Función de la proteína CarF en la transducción de la señal luminosa en la bacteria "Myxococcus xanthus" y su conservación en eucariotas superiores

Resumen

The membrane protein CarF plays a key role in one of the two pathways that operate in Myxococcus xanthus to sense blue light and defend the bacterium against photooxidative stress, which stems from the photoexcitation of protoporphyrin IX and consequent generation of singlet oxygen (1O2). We have recently discovered that CarF is the plasmanylethanolamine desaturase (PEDS1) that generates the vinyl-ether bond in plasmalogens, and that this special class of glycerophospholipids somehow signals photooxidative stress. The overall goal of this work has been to delve in-depth into the mode of action of CarF and its homologs in animals, the biosynthesis of plasmalogens and its ether bond precursor in M. xanthus, and the novel molecular mechanism of light perception via plasmalogens. CarF contains twelve cytoplasmic histidines, eight of which are conserved in all homologs (from myxobacteria, Leptospiraceae and Alphaproteobacteria among bacteria; from animals and plants among eukaryotes). Our analysis revealed a good correlation between the extent of conservation of these histidines and their functional importance. Among the histidines that are not conserved in all homologs, H113 deserves a special mention, as it is essential for CarF function and is conserved only in animals, Myxoccocales and Leptospiraceae homologs. Some of the histidines in CarF may participate in the formation of a diiron cluster active site. Consistent with this, the CarF sample, purified after heterologous expression and solubilization in Foscholine-12, was shown to contain iron (~2:1 Fe:CarF). This purified CarF, which exhibits some PEDS1 activity, displayed a tendency to form homooligomers whose functional relevance is unknown. To obtain purified CarF better suited for future studies, diverse strategies were tested: expression of truncated versions, expression of CarF in a cellular environment with ether lipids, expression of protein homologs, and CarF solubilization using polymer nanodiscs. Despite the enormous evolutionary distance between myxobacteria and animals, all animal homologs tested, including the human one, complemented the lack of CarF in M. xanthus. By contrast, none of the plant homologs or that from Alphaproteobacteria analyzed could do so. Thus, as with CarF, its animal homologs correspond to the PEDS1, whose identity had remained unknown over almost 50 years. Unlike mammals, M. xanthus has two pathways available to synthesize the precursors of plasmalogens: a main pathway, widely distributed among myxobacteria, involving the multidomain protein ElbD (and, possibly, other gene products of the elbA-E operon); an ancillary pathway, restricted to the genus Myxococcus, in which the three gene products of the MXAN_1676-1674 operon participate. Experimental and comparative analyses allow us to propose the possible steps wherein the genes of each operon act in the corresponding pathway for the biosynthesis of the ether lipid precursors. The work accomplished demonstrates that the sole determinant in plasmalogens indispensable for their role in M. xanthus is the vinyl ether bond. Susceptibility of this bond to cleavage by 1O2, yielding a fatty aldehyde and a lysophospholipid, could explain how plasmalogens signal photooxidative stress. Nonetheless, whereas such a degradation of plasmalogens by 1O2 was clearly detected in vitro, this cleavage was more subtle in vivo. It may thus be speculated that M. xanthus may have developed a highly sensitive system to sense tiny amounts of lysed plasmalogen to trigger the photoprotective response and evade the toxic effects that may be generated by an excess of plasmalogen degradation products. To address the study of this complex mechanism, we have initiated efforts to deploy more sophisticated techniques such as “click chemistry”.