As a strategy to find efficient lignocellulose degrading enzymes/microorganisms for sugarcane biomass pretreatment purposes, 118 culturable bacterial strains were isolated from intestines of sugarcane-fed larvae of the moth Diatraea saccharalis. All strains were tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays or by growing bacteria on sugarcane biomass as sole carbon sources. Out of the 118 strains isolated thirty eight were found to possess cellulose degrading activity and phylogenetic studies of the 16S rDNA sequence revealed that all cellulolytic strains belonged to the phyla γ-Proteobacteria, Actinobacteria and Firmicutes. Within the three phyla, species belonging to five different genera were identified (Klebsiella, Stenotrophomonas, Microbacterium, Bacillus and Enterococcus). Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella. Good cellulolytic activity correlated with high extracellular protein concentrations. In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates. The results of this study indicate the possibility to find efficient cellulose degrading enzymes and microorganisms from intestines of insect larvae feeding on sugarcane and their possible application in industrial processing of sugarcane biomass such as second generation biofuel production.
E7 is the major transforming activity in human papillomaviruses, a causal agent for cervical cancer. HPV16 E7 is a small protein with a natively unfolded domain for which dozens of specific cellular targets were described, and represents a prototypical oncoprotein among small DNA tumor viruses. The protein can form spherical oligomers with amyloid-like properties and chaperone activity. Conformation specific antibodies locate endogenous oligomeric E7 species in the cytosol of 3 model cell lines, strongly colocalizing with amyloid structures and dimeric E7 localizes to the nucleus. The cytosolic oligomeric E7 appear as the most abundant species in all cell systems tested. We show that nuclear E7 levels are replenished dynamically from the cytosolic pool and do not result from protein synthesis. Our results suggest that long-term events related to de-repression of E7 would cause accumulation of excess E7 into oligomeric species in the cytosol. These, together with the known target promiscuity of E7, may allow interactions with many of the non-pRb dependent targets described. This hypothesis is further supported by the detection of E7 oligomers in the cytosol of cancerous cells from tissue biopsies. ' UICCKey words: cancer; oncoprotein; papillomavirus; E7; amyloid Small DNA tumor viruses share the requirement of the cellular DNA replication machinery to amplify their genome when they must force cells into the S phase. 1 The main proteins behind the cell transforming activity are E1A in adenovirus, large T-antigen in SV40 and the E7 oncoprotein in papillomavirus. 2 Although the large T-antigen polypeptide is multidomain and multifunctional, the papillomavirus E7 protein is small with not known biochemical functions other than protein-protein interactions. Besides sequence homology, they also share the property of sequestering and destabilizing the retinoblastoma (pRb) family of proteins, which are essential tumor suppressors that regulate the cell cycle. [3][4][5] Human papillomaviruses (HPV) infect epithelia and are causal agents for benign and malignant diseases, where the most widespread is cervical cancer in women. 6 Two virus-like particle-based vaccines proved to be highly efficacious for preventing the infection. 7 However, these vaccines are prophylactic and there is still the need of treatment of the female world population between 15 and 75 years old and, with 5 million estimated cancer deaths over the next 20 years due to existing HPV infections. 8 Of the 8 proteins that are encoded by its small 8 kb DNA genome, 2 proteins, E6 and E7 cooperate for transformation by HPV. 9,10 The primary events behind transformation by E6 is binding and degradation of p53. 11 Similarly, E7 was early described to bind pRb, 10 promoting its degradation via the ubiquitin proteasome system, 12 with counterparts in other DNA tumor viruses, thus validating a common strategy. 13 However, E7 is the major transforming protein in HPV, since its interaction with hypophosphorylated pRb causes disruption of growth-suppressive pRb-E2F...
The E6 oncoproteins of high-risk HPV types 16 and 18 are involved in the development of cervical cancer. Besides its determinant role in carcinogenic progression, HPV E6 oncoprotein has also been instrumental in elucidating fundamental aspects of p53 function and its ubiquitin-proteasome degradation, with counterpart activities in various DNA tumor viruses. Establishing the conformational state and cellular distribution unequivocally for the endogenous protein in HPV-transformed cell lines derived from carcinomas is essential for understanding the underlying mechanism. Recombinant E6 from high-risk strains 16 and 18 folds into soluble oligomers of approximately 1.2 MDa, which are thermostable and display cooperative loss of tertiary and secondary structure upon chemical denaturation. Antibodies raised against these assemblies locate E6 evenly distributed in the cells. By depleting the polyclonal serum by immunoblocking with monomeric E6, the nuclei of Hela and CaSki cells become completely devoid of label, indicating that monomeric species are mainly localized in the nucleus and that both monomers and oligomers share epitopes. The monomeric species promote degradation of p53 by the proteasome, which correlates with the nuclear localization we describe. In contrast, the oligomeric E6 does not promote p53 degradation, in agreement with its cytoplasmic localization inferred from the immunoneutralization experiments. Our results indicate that the cytoplasmic species contain conformational epitopes that may arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets, those involving PDZ binding domains, which requires further investigation.
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