The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics

Cyclic AMP (cAMP)-dependent phosphorylation has been reported to exert biological effects in both the mitochondrial matrix and outer mitochondrial membrane (OMM). However, the kinetics, targets, and effectors of the cAMP cascade in these organellar domains remain largely undefined. Here we used sensitive FRET-based sensors to monitor cAMP and protein kinase A (PKA) activity in different mitochondrial compartments in real time. We found that cytosolic cAMP did not enter the matrix, except during mitochondrial permeability transition. Bicarbonate treatment (expected to activate matrix-bound soluble adenylyl cyclase) increased intramitochondrial cAMP, but along with membrane-permeant cAMP analogues, failed to induce measureable matrix PKA activity. In contrast, the OMM proved to be a domain of exceptionally persistent cAMP-dependent PKA activity. Although cAMP signaling events measured on the OMM mirrored those of the cytosol, PKA phosphorylation at the OMM endured longer as a consequence of diminished control by local phosphatases. Our findings demonstrate that mitochondria host segregated cAMP cascades with distinct functional and kinetic signatures.     

Inhibitor analysis of protein phosphorylation/dephosphorylation in maize mitochondria in relation to redox conditions

The role of protein phosphorylation/ dephosphorylation in the redox regulation of mitochondrial functioning was investigated. Incubation of isolated mitochondria of maize (Zea mays L.) in the presence of γ-³²P-ATP revealed phosphorylation of polypeptides with mol wt of 66, 60, 55, 48/50 doublet, 45, 29, 22, and 19 kD. The presence in the incubation medium of oxidized glutathione significantly reduced the level of protein phosphorylation. The addition of reduced glutathione diminished phosphorylation of proteins with mol wt of 60 and 48/50 kD and slightly increased phosphorylation of proteins with mol wt of 66, 55, and 45 kD. The reducing agent, sodium dithionite decreased phosphorylation of proteins with mol wt of 60, 45, 29, 22, and 19 kD but increased phosphorylation of 55 kD protein. The inhibitors of protein kinases and protein phosphatases significantly modified the effects of redox agents. For example, simultaneous action of an oxidant K₃[Fe(CN)₆] and NaF enhanced phosphorylation level compared to separate treatments with these agents. The combined application of sodium dithionite and NaF elevated phosphorylation level of 55 kD protein. Phosphoprotein with mol wt of about 66 kD was identified immunochemically as a heat shock protein (HSP 60). The results indicate the presence in mitochondria of redox-sensitive protein kinases and protein phosphatases. Differential changes in the pattern of mitochondrial phosphoproteins under the action of various redox agents suggest that phosphorylation is probably involved in the transduction of redox signal in plant mitochondria.     

Mitochondrial organization and motility probed by two-photon microscopy in cultured mouse brainstem neurons

Two-photon microscopy of rhodamine 123-labeled mitochondria revealed that mitochondria of neurons cultured from mouse respiratory center form functionally coupled, dynamically organized aggregates such as chains and clusters, while single mitochondria were rarely seen. Mitochondrial chain structures predominate in dendrites, while irregularly shaped mitochondrial clusters are mostly found in the soma. Both types of mitochondrial structures showed chaotic Brownian motions and the mitochondrial chains also revealed well-directed movements. The latter dislocations were arrested upon mitochondrial depolarization or blockade of mitochondrial ATP synthesis. Depolymerization of microtubules by colchicine or nocodazole or inhibition of protein phosphatases by calyculin A disrupted mitochondrial chains and the mitochondria accumulated in the soma. Forskolin and IBMX reversibly blocked directed movements of mitochondria, but did not affect their overall spatial distribution. Thus, protein phosphorylation seems to control both mitochondrial transport and organization. Protein phosphorylation downstream of enhanced cytosolic cAMP levels apparently regulates the transition from motile to non-motile mitochondria, while phosphorylation resulting from inhibition of types 1 and 2A protein phosphatases massively disturbs mitochondrial organization. The complex phosphorylation processes seem to control the close interaction of mitochondria and cytoskeleton which may guarantee that mitochondria are immobilized at energetic hot spots and rearranged in response to changes in local energy demands.     

线粒体蛋白质组及蛋白质量控制

线粒体是哺乳动物细胞内重要细胞器,不仅通过氧化磷酸化产生ATP为细胞提供能量,也参与调节钙离子稳态、活性氧(reactive oxygen species,ROS)的产生、细胞应激反应和细胞死亡等过程,其功能障碍不仅导致多种人类疾病的发生,而且也能降低动物卵母细胞质量和早期胚胎发育能力.大量证据表明,线粒体的功能依赖于...     

Thermal Sensitivity of Mitochondrial Respiration Efficiency and Protein Phosphorylation in the Clam Mercenaria mercenaria

The mitochondria of intertidal invertebrates continue to function when organisms are exposed to rapid substantial shifts in temperature. To test if mitochondrial physiology of the clam Mercenaria mercenaria is compromised under elevated temperatures, we measured mitochondrial respiration efficiency at 15°C, 18°C, and 21°C using a novel, high-throughput, microplate respirometry methodology developed for this study. Though phosphorylating (state 3) and resting (state 4) respiration rates were unaffected over this temperature range, respiratory control ratios (RCRs: ratio of state 3 to state 4 respiration rates) decreased significantly above 18°C (p < 0.05). The drop in RCR was not associated with reduction of phosphorylation efficiency, suggesting that, while aerobic scope of mitochondrial respiration is limited at elevated temperatures, mitochondria continue to efficiently produce adenosine triphosphate. We further investigated the response of clam mitochondria to elevated temperatures by monitoring phosphorylation of mitochondrial protein. Three proteins clearly demonstrated significant time- and temperature-specific phosphorylation patterns. The protein-specific patterns of phosphorylation may suggest that a suite of protein kinases and phosphatases regulate mitochondrial physiology in response to temperature. Thus, while aerobic scope of clam mitochondrial respiration is reduced at moderate temperatures, specific protein phosphorylation responses reflect large shifts in function that are initiated within the organelle at higher temperatures.     

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.     

Phosphorylation/dephosphorylation of maize mitochondrial proteins with different localization

Phosphorylation and dephosphorylation of the proteins residing in the outer mitochondrial membrane, mitoplasts and whole mitochondria of maize (Zea mays L.) were investigated in order to reveal the possible participation of these processes in mitochondrial signaling. A mitochondrial protein of around 57 kD was identified by immunocytochemistry as α-subunit of the F₁-ATPase complex. In isolated mitochondria of maize, phosphorylation of this protein could be visualized only after treating mitochondria with endotholl, an inhibitor of the PP1a and PP2A protein phosphatases. A phosphorylated protein of 46.6 kD was identified as β-subunit of the F₁-ATPase complex. Ca²⁺ is the most common second messenger participating in mitochondrial signaling. We conclude that the transmission of the Ca²⁺ signal to the plant mitochondria occurs via proteins of the outer mitochondrial membrane. The systems perceiving this signal could include the protein phosphatases residing in the outer mitochondrial membrane, which preferentially dephosphorylate the proteins in the inner membrane.     

The Involvement of Protein Phosphorylation/Dephosphorylation in the Redox Control of Translation in Cereal Mitochondria

A comparative analysis of a dependence of protein synthesis in mitochondria of cultivated (Zea mays) and wild (Elymus sibiricus) cereals on redox conditions showed that the addition of oxidized glutathione enhanced and the addition of reduced glutathione suppressed translation in organello. Inhibitors of protein kinases and protein phosphatases modified substantially the effects of redox agents on protein synthesis in mitochondria. It is supposed that protein phosphorylation in mitochondria may be a mechanism mediating the interrelation between the redox state of the respiratory chain and the activity of mitochondrial translation.     

膜蛋白ATAD3A在线粒体质量控制中的作用

线粒体质量控制对于线粒体网络的稳态和线粒体功能的正常发挥具有重要意义。三磷酸腺苷酶家族蛋白3A(ATAD3A)是同时参与调节线粒体结构功能、线粒体动力学和线粒体自噬等重要生物学过程的线粒体膜蛋白之一。近期研究表明,ATAD3A既可与Mic60/Mitofilin和线粒体转录因子A (TFAM)等因子相互作用以维持线粒体嵴的形态和氧化磷酸化功能,又能与发动蛋白相关蛋白1 (Drp1)结合而正性/负性调节线粒体分裂,还可作为线粒体外膜转位酶（TOM）复合物和线粒体内膜转位酶（TIM）复合物之间的桥接因子而介导PTEN诱导激酶（PINK1）输入线粒体进行加工,显示出促自噬或抗自噬活性。本文对ATAD3A在调控线粒体质量控制中的作用及其机制进行了综述。     

Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.     

Mitochondrial protein tyrosine nitration

Mitochondria are primary loci for the intracellular formation and reactions of reactive oxygen and nitrogen species including superoxide (O???), hydrogen peroxide (H?O?) and peroxynitrite (ONOO?). Depending on formation rates and steady-state levels, the mitochondrial-derived short-lived reactive species contribute to signalling events and/or mitochondrial dysfunction through oxidation reactions. Among relevant oxidative modifications in mitochondria, the nitration of the amino acid tyrosine to 3-nitrotyrosine has been recognized in vitro and in vivo. This post-translational modification in mitochondria is promoted by peroxynitrite and other nitrating species and can disturb organelle homeostasis. This study assesses the biochemical mechanisms of protein tyrosine nitration within mitochondria, the main nitration protein targets and the impact of 3-nitrotyrosine formation in the structure, function and fate of modified mitochondrial proteins. Finally, the inhibition of mitochondrial protein tyrosine nitration by endogenous and mitochondrial-targeted antioxidants and their physiological or pharmacological relevance to preserve mitochondrial functions is analysed.     

Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics

Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.     

Control of mitochondria dynamics and oxidative metabolism by cAMP, AKAPs and the proteasome

Mitochondria are highly specialized organelles and major players in fundamental aspects of cell physiology. In yeast, energy metabolism and coupling of mitochondrial activity to growth and survival is controlled by the protein kinase A pathway. In higher eukaryotes, modulation of the so-called A-kinase anchor protein (AKAP) complex regulates mitochondrial dynamics and activity, adapting the oxidative machinery and the metabolic pathway to changes in physiological demand. Protein kinases and phosphatases are assembled by AKAPs within transduction units, providing a mechanism to control signaling events at mitochondria and other target organelles. Ubiquitin-mediated proteolysis of signal transducers and effectors provides an additional layer of complexity in the regulation of mitochondria homeostasis. Genetic evidence indicates that alteration of one or more components of these biochemical pathways leads to mitochondrial dysfunction and human diseases. In this review, we focus on the emerging role of AKAP scaffolds and the proteasome pathway in the control of oxidative metabolism, organelle dynamics and the mitochondrial signaling network. These aspects are crucial elements for maintaining a proper energy balance and cellular lifespan.     

Mitochondrial DNA diseases: Histological and cellular studies

Large-scale deletions and tRNA point mutations in mitochondrial DNA (mtDNA) are associated with a variety of different mitochondrial encephalomyopathies. Skeletal muscle in these patients shows a typical pathology, characterized by the focal accumulation of large numbers of morphologically and biochemically abnormal mitochondria (ragged-red fibers). Both mtDNA deletions and tRNA point mutations impair mitochondrial translation and produce deficiencies in oxidative phosphorylation. However, mutant and wild-type mtDNAs co-exist (mtDNA heteroplasmy) and the translation defect is not expressed until the ratio of mutant: wild-type mtDNAs exceeds a specific threshold. Below the threshold the phenotype can be rescued by intramitochondrial genetic complementation. The mosaic expression of the skeletal muscle pathology is thus determined by both the cellular and organellar distribution of mtDNA mutants.     

Protein phosphorylation in plant mitochondria

Reversible phosphorylation of proteins is one of the most common regulatory mechanisms in eukaryotic cells and it can affect virtually any property of a protein. We predict that plant mitochondria possess 50–200 protein kinases (PKs), at least as many target proteins and 10–30 protein phosphatases although all will not be expressed at the same time in the same cell type or tissue. Presently available high-throughput methods for the identification of phosphoproteins and their phosphorylation sites are first reviewed and a number of useful databases listed. We then discuss the known phosphoproteins, PKs and phosphatases in plant mitochondria and compare with yeast and mammalian mitochondria. Three case stories—respiratory chain complex I, pyruvate dehydrogenase and formate dehydrogenase—are briefly considered before a final treatment of mitochondrial protein phosphorylation in intracellular signal transduction and programmed cell death.     

An integrated perspective and functional impact of the mitochondrial acetylome

Growing evidence suggests that a range of reversible protein post-translational modifications such as acetylation regulates mitochondria signalling, impacting cellular homeostasis. However, the extent of this type of regulation in the control of mitochondria functionality is just beginning to be discovered, aided by the availability of high-resolution mass spectrometers and bioinformatic tools. Data mining from literature on protein acetylation profiling focused on mitochondria isolated from tissues retrieved more than 1395 distinct proteins, corresponding to more than 4858 acetylation sites. ClueGo analysis of identified proteins highlighted oxidative phosphorylation, tricarboxylic acid cycle, fatty acid oxidation and amino acid metabolism as the biological processes more prone to regulation through acetylation. This review also examines the physiological relevance of protein acetylation on the molecular pathways harbored in mitochondria under distinct pathophysiological conditions as caloric restriction and alcohol-induced liver damage. This integrative perspective will certainly help to envisage future studies targeting the regulation of mitochondrial functionality.     

Protein modification and replicative senescence of WI‐38 human embryonic fibroblasts

Oxidized proteins as well as proteins modified by the lipid peroxidation product 4‐hydroxy‐2‐nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins build up and potentially affect cellular function during replicative senescence of WI‐38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresis‐based proteomic approach coupled with immunodetection of HNE‐, AGE‐modified and carbonylated proteins. Thirty‐seven proteins targeted for either one of these modifications were identified by mass spectrometry and are involved in different cellular functions such as protein quality control, energy metabolism and cytoskeleton. Almost half of the identified proteins were found to be mitochondrial, which reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE‐modified proteins could be explained by the senescence‐associated decreased activity of glyoxalase‐I, the major enzyme involved in the detoxification of the glycating agents methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age‐related build‐up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP‐stimulated Lon‐like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified mitochondrial proteins.     

Roles of sirtuins in the regulation of antioxidant defense and bioenergetic function of mitochondria under oxidative stress

Abstract

In addition to serving as the power house of mammalian cells, mitochondria are crucial for the maintenance of cellular homeostasis in response to physiological or environmental changes. Several lines of evidence suggest that posttranslational modification (PTM) of proteins plays a pivotal role in the regulation of the bioenergetic function of mitochondria. Among them, reversible lysine acetylation of mitochondrial proteins has been established as one of the key mechanisms in cellular response to energy demand by modulating the flux of a number of key metabolic pathways. In this article, we focus on the role of Sirt3-mediated deacetylation in: (1) flexibility of energy metabolism, (2) activation of antioxidant defense, and (3) maintenance of cellular redox status in response to dietary challenge and oxidative stress. We suggest that oxidative stress-elicited down-regulation of Sirt3 plays a role in the pathophysiology of diabetes, cardiac hypotrophy, mitochondrial diseases, and age-related diseases. Besides, the physiological role of newly identified lysine acylation mediated by Sirt5 and its biochemical effects on oxidative metabolism are also discussed. Moreover, we have integrated the regulatory function of several protein kinases that are involved in the phosphorylation of mitochondrial enzymes during oxidative stress. Finally, the functional consequence of the synergistic regulation through diverse protein modifications is emphasized on the maintenance of the bioenergetic homeostasis and metabolic adaptation of the animal and human cells. Together, we have provided an updated review of PTM in mitochondrial biology and their implications in aging and human diseases through an intricate regulation of energy metabolism under oxidative stress.