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

Unravelling the molecular mechanisms of regulation of plant type 2B Ca2+-ATPases using Arabidopsis thaliana plasma membrane isoform ACA8 as a model system

Type 2B Ca2+-ATPases of plants (ACAs) have an extended cytosolic N-terminus containing an auto-inhibitory domain which by interacting with the catalytic head hampers pump activity. Using the Arabidopsis thaliana plasma membrane isoform ACA8 as a model, we have shown that fine-tuning of plant type 2B Ca2+-ATPases depends on multiple molecular mechanisms. Calmodulin (CaM) is the best known regulator of type 2B Ca2+-ATPases: CaM-binding to ACA8 at two sites in the N-terminus suppresses auto-inhibition and determines both an increase of Vmax and a decrease of the K0.5 for free Ca2+. Beside CaM, acidic phospholipids (APL) \u2013 as e.g. phosphatidylinositol-4P \u2013 stimulate ACA8 activity via a dual mechanism, involving different APL binding sites. APL binding to the N-terminus suppresses its auto-inhibitory action similarly to CaM, while binding to another, yet unidentified, site further increases the enzyme affinity for Ca2+. In addition, the N-terminus of ACA8 contains several Ser residue

PPI1, UNA NUOVA PROTEINA CAPACE DI INTERAGIRE CON IL DOMINIO C-TERMINALE DELL&apos; H+-ATPASI DEL PLASMALEMMA

Il dominio autoinibitorio C-terminale dell' H+-ATPasi del plasmalemma costituisce il principale sito di regolazione dell'enzima: la sua rimozione proteolitica, cos\uec come il legame di proteine 14-3-3 ad una sequenza contenuta in questa regione, causano un aumento dell'attivit\ue0 enzimatica. Utilizzando il metodo del doppio ibrido abbiamo identificato una proteina di 612 aa (Ppi1, proton pump interactor), la cui porzione N-terminale (88aa) interagisce con il dominio C-terminale dell'H+-ATPasi, isoforma AHA1. Diversi geni di Arabidopsis thaliana e alcuni EST di diverse specie vegetali mostrano una omologia di sequenza significativa (50-70% in porzioni di 200-600 aa) con Ppi1. La regione N-terminale di Ppi1 \ue8 stata espressa in E. coli come proteina di fusione con la GST o con una coda di istidine (His6-Ppi). In esperimenti di overlay, entrambe le proteine di fusione si legano all'H+-ATPasi immunoprecipitata da una frazione di plasmalemma purificata da cellule in coltura di A. thaliana His6-Ppi stimola l'attivit\ue0 dell'H+-ATPasi; lo stimolo \ue8 maggiore a pH 6.4 (massimo stimolo a 20 mM His-Ppi) che a pH 7.3, dove lo stimolo non \ue8 saturato neppure alla concentrazione di 60 mM. L'attivazione indotta da Ppi1 \ue8 sinergica con quella indotta da fusicoccina e da tripsina; inoltre His6-Ppi si lega all'H+-ATPasi trattata con tripsina. Il legame dell'attivatore avviene perci\uf2 ad un sito diverso da quello delle 14-3-3 e a monte del sito di taglio della tripsina