Metastasis is a complex, multistep process by which a cancer cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary cancer. A deeper understanding of the molecular events underlying metastasis will provide information that will be useful for the development of new diagnostic and therapeutic strategies. The B16 and B16F10 mouse melanoma cell lines are widely used as model system for studying many aspects of cancer biology including metastasis. Compared with B16, which has a low metastatic potential, the highly metastatic cell line B16F10 displayed a higher metastatic ability along with higher expression levels of the metastasis-associated phosphatase of regenerating liver-3 (PRL-3). B16 cells transfected with PRL-3 cDNA (B16-PRL3) had metastatic abilities comparable to those of Bl16F10 cells. To study the molecular mechanisms that underlie metastasis, the proteomes of the B16, B16F10, and B16-PRL3 cell lines were compared using two-dimensional differential in-gel electrophoresis. Proteins that varied significantly in levels between these cell lines were selected and identified using mass spectrometry. Interestingly, many proteins, especially those present in membrane fractions, were similarly up- or downregulated in both the Bl16F10 and B16-PRL3 cells lines compared to B16 cell lines. The list of similarly regulated proteins included heat shock protein 70, fascin-1, septin-6, ATP synthase beta subunit, and bone morphogenic protein receptor type IB. These proteins may play a causal role in PRL-3-mediated metastasis. These investigations open an avenue for the further characterization of the molecular mechanisms that underlie metastasis.
Parkinson's disease (PD) is one of the most common neurodegenerative diseases caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD is still unclear, the death of dopaminergic neurons during PD progression was revealed to be associated with abnormal aggregation of α-synuclein, elevation of oxidative stress, dysfunction of mitochondrial functions, and increased neuroinflammation. In this study, the effects of Licochalcone D (LCD) on MG132-induced neurotoxicity in primitive neural stem cells (pNSCs) derived from reprogrammed iPSCs were investigated. A cell viability assay showed that LCD had anti-apoptotic properties in MG132-induced oxidative-stressed pNSCs. It was confirmed that apoptosis was reduced in pNSCs treated with LCD through 7-AAD/Annexin Ⅴ staining and cleaved caspase3. These effects of LCD were mediated through an interaction with JunD and through the EGFR/AKT and JNK signaling pathways. These findings suggest that LCD could be a potential antioxidant reagent for preventing disease-related pathological phenotypes of PD.
Ubiquitination is a post translational modification which mostly links with proteasome dependent protein degradation. This process has been known to play pivotal roles in the number of biological events including apoptosis, cell signaling, transcription and translation. Although the process of ubiquitination has been studied extensively, the mechanism of polyubiquitination by multi protein E3 ubiquitin ligase, SCF complex remains elusive. In the present study, we identified UbcH5a as a novel stimulating factor for poly-ubiquitination catalyzed by $SCF^{hFBH1}$ using biochemical fractionations and MALDI-TOF. Moreover, we showed that recombinant UbcH5a and Cdc34 synergistically stimulate $SCF^{hFBH1}$ catalyzed polyubiquitination in vitro. These data may provide an important cue to understand the mechanism how the SCF complex efficiently polyubiquitinates target substrates.
We have developed a method for rapid activity staining of proteins with glutathione peroxidase (GPx) activity after 2-D gel electrophoresis. After separating proteins extracted from yeast, or mouse red blood cells, by two-dimensional gel electrophoresis, SDS was removed and the gel was submerged in a Tris-HCl buffer containing glutathione and hydrogen peroxide, followed by incubation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phenazine methosulfate (PMS). After this proteins with GPx activity appeared as clear zones on a purple background. This relatively simple activity staining method could be useful for rapid screening of proteins with GPx activity in cell extracts.
Abstract Deer antlers are the only mammalian organs capable of repeated regeneration. Although antlers are known to develop from pedicles, which arise from antlerogenic cells of cranial periosteum, their developmental process is not fully elucidated. For example, while endocrine and environmental factors influence the antler development, it is still unclear which signaling pathways are involved in the transduction of such stimuli. To study the developmental process of antlers and identify proteins functioning in their growth, we have established proteome maps of red deer ( Cervus elaphus ) antlers. With two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization mass spectrometry, we analyzed more than 800 protein spots and identified ∼ 130 individual proteins derived from the growing tip of antlers. The overall profile of the antler proteome was dissimilar to those of other types of tissue. Also comparison of proteomes derived from proximal bony tissue and the growing tip of antlers revealed substantial differences. Moreover several cell growth or signaling‐related proteins are expressed exclusively in the growing tip, suggesting that these proteins function in the growth and differentiation of antlers. Currently, using the antler proteome maps, we are actively searching for the regulatory factor(s) that may control the antler development.
Abstract Intracellular serine protease‐1 (Isp‐1) is a major intracellular serine protease of Bacillus subtilis , whose functions still remain largely unknown. Furthermore, physiological substrates are yet to be determined. To identify Isp‐1 substrates, we digested extract obtained from an Isp‐1 deficient Bacillus mutant with purified Isp‐1 and examined eliminated or decreased spots by two‐dimensional gel and matrix‐assisted laser desorption/ionization‐time of flight analyses. Proteins degraded by Isp‐1, termed the Isp‐1 degradome, are involved in a variety of cellular functions such as DNA packing, genetic competence, and protein secretion. From the degradome we selected ClpC and EF‐Tu as putative Isp‐1 substrates and studied their in vitro degradation. ClpC and EF‐Tu contain putative cleavage sites for Isp‐1. N ‐terminal sequencing of in vitro proteolytic fragments of ClpC and EF‐Tu revealed that these sites are indeed recognized and cleaved by Isp‐1. Moreover, the cellular levels of ClpC and EF‐Tu were dramatically reduced at the late stationary phase, where the expression level of Isp‐1 was greatly increased. These results suggest that the regulated proteolysis of ClpC by Isp‐1 plays an important role in the stationary phase adaptive response. This degradomic approach could provide a powerful tool for finding physiological substrates of many proteolytic enzymes whose functions remain to be determined.
Abstract Human genes exist as numerous copy number variations (CNV) and single-nucleotide polymorphisms (SNP) that exert control over the majority of the bodies’ core functions. By virtue of numerous genome-wide association studies (GWAS), we now know that CNVs and SNPs can coexist in certain genomic regions, amplifying expression, regulation, and susceptibility effects. Because of the significant impact of CNVs and SNPs, research groups worldwide have eagerly sought an adequate method for measuring both simultaneously only to end as measured separately. To overcome the current limitations, we developed a polymerase chain reaction (PCR) based novel approach which combines primers designed by the STexS method with multiplex probes used in the STexS II method. The method successfully measured both CNVs and SNPs of CYP2A6 and CYP2A7 in a single quantitative polymerase chain reaction (qPCR). Further testing of 100 human genomic DNA samples enabled calculation of the overall frequency of [T] and [G] components within an East Asian population, yielding results that were not only highly congruent with a previous NIH database, but also correcting it. The novel method should redefine genetic profiling and could provide a means to not only successfully predict one’s genetic characteristics, but further enhance personalized medicine and individualized treatments.
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