical biomarkers such as βIII-tubulin isotypes, a potential marker of drug resistance and tumor progression. This paper raises the challenge of protein isotypes characterization for early cancer detection and treatment monitoring.
Calcium‐binding proteins, such as S‐100, dimerize readily, and this phenomenon plays an important role in their regulation of target enzymes [Krebs, J., Quadroni, M. & Van Eldik, L.J. (1995) Nat. Struct. Biol. 2 , 711–714; Kilby, P.M., Van Eldik, L.J. & Roberts, G.C. (1996) Structure 4 , 1041–1052]. We have investigated by Fourier‐transform ion cyclotron resonance (FTICR) MS the conformational states of the calcium‐binding protein calmodulin, and present clear evidence for a calmodulin dimer formed as a result of noncovalent interactions between folded monomers. Ultra‐high‐resolution electrospray ionization (ESI) mass spectra for calmodulin, obtained with a 9.4 T FTICR mass spectrometer, are presented. With the use of denaturing solutions (1 : 1 acetonitrile/water + 1% formic acid), relatively high charge states (20 < z < 10) of monomeric calmodulin ions were detected, whereas when calmodulin was electrosprayed from buffer, monomers ions with only 5–10 charges were detected. CD measurements for calmodulin in buffered solution revealed that its α‐helical content was significantly higher than that for calmodulin in acetonitrile/water solutions, consistent with a proposition that changes in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding. Under buffered conditions, noncovalently bound calmodulin dimers were observed by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were consistent with the proposed calmodulin dimer–monomer equilibrium. The ultra‐high mass resolution achieved with the 9.4 T FTICR mass spectrometer allowed unequivocal identification of the noncovalent, as opposed to covalent, character of the calmodulin dimer.
Glioblastoma multiforme is one of the most common intracranial tumors encountered in adults. This tumor of very poor prognosis is associated with a median survival rate of approximately 14 months. One of the major issues to better understand the biology of these tumors and to optimize the therapy is to obtain the molecular structure of glioblastoma. MALDI molecular imaging enables location of molecules in tissues without labeling. However, molecular identification in situ is not an easy task. In this paper, we used MALDI imaging coupled to in-source decay to characterize markers of this pathology. We provided MALDI molecular images up to 30 μm spatial resolution of mouse brain tissue sections. MALDI images showed the heterogeneity of the glioblastoma. In the various zones and at various development stages of the tumor, using our top-down strategy, we identified several proteins. These proteins play key roles in tumorigenesis. Particular attention was given to the necrotic area with characterization of hemorrhage, one of the most important poor prognosis factors in glioblastoma.
Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins. Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized. Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes. The msrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused. The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations. Our in vivo study revealed that msrB is required for cadmium resistance of E. coli, a carcinogenic compound that induces oxidative stress. Our in vitro studies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turn-over rates of 0.6 min(-1) and 20 min(-1), respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin. A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity.
Vinca alkaloids vinblastine and vincristine and some of their derivatives such as vinorelbine are widely used in therapy of leukemia and several solid tumors. Their action is associated with alterations of the mitotic spindle functions that prevent the cell cycle progression and lead to mitotic block. A number of studies show that some Vinca alkaloids inhibit CaM−target interaction. The newest microtubule inhibitor, vinflunine (Javlor), currently in clinical trials, is remarkably more active than vinblastine against a number of tumors. Moreover, vinflunine is significantly less toxic than other Vinca alkaloids. The high antitumor activity of this molecule is not well understood since it binds to tubulin with an overall affinity several-fold lower than that of vinblastine or vincristine. In this study, we examined the interaction of Ca2+−CaM with vinflunine, vinblastine, and stable tubule only polypeptide (STOP) by using a combination of thermodynamic and mass spectrometric approaches. We characterized the influence of Vinca alkaloids on Ca2+−CaM−STOP complex formation. Our results revealed different binding modes to Ca2+−CaM for vinflunine and vinblastine, highlighting that adding fluorine atoms on the cleavamine moiety of the Vinca alkaloid molecule is critical for the localization of the drug on calmodulin. We demonstrate that vinflunine is a better inhibitor for STOP binding to calmodulin than vinblastine. We suggest that vinflunine action on calmodulin can have an effect on microtubule dynamics. These data may contribute to a better understanding of the superior antitumor efficiency and lower toxicity of vinflunine.
In anaerobiosis, Escherichia coli can use trimethylamine N-oxide (TMAO) as a terminal electron acceptor. Reduction of TMAO in trimethylamine (TMA) is mainly performed by the respiratory TMAO reductase. This system is encoded by the torCAD operon, which is induced in the presence of TMAO. This regulation involves a two-component system comprising TorS, an unorthodox histidine kinase, and TorR, a response regulator. A third protein, TorT, sharing homologies with periplasmic binding proteins, plays a key role in this regulation because disruption of the torT gene abolishes tor expression. In this study we showed that TMAO protects TorT against degradation by the GluC endoproteinase and modifies its temperature-induced CD spectrum. We also isolated a TorT negative mutant that is no longer protected by TMAO from degradation by GluC. Isothermal titration calorimetry confirmed that TorT binds TMAO with a binding constant of 150 mum. Therefore, we conclude that TorT binds TMAO and that this binding promotes a conformational change of TorT. We also showed that TorT interacts with the periplasmic domain of TorS in both the presence and absence of TMAO but the TorT-TMAO complex induces a higher GluC protection of TorS than TorT alone. These results support the idea that TMAO binding to TorT induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. We identified several homologues of the TorT protein that define a new family of periplasmic binding proteins. We thus propose that the members of this family bind TMAO or related compounds and that they are involved in signal transduction or even substrate transport.
