Messenger (Los Angeles, Calif. : Print)最新文献

Lysosomes have emerged in the last decade as an immensely important intracellular site of Ca²⁺ storage and signalling. More recently there has been an increase in the number of new ion channels found to be functional on lysosomes and the potential roles that these signalling pathways might play in fundamental cellular processes are being uncovered. Defects in lysosomal function have been shown to result in changes in lysosomal Ca²⁺ homeostasis and ultimately can result in cell death. Several neurodegenerative diseases, from rare lysosomal storage diseases through to more common diseases of ageing, have recently been identified as having alterations in lysosomal Ca²⁺ homeostasis that may play an important role in neuronal excitotoxicity and ultimately cell death. This review will critically summarise these recent findings.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been implicated as an initial Ca²⁺ trigger in T cell Ca²⁺ signalling, but its role in formation of the immune synapse in CD4⁺ effector T cells has not been analysed. CD4⁺ T cells are activated by the interaction with peptide-MHCII complexes on the surface of antigen-presenting cells. Establishing a two-cell system including primary rat CD4⁺ T cells specific for myelin basic protein and rat astrocytes enabled us to mirror this activation process in vitro and to analyse Ca²⁺ signalling, cell shape changes and motility in T cells during formation and maintenance of the immune synapse. After immune synapse formation, T cells showed strong, antigen-dependent increases in free cytosolic calcium concentration ([Ca²⁺] _i ). Analysis of cell shape and motility revealed rounding and immobilization of T cells depending on the amplitude of the Ca²⁺ signal. NAADP-antagonist BZ194 effectively blocked Ca²⁺ signals in T cells evoked by the interaction with antigen-presenting astrocytes. BZ194 reduced the percentage of T cells showing high Ca²⁺ signals thereby supporting the proposed trigger function of NAADP for global Ca²⁺ signalling. Taken together, the NAADP signalling pathway is further confirmed as a promising target for specific pharmacological intervention to modulate T cell activation.

Recent interest in two-pore channels (TPCs) has resulted in a variety of studies dealing with the functional role and mechanism of action of these endo-lysosomal proteins in diverse physiological processes. With the availability of mouse lines harbouring mutant alleles for Tpcnl and/or Tpcn2 genes, several studies have made use of them to validate, consolidate and discover new roles for these channels not only at the cellular level but, importantly, also at the level of the whole organism. The different mutant mouse lines that have been used were derived from distinct genetic manipulation strategies, with the aim of knocking out expression of TPC proteins. However, the expression of different residual TPC sequences predicted to occur in these mutant mouse lines, together with the varied degree to which the effects on Tpcn expression have been studied, makes it important to assess the true knockout status of some of the lines. In this review we summarize these Tpcn mutant mouse lines with regard to their predicted effect on Tpcn expression and the extent to which they have been characterized. Additionally, we discuss how results derived from studies using these Tpcn mutant mouse lines have consolidated previously proposed roles for TPCs, such as mediators of NAADP signalling, endo-lysosomal functions, and pancreatic β cell physiology. We will also review how they have been instrumental in the assignment of new physiological roles for these cation channels in processes such as membrane electrical excitability, neoangiogenesis, viral infection and brown adipose tissue and heart function, revealing, in some cases, a specific contribution of a particular TPC isoform.

Two-pore channels are members of the voltage-gated ion channel superfamily. They localise to the endolysosomal system and are likely targets for the Ca²⁺ mobilising messenger NAADP. In this brief review, we relate mutagenesis of the TPC pore to a recently published homology model and discuss how pore mutants are informing us of TPC function. Molecular physiology of these ubiquitous proteins is thus emerging.

A cytotoxic T-lymphocyte (CTL) kills an infected or tumorigenic cell by Ca²⁺-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. However, these granules are more than reservoirs of secretory cytolytic proteins but may also serve as unique Ca²⁺ signaling hubs that autonomously generate their own signals for exocytosis. This review discusses a selective role for the Ca²⁺-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) and its molecular targets, two-pore channels (TPCs), in stimulating exocytosis. Given that TPCs reside on the exocytotic granules themselves, these vesicles generate as well as respond to NAADP-dependent Ca²⁺ signals, which may have wider implications for stimulus-secretion coupling, vesicular fusion, and patho-physiology.

Here we describe the successful synthesis of cyclic ADP-4-thioribose (cADPtR, 3), designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca²⁺-mobilizing second messenger, in which the key N1-β-thioribosyladenosine structure was stereoselectively constructed by condensation between the imidazole nucleoside derivative 8 and the 4-thioribosylamine 7 via equilibrium in 7 between the α-anomer (7α) and the β-anomer (7β) during the reaction course. cADPtR is, unlike cADPR, chemically and biologically stable, while it effectively mobilizes intracellular Ca²⁺ like cADPR in various biological systems, such as sea urchin homogenate, NG108-15 neuronal cells, and Jurkat T-lymphocytes. Thus, cADPtR is a stable equivalent of cADPR, which can be useful as a biological tool for investigating cADPR-mediated Ca²⁺-mobilizing pathways.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), two intracellular Ca²⁺ mobilizing second messengers, have been recognized as a fundamental signaling mechanism regulating a variety of cell or organ functions in different biological systems. Here we reviewed the literature regarding these ADP-ribosylcyclase products in vascular cells with a major focus on their production, physiological roles, and related underlying mechanisms mediating their actions. In particular, several hot topics in this area of research are comprehensively discussed, which may help understand some of the controversial evidence provided by different studies. For example, some new models are emerging for the agonist receptor coupling of CD38 or ADP-ribosylcyclase and for the formation of an acidic microenvironment to facilitate the production of NAADP in vascular cells. We also summarized the evidence regarding the NAADP-mediated two-phase Ca²⁺ release with a slow Ca²⁺-induced Ca²⁺ release (CICR) and corresponding physiological relevance. The possibility of a permanent structural space between lysosomes and sarcoplasmic reticulum (SR), as well as the critical role of lysosome trafficking in phase 2 Ca²⁺ release in response to some agonists are also explored. With respect to the molecular targets of NAADP within cells, several possible candidates including SR ryanodine receptors (RyRs), lysosomal transient receptor potential-mucolipin 1 (TRP-ML1) and two pore channels (TPCs) are presented with supporting and opposing evidence. Finally, the possible role of NAADP-mediated regulation of lysosome function in autophagy and atherogenesis is discussed, which may indicate a new direction for further studies on the pathological roles of cADPR and NAADP in the vascular system.

NAADP is a potent Ca²⁺ mobilizing messenger [1-3]. Since its discovery in 1995 [4] a considerable volume of literature has shown that NAADP couples cell stimulation to endolysosomal Ca²⁺ release and thereby the regulation of many cellular functions [5]. However definition of its molecular mechanism of action has proved far from easy. Since 2009, a consensus emerged as several independent groups coalesced upon the two-pore channel (TPC) family as NAADP-activated channels essential for Ca²⁺ release from endolysosomal Ca²⁺ stores [6-8]. However this view has been recently challenged by data clearly showing that TPCs function as Na⁺-selective channels apparently insensitive to NAADP [9;10]. Given the two fundamental characteristics defining an ion channel comprise the opening stimulus and the nature of the permeant ions, scrutiny of these seeming irreconcilable viewpoints is essential. The purpose of this commentary is to distil the remaining consensus while interrogating these divergent viewpoints. From this analysis, critical experimental needs are identified.

A potential extracellular target for inositol phosphates and analogues with anticancer properties is identified. Proteins from detergent-solubilised HeLa cell lysates bound to a novel affinity column of myo-inositol 1,3,4,5,6-pentakisphosphate (InsP₅) coupled to Affigel-10. One high-affinity ligand was fibrinogen Bβ. Inositol phosphates and analogues were able to elute purified fibrinogen from this matrix. InsP₅ and the inositol phosphate mimic biphenyl 2,3',4,5',6-pentakisphosphate (BiPhP₅) bind fibrinogen in vitro, and block the effects of fibrinogen in A549 cell-based assays of proliferation and migration. They are also able to prevent the fibrinogen-mediated activation of phosphatidylinositol 3-kinase. These effects of fibrinogen appear to be mediated through the intercellular adhesion molecule-1 (ICAM-1), as cells not expressing ICAM-1 fail to respond. In contrast, myo-inositol hexakisphosphate and the epimeric scyllo-inositol 1,2,3,4,5-pentakisphosphate were without effect. These findings are consistent with earlier reports that higher inositol phosphates have anticancer properties. This new mechanism of action and target for these extracellular inositol phosphates to have their effects allows a re-evaluation of earlier data.