Purine catabolic pathway in Bacillus subtilis is consisted of more than 14 genes. Among these genes, pucL and pucM are required for uricase activity. While PucL is known to encode the uricase itself, the function of PucM is still unclear although this protein is also indispensable for uric acid decomposition. Here, we provide evidence that PucM, a transthyretin-related protein, functions to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Based on these results, we propose that transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as a hydroxyisourate hydrolase.
Human papillomaviruses (HPVs) have been recognized as the primary cause of cervical cancer. HPV 16 E7 binds to tumor suppressor retinoblastoma protein, and interferes with its function, causing release of the transcription factor E2F, which influences expression of cell cycle-related genes. This study was performed to identify the genes and proteins modulated by the HPV E7 oncogene. An HPV-negative cervical cancer cell line (C33A) was prepared to establish a stable cell line expressing E7. In order to analyze the target molecules modulated by E7 expression, we used two approaches: matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and DNA microarrays. Forty-seven spots were identified in C33A/E7 by two-dimensional electrophoresis and MALDI/TOF MS. Protein disulfide isomerase A3, integrase interactor 1 protein, growth inhibitory protein, glutathione S-transferase P, and vav proto-oncogene were down-regulated, whereas heat shock 60 kDa protein 1, Ku70 binding protein, alpha enolase, 26S proteasome subunit were up-regulated. A genomic approach using a microarray kit showed that IL-12R beta 1, cytochrome c, and tumor necrosis factor receptor II were induced by the E7 oncogene. These results suggest that E7 can evade immune surveillance by suppressing or inducing these cell signaling factors, cell cycle regulators, and chaperones.
Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.
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