A functional calcium-transporting ATPase encoded by chlorella viruses

Calcium-transporting ATPases (Ca2+ pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca2+ pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca2+ pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of the triple yeast mutant K616 confirmed that M535L transports calcium ions and, unusually for group IIB pumps, also manganese ions. In vitro assays show basal ATPase activity. This activity is inhibited by vanadate, but, unlike that of other Ca2+ pumps, is not significantly stimulated by either calcium or manganese. The enzyme forms a 32P-phosphorylated intermediate, which is inhibited by vanadate and not stimulated by the transported substrate Ca2+, thus confirming the peculiar properties of this viral pump. To our knowledge this is the first report of a functional P-type Ca2+-transporting ATPase encoded by a virus

The structural bases for agonist diversity in an Arabidopsis thaliana glutamate receptor-like channel

Arabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for L-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology

Cellular Ca2+ signals generate defined pH signatures in plants

Calcium ions (Ca2+) play a key role in cell signaling across organisms. The question of how a simple ion can mediate specificity has spurred research into the role of Ca2+ signatures and their encoding and decoding machinery. Such studies have frequently focussed on Ca2+ alone and our understanding of how Ca2+ signalling is integrated with other responses remains poorly understood. Using in vivo imaging with different genetically-encoded fluorescent sensors in Arabidopsis cells we show that Ca2+ transients do not occur in isolation but are accompanied by pH changes in the cytosol. We estimate the degree of cytosolic acidification at up to 0.25 pH units in response to external ATP in seedling root tips. We validated this pH-Ca2+ link for distinct stimuli. Our data suggest that the association with pH may be a general feature of Ca2+ transients that depends on the transient characteristics and the intracellular compartment. These findings suggest a fundamental link between Ca2+ and pH dynamics in plant cells, generalizing previous observations of their association in growing pollen tubes and root hairs. Ca2+ signatures act in concert with pH signatures, possibly providing an additional layer of cellular signal transduction to tailor signal specificity

BTN3A2 Expression in Epithelial Ovarian Cancer Is Associated with Higher Tumor Infiltrating T Cells and a Better Prognosis

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR = 0.651, p = 0.006 and HR = 0.642, p = 0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR = 1.355 p = 0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells

Abscisic acid stimulates the expression of two isoforms of plasma membrane Ca2+-ATPase in Arabidopsis thaliana seedlings

At-ACA8 and At-ACA9 are two plasma membrane (PM) Ca2+-ATPases of Arabidopsis thaliana. In this article the expression of At-ACA8, At-ACA9, and of At-ACA10, a third isoform of Ca2+-ATPase closely related to PM Ca2+-ATPases, was analyzed and the effect of the hormone abscisic acid (ABA) on the expression level of PM Ca2+-ATPase specific transcripts was investigated. In adult plants of A. thaliana, At-ACA8 and At-ACA10 are expressed in all organs considered whereas At-ACA9 is expressed only in flowers. All isoforms of PM Ca2+-ATPases can be detected in young seedlings but the amt. of At-ACA9 mRNA is much lower than those of At-ACA8 and At-ACA10. ABA markedly and rapidly stimulates the expression of both At-ACA8 and At-ACA9 genes in young seedlings but not that of At-ACA10. ABA also increases the level of At-ACA8 protein at the PM, suggesting a role for PM Ca2+-ATPases in ABA signalling. [on SciFinder (R)

Intracellular localisation of PPI1 (proton pump interactor, isoform 1), a regulatory protein of the plasma membrane H(+)-ATPase of Arabidopsis thaliana.

PPI1 (proton pump interactor isoform 1) is a novel protein able to interact with the C-terminal autoinhibitory domain of the Arabidopsis thaliana plasma membrane (PM) H(+)-ATPase. In vitro, PPI1 binds the PM H(+)-ATPase in a site different from the known 14-3-3 binding site and stimulates its activity. In this study, we analysed the intracellular localisation of PPI1. The intracellular distribution was monitored in A. thaliana cultured cells by immunolocalisation using an antiserum against the PPI1 N-terminus and in Vicia faba guard cells and epidermal cells by transient expression of a GFP::PPI1 fusion. The results indicate that the bulk of PPI1 is localised at the endoplasmic reticulum, from which it might be recruited to the PM for interaction with the H(+)-ATPase in response to as yet unidentified signals