Depletion of YhcB, an inner membrane protein of Escherichia coli, inhibited the growth of rodZ deletion mutant showing that the loss of both YhcB and RodZ is synthetically lethal. Furthermore, YhcB was demonstrated to interact with RodZ as well as several other proteins involved in cell shape maintenance and an inner membrane protein YciS of unknown function, using bacterial two-hybrid system. These observations seem to indicate that YhcB is involved in the biogenesis of cell envelope and the maintenance of cell shape together with RodZ.
The present study aimed to clarify the association between kinesin family member 11 (KIF11) and human breast cancer, and the effect of KIF11 on breast cancer cell progression. Western blot analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, retroviral infection, immunohistochemistry staining, MTT assay, anchorage-independent growth ability assay and tumorigenicity assay were all used in the present study. Western blot and RT-qPCR analysis revealed that the expression of KIF11 was markedly increased in malignant cells compared with that in non-tumorous cells at the mRNA and protein level. Immunohistochemical analysis revealed that KIF11 expression was upregulated in 256/268 (95.8%) paraffin-embedded archival breast cancer biopsies. Statistical analysis demonstrated a significant association between the upregulation of KIF11 expression and the progression of breast cancer. Multivariate analysis revealed that KIF11 upregulation represents an independent prognostic indicator for the survival of patients with breast cancer. Tumorigenicity experiments were further used to evaluate the effect of KIF11 in non-obese diabetic/severe combined immunodeficient mice. Silencing endogenous KIF11 by short hairpin RNAs inhibited the proliferation of breast cancer cells in vitro and in vivo. The present results suggest that KIF11 may serve an important function in the proliferation of breast cancer and may represent a novel and useful prognostic marker for breast cancer.
RodZ (YfgA) is a membrane protein well conserved among bacterial species and important in the determination of cell shape and motility, although the molecular mechanism involved is not well established. We have characterized a DeltarodZ mutant and show that defective peptidoglycan synthesis might be the primary effect of the deletion. A motile pseudorevertant of DeltarodZ isolated possessed a near rod-shaped cell morphology, indicating that RodZ is not absolutely required for the elongation of the lateral cell wall and the synthesis of functional flagella.
Purpose: Our previous studies have shown that kinesin family member 11 (KIF11) is markedly overexpressed in human breast cancer cells or tissues and positively correlated with distant metastasis and prognosis in patients with breast cancer, suggesting an important role in the regulation of cancer stem cells. Herein, we examined the role of KIF11 in breast cancer stem cells. Methods: In the current study, we validated our previous findings through analysis of data collected in The Cancer Genome Atlas. Endogenous KIF11 was stably silenced in MCF-7 and SKBR-3 cells. Flow cytometry was used to measure the proportion of side-population (SP) cells. Mammosphere culture and tumor implantation experiments in immunodeficient mice were used to assess the self-renewal ability of breast cancer cells. Real-time polymerase chain reaction, western blot, immunofluorescence staining, luciferase reporter assays and Wnt agonist treatment were conducted to investigate the signaling pathways regulated by KIF11. Results: We found that the expression level of KIF11 was positively correlated with stem cell-enrichment genes. The proportion of SP cells was significantly reduced in KIF11-silenced cells. Silencing endogenous KIF11 not only reduced the size and number of mammospheres in vitro, but also reduced the ability of breast cancer cells to form tumors in mice. Simultaneously, we found that KIF11 was involved in regulating the activation of the Wnt/βcatenin signaling pathway. Conclusion: Endogenous KIF11 enhances the self-renewal of breast cancer cells by activating the Wnt/β-catenin signaling pathway, thereby enhancing the characteristics of breast cancer stem cells.
The function of inner membrane protein YciB in Escherichia coli has not been identified. In this study, the membrane topology of the protein that contains five transmembrane domains was clarified. YciB was found to interact with various proteins involved in cell elongation and cell division using a bacterial two-hybrid system. It was also found that the deletion mutant of yciB is susceptible to the low osmolarity. These observations together with previous reports indicate that YciB is involved in synthesis of the cell envelope by interacting with cell elongation and cell division complexes.Key words Escherichia coli, membrane topology, two-hybrid analysis, YciB.Bacterial cell envelopes that consist of an IM and the cell wall are the principal stress-bearing and shape-maintaining elements of cells; their integrity is of critical importance to cell viability. The E. coli genome contains over 800 genes (1) that encode IM proteins; however, many of them have not yet been well characterized and their functions remain unknown. We recently identified that one such gene, yciB, is a gene that is required for normal biofilm formation (2) and interacts genetically with rodZ, a gene that is important for maintaining rodtype morphology (3-5). These findings suggest that YciB has a role in cell envelope synthesis.YciB is predicted to be a multi-pass IM protein with five transmembrane domains (Fig. 1a); however, this prediction has not experimentally been examined. Therefore, we first investigated the membrane topology of YciB using a dual pho-lac reporter system (6). In this system, the color on the indicator plate of E. coli strain DH5a (phoA -lacZ -DM15) carrying the pKTop plasmid shows whether the C-terminus of the protein expressed from the cloned gene resides in the cytoplasm or in the periplasm. According to the predicted structure of YciB, we designed serial mutants with deletions from the Cterminus, as shown in Figure 1a, and cloned each fragment into pKTop. The resultant pKTop clones (Table S1) were introduced into DH5a and the topology of the fusion proteins analyzed. Transformants expressing YciB 147 and YciB 75 fusion proteins exhibited blue color, indicating strong phosphatase but low b-galactosidase activity, which indicates that the C-termini of these positions are localized on the periplasmic side of the inner membrane. In contrast, full length YciB, YciB 118 , YciB 48 fusion proteins showed high b-galactosidase activity but low phosphatase activity (red color), showing that the C-termini of these proteins are located in the cytoplasm (Fig. 1b). We also constructed a pKTop recombinant expressing YciB 42-179 fusion. This fusion protein lacks the N-terminal region that is predicted to be periplasmic and the first transmembrane domain. We found that the C-terminus of this mutant is cytoplasmic, as is the full length YciB. Similarly, YciB 42-147 fusion that we deleted at both the N-and C-termini gave the same topological result (blue color) as YciB 147 . These results suggest that neither the N-terminal no...
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