Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

Characterization of the human dynein light chain Rp3 and its use as a non-viral gene delivery vector.

Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus. In the context of non-viral gene delivery, Rp3-Db is expected to simultaneously interact with DNA and dynein, thereby enabling a more rapid and efficient transport of the genetic material across the cytoplasm. We successfully purified recombinant Rp3 and obtained a low-resolution structural model using small-angle X-ray scattering. Additionally, we observed that Rp3 is a homodimer under reducing conditions and remains stable over a broad pH range. The ability of Rp3 to interact with the dynein intermediate chain in vitro was also observed, indicating that the recombinant Rp3 is correctly folded and functional. Finally, Rp3-Db was successfully expressed and purified and exhibited the ability to interact with pDNA and mediate the transfection of cultured HeLa cells. Rp3-Db was also capable of interacting in vitro with dynein intermediate chains, indicating that the addition of the N-terminal DNA-binding domain does not compromise its function. The transfection level observed for Rp3-Db is far superior than that reported for protamine and is comparable to that of the cationic lipid Lipofectamine™. This report presents an initial characterization of a non-viral delivery vector based on the dynein light chain Rp3 and demonstrates the potential use of modified human light chains as gene delivery vectors.

Proteomic response of Moniliophthora perniciosa exposed to pathogenesis-related protein-10 from Theobroma cacao.

TcPR-10, a member of the pathogenesis-related protein 10 family, was identified in EST library of interactions between Theobroma cacao and Moniliophthora perniciosa. TcPR-10 has been shown to have antifungal and ribonuclease activities in vitro. This study aimed to identify proteins that are differentially expressed in M. perniciosa in response to TcPR-10 through a proteomic analysis. The fungal hyphae were subjected to one of four treatments: control treatment or 30-, 60- or 120-min treatment with the TcPR-10 protein. Two-dimensional maps revealed 191 differentially expressed proteins, 55 of which were identified by mass spectrometry. The proteins identified in all treatments were divided into the following classes: cell metabolism, stress response, zinc binding, phosphorylation mechanism, transport, autophagy, DNA repair, and oxidoreductases. The predominant class was stress-response proteins (29%), such as heat shock proteins; these proteins exhibited the highest expression levels relative to the control treatment and are known to trigger defense mechanisms against cytotoxic drugs as well as TcPR-10. Oxidoreductases (25%) were overexpressed in the control and in 30-min treatments but exhibited reduced expression at 120 min. These proteins are involved in the repair of damage caused by oxidative stress due to the contact with TcPR- 10. Consistent with the antifungal activity of TcPR-10, several proteins identified were related to detoxification, autophagy or were involved in mechanisms for maintaining fungal homeostasis, such as ergosterol biosynthesis. These results show that the sensitivity of the fungus to TcPR-10 involves several biochemical routes, clarifying the possible modes of action of this antifungal protein.