ABSTRACT
The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.
Subject(s)
Base Sequence , Cytomegalovirus Infections/virology , Cytomegalovirus/genetics , Genome, Viral , Recombination, Genetic , DNA, Viral/genetics , Databases, Nucleic Acid , Datasets as Topic , Evolution, Molecular , Genes, Viral , Genetic Variation , Genome, Viral/genetics , Genotype , High-Throughput Nucleotide Sequencing , Humans , Mutation , Sequence Analysis, DNA , Whole Genome SequencingABSTRACT
Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The plasma membrane calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here, we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, upregulation in the expression of the pro-apoptotic protein Fas Ligand and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Calcineurin/physiology , Paclitaxel/pharmacology , Plasma Membrane Calcium-Transporting ATPases/physiology , Cell Line, Tumor , Fas Ligand Protein/analysis , Genes, p53 , Humans , NFATC Transcription Factors/physiology , Protein Binding/drug effects , Signal TransductionSubject(s)
Child Health Services/methods , Fetal Growth Retardation/diagnosis , Fetal Growth Retardation/etiology , Infant, Newborn, Diseases/diagnosis , Infant, Newborn, Diseases/mortality , Female , Fetal Growth Retardation/therapy , Humans , Infant, Newborn , Infant, Newborn, Diseases/therapy , Male , PregnancyABSTRACT
AIMS: Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular physiology. Endothelial NO is mainly produced by the endothelial nitric oxide synthase (eNOS) enzyme. eNOS enzymatic activity is regulated at several levels, including Ca(2+)/calmodulin binding and the interaction of eNOS with associated proteins. There is emerging evidence indicating a role for the plasma membrane calcium ATPase (PMCA) as a negative regulator of Ca(2+)/calmodulin-dependent signal transduction pathways via its interaction with partner proteins. The aim of our study was to investigate the possibility that the activity of eNOS is regulated through its association with endothelial PMCA. METHODS AND RESULTS: We show here a novel interaction between endogenous eNOS and PMCA in human primary endothelial cells. The interaction domains were located to the region 735-934 of eNOS and the catalytic domain of PMCA. Ectopic expression of PMCA in endothelial cells resulted in an increase in phosphorylation of the residue Thr-495 of endogenous eNOS. However, disruption of the PMCA-eNOS interaction by expression of the PMCA interaction domain significantly reversed the PMCA-mediated effect on eNOS phosphorylation. These results suggest that eNOS activity is negatively regulated via interaction with PMCA. Moreover, NO production by endothelial cells was significantly reduced by ectopic expression of PMCA. CONCLUSION: Our results show strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways.
Subject(s)
Endothelial Cells/enzymology , Nitric Oxide Synthase Type III/metabolism , Plasma Membrane Calcium-Transporting ATPases/metabolism , Calcineurin/metabolism , Cells, Cultured , Cyclic GMP/metabolism , Down-Regulation , Enzyme Activation , Humans , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/genetics , Phosphorylation , Plasma Membrane Calcium-Transporting ATPases/genetics , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Signal Transduction , TransfectionABSTRACT
Plasma membrane calcium/calmodulin-dependent ATPases (PMCAs) are high affinity calcium pumps that extrude calcium from the cell. Emerging evidence suggests a novel role for PMCAs as regulators of calcium/calmodulin-dependent signal transduction pathways via interaction with specific partner proteins. In this work, we demonstrate that endogenous human PMCA2 and -4 both interact with the signal transduction phosphatase, calcineurin, whereas, no interaction was detected with PMCA1. The strongest interaction was observed between PMCA2 and calcineurin. The domain of PMCA2 involved in the interaction is equivalent to that reported for PMCA4b. PMCA2-calcineurin interaction results in inhibition of the calcineurin/nuclear factor of activated T-cells signalling pathway.