Anaplasma phagocytophilum Ats-1 Is Imported into Host Cell Mitochondria and Interferes with Apoptosis Induction

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, infects human neutrophils and inhibits mitochondria-mediated apoptosis. Bacterial factors involved in this process are unknown. In the present study, we screened a genomic DNA library of A. phagocytophilum for effectors of the type IV secretion system by a bacterial two-hybrid system, using A. phagocytophilum VirD4 as bait. A hypothetical protein was identified as a putative effector, hereby named Anaplasma translocated substrate 1 (Ats-1). Using triple immunofluorescence labeling and Western blot analysis of infected cells, including human neutrophils, we determined that Ats-1 is abundantly expressed by A. phagocytophilum, translocated across the inclusion membrane, localized in the host cell mitochondria, and cleaved. Ectopically expressed Ats-1 targeted mitochondria in an N-terminal 17 residue-dependent manner, localized in matrix or at the inner membrane, and was cleaved as native protein, which required residues 55–57. In vitro-translated Ats-1 was imported in a receptor-dependent manner into isolated mitochondria. Ats-1 inhibited etoposide-induced cytochrome c release from mitochondria, PARP cleavage, and apoptosis in mammalian cells, as well as Bax-induced yeast apoptosis. Ats-1(55–57) had significantly reduced anti-apoptotic activity. Bax redistribution was inhibited in both etoposide-induced and Bax-induced apoptosis by Ats-1. Taken together, Ats-1 is the first example of a bacterial protein that traverses five membranes and prevents apoptosis at the mitochondria

The creatine kinase system and pleiotropic effects of creatine

The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans

Mitochondrial channels: ion fluxes and more.

The field of mitochondrial ion channels has recently seen substantial progress, including the molecular identification of some of the channels. An integrative approach using genetics, electrophysiology, pharmacology, and cell biology to clarify the roles of these channels has thus become possible. It is by now clear that many of these channels are important for energy supply by the mitochondria and have a major impact on the fate of the entire cell as well. The purpose of this review is to provide an up-to-date overview of the electrophysiological properties, molecular identity, and pathophysiological functions of the mitochondrial ion channels studied so far and to highlight possible therapeutic perspectives based on current information

Mitochondrial regulation of cytosolic Ca2+ signals in smooth muscle

The cytosolic Ca2+ concentration ([Ca2+]c) controls virtually every activity of smooth muscle, including contraction, migration, transcription, division and apoptosis. These processes may be activated by large (>10 μM) amplitude [Ca2+]c increases, which occur in small restricted regions of the cell or by smaller (<1 μM) amplitude changes throughout the bulk cytoplasm. Mitochondria contribute to the regulation of these signals by taking up Ca2+. However, mitochondria’s reported low affinity for Ca2+ is thought to require the organelle to be positioned close to ion channels and within a microdomain of high [Ca2+]. In cultured smooth muscle, mitochondria are highly dynamic structures but in native smooth muscle mitochondria are immobile, apparently strategically positioned organelles that regulate the upstroke and amplitude of IP3-evoked Ca2+ signals and IP3 receptor (IP3R) cluster activity. These observations suggest mitochondria are positioned within the high [Ca2+] microdomain arising from an IP3R cluster to exert significant local control of channel activity. On the other hand, neither the upstroke nor amplitude of voltage-dependent Ca2+ entry is modulated by mitochondria; rather, it is the declining phase of the transient that is regulated by the organelle. Control of the declining phase of the transient requires a high mitochondrial affinity for Ca2+ to enable uptake to occur over the normal physiological Ca2+ range (<1 μM). Thus, in smooth muscle, mitochondria regulate Ca2+ signals exerting effects over a large range of [Ca2+] (∼200 nM to at least tens of micromolar) to provide a wide dynamic range in the control of Ca2+ signals

Mitochondrial Permeability Transition in the CNS - Composition, Regulation, and Pathophysiological Relevance

Mitochondrial permeability transition (MPT) is induced in isolated brain mitochondria by calcium and oxidants and is inhibited by adenine nucleotides. When induced, MPT is associated with equilibration of solutes of <1500 Da across the inner mitochondrial membrane. A persistent induction of MPT depolarizes the inner membrane and causes cessation of ATP synthesis, swelling of the matrix, and bursting of the mitochondrial membranes. The rupture of the membranes releases calcium stored in the mitochondrial matrix and apoptogenic factors from the intermembrane space, leading to cell death. MPT has been implicated in acute brain injury and neurodegenerative disease since inhibitors of MPT such as cyclosporin A (CsA) are brain protective. Whether MPT has a physiological role is unclear, but MPT may be important in calcium homeostasis under conditions of excessive neuronal activity