Glycoside Hydrolases in Plant Cell Wall Proteomes: Predicting Functions That Could Be Relevant for Improving Biomass Transformation Processes

Glycoside hydrolases (GHs) are enzymes that are able to rearrange the plant cell wall polysaccharides, being developmental- and stress-regulated. Such proteins are used in enzymatic cocktails for biomass hydrolysis in the second-generation ethanol (E2G) production. In this chapter, we investigate GHs identified in plant cell wall proteomes by predicting their functions through alignment with homologous plant and microorganism sequences and identification of functional domains. Up to now, 49 cell wall GHs were identified in sugarcane and 114 in Brachypodium distachyon. We could point at candidate proteins that could be targeted to lower biomass recalcitrance. We more specifically addressed several GHs with predicted cellulase, hemicellulase, and pectinase activities, such as β-xylosidase, α and β-galactosidase, α-N-arabinofuranosidases, and glucan β-glucosidases. These enzymes are among the most used in enzymatic cocktails to deconstruct plant cell walls. As an example, the fungi arabinofuranosidases belonging to the GH51 family, which were also identified in sugarcane and B. distachyon, have already been associated to the degradation of hemicellulosic and pectic polysaccharides, through a peculiar mechanism, probably more efficient than other GH families. Future research will benefit from the information available here to design plant varieties with self-disassembly capacity, making the E2G more cost-effective through the use of more efficient enzymes

Morphological alterations and gene and protein expression profiling of bladder tumor cells after treatment with gemcitabine.

Chemical agents used in cancer therapy are associated with cell cycle arrest, activation or deactivation of mechanisms\ud associated to DNA repair and apoptosis. However, due to the complexity of biological systems, the molecular\ud mechanisms responsible for these activities are not fully understood. Thus, studies about gene and protein expression\ud have shown promising results for understanding the mechanisms related to cellular responses and regression of cancer\ud after chemotherapy. This study aimed to evaluate the gene and protein expression profiling in bladder transitional cell\ud carcinoma (TCC) with different TP53 status after gemcitabine (1.56 μM) treatment. The RT4 (grade 1, TP53 wild\ud type), 5637 (grade 2, TP53 mutated) and T24 (grade 3, TP53 mutated) cell lines were used. PCR arrays and mass\ud spectrometry were used to analyze gene and protein expression, respectively. Morphological alterations were observed\ud using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of PCR array\ud showed that gemcitabine activity was mainly related to CDKN1A, GADD45A and SERTDA1 overexpression, and BAX\ud overexpression only in the wild type TP53 cells. Mass spectrometry demonstrated that gemcitabine modulated the protein\ud expression, especially those from genes related to apoptosis, transport of vesicles and stress response. Analyses using SEM\ud and TEM showed changes in cell morphology independently on the cell line studied. The observed decreased number of\ud microvillus suggests low contact among the cells and between cell and extracellular matrix; irregular forms might indicate\ud actin cytoskeleton deregulation; and the reduction in the amount of organelles and core size might indicate reduced\ud cellular metabolism. In conclusion, independently on TP53 status or grade of bladder tumor, gemcitabine modulated\ud genes related to the cell cycle and apoptosis, that reflected in morphological changes indicative of future cell death.FAPESPCNP

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo - FAPESP [04/11799-0, 2008/52691-9]Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo FAPES

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo - FAPESP [04/11799-0, 2008/52691-9]Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo FAPES