Project description:Chemotaxis is used by free-living motile bacteria to swim towards nutrient sources or away from repellents, and to navigate the environment to locate niches optimal for growth and survival. Multiple chemotaxis systems have been identified in different bacterial species, including Azospirillum brasilense. In A. brasilense, chemotaxis is mediated by two distinct chemotaxis pathways, named Che1 and Che4, that physically interact to form mixed chemotaxis signaling arrays. Signaling from the Che1 and Che4 pathways control transient increases in swimming speed and swimming reversals, respectively, during chemotaxis. In A. brasilense, chemotaxis is tightly linked to energy metabolism with this coupling occurring through the sensory input of several energy-sensing chemoreceptors and through the control of chemoreceptor activity by the c-di-GMP second messenger. Previous work has demonstrated that chemotaxis in A. brasilense also affects unrelated cellular functions including cell-to-cell clumping and flocculation. However, the molecular mechanism for these effects is not known. Here, we identify additional effects of mutations abolishing Che1 (cheA1 mutant), Che4 (cheA4 mutant) or both Che1 and Che4 (cheA1/cheA4 mutant) function on nitrogen and carbon metabolism and use whole cell proteome and metabolome mass spectrometry to further characterize the interplay between chemotaxis and metabolism. We found that CheA1 mediates most changes in chemoreceptor arrays composition and also affects small molecules signaling while a mutant lacking CheA4 displays changes in nitrogen metabolism, including nitrate assimilation and nitrogen fixation. In contrast, the mutant lacking both CheA1 and CheA4, which lacks chemotaxis and does not form chemotaxis signaling arrays, displays distinct and non-overlapping changes that suggest the assembly of chemotaxis signaling arrays modulates energy and carbon metabolism. Together, the results suggest distinct roles for CheA1, CheA4 and chemotaxis signaling arrays in modulating chemotaxis and metabolism, likely through control of distinct global regulatory networks.

Project description:Gonadotrophin-releasing hormone (GnRH) significantly inhibits proliferation of a proportion of cancer cell lines by activating GnRH receptor-G protein signaling. Therefore, manipulation of GnRH receptor signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GnRH receptor activation in sensitive cells (HEK293-GnRHR, SCL60) in in vitro and in vivo settings, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic SCL60 response to the GnRH super-agonist Triptorelin. Early and mid-phase changes (0.5-1.0 h) comprised mainly transcription factors. Later changes (8-24 h) included a GnRH target gene, CGA, and up or down-regulation of transcripts encoding signaling and cell division machinery. Pathway analysis exposed identified altered mitogen-activated protein kinase and cell cycle pathways, consistent with occurrence of G2/M arrest and apoptosis. NFκB pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenografts in vivo during Triptorelin anti-proliferation. Increased phosphorylated NFκB (p65) occurred in SCL60 in vitro, and p-NFκB and IκBε were higher in treated xenografts than controls after 4 days Triptorelin. NFκB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GnRH receptor signaling, identifies potential anti-proliferative target genes and implicates the NFκB survival pathway as a node for enhancing GnRH agonist-induced anti-proliferation. 55 samples: 35 SCL60 (15 Control, 20 Treated), 20 HEK293 (12 Control, 8 Treated). Samples collected after 0, 0.5, 1, 2, 8 or 24h after treatment with Triptorelin (100nM) or vehicle control (20% Propylene Glycol solution). SCL60 cells are HEK293 cells stably transfected with a high level of functional rat GnRHR.

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Homo sapiens

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