Mitophagy or how to control the Jekyll and Hyde embedded in mitochondrial metabolism: implications for melanoma progression and drug resistance

Proteins and pathways that control cell fate are placed under intense scrutiny. The same tight regulation applies to essential organelles that can both sustain cell survival or promote self‐degradation programs. Mitochondria are perhaps the prime example of cellular machineries with split functions (personalities). As a main source of ATP, mitochondria represent the main powerhouse of eukaryotic cells. However, mitochondrial respiration has the hidden complication of the production of potentially harmful reactive oxygen species (ROS). Moreover, mitochondria holds an armamentarium of stress‐response factors, which depending on the context, may lead to pro‐inflammatory signals, and to various forms of cell death, ranging from apoptosis to necrosis. A main clearance mechanism to eliminate superfluous, damaged or hyperactive mitochondria is selective mitophagy. Mitophagy, in fact, is emerging as a key quality‐control mechanism in cancer cells. Specifically, malignant transformation has been found to induce marked changes in mitochondrial dynamics and structure. Moreover, a key hallmark of tumor progression is metabolic reprogramming, which further deregulates ROS content and renders cells more susceptible to mitochondrial perturbations. Despite its increasing relevance in cancer biology, the field of mitophagy remains virtually unexplored in melanoma. However, given unique antioxidant mechanisms in melanocytic cells (e.g., linked to melanin) and the idiosyncratic interplay between ROS and hypoxia (both mitophagy inducers) in melanoma, this tumor type represents an ideal scenario for physiological studies of mitochondrial turnover. This perspective summarizes proof of concept for in‐depth basic and translational studies of mitophagy in melanoma. Particular emphasis is dedicated to new opportunities for gene discovery and drug design in this still aggressive disease.     

A promoter-swap strategy between the AtALMT and AtMATE genes increased Arabidopsis aluminum resistance and improved carbon-use efficiency for aluminum resistance

The primary mechanism of Arabidopsis aluminum (Al) resistance is based on root Al exclusion, resulting from Al-activated root exudation of the Al(3+) -chelating organic acids, malate and citrate. Root malate exudation is the major contributor to Arabidopsis Al resistance, and is conferred by expression of AtALMT1, which encodes the root malate transporter. Root citrate exudation plays a smaller but still significant role in Arabidopsis Al resistance, and is conferred by expression of AtMATE, which encodes the root citrate transporter. In this study, we demonstrate that levels of Al-activated root organic acid exudation are closely correlated with expression of the organic acid transporter genes AtALMT1 and AtMATE. We also found that the AtALMT1 promoter confers a significantly higher level of gene expression than the AtMATE promoter. Analysis of AtALMT1 and AtMATE tissue- and cell-specific expression based on stable expression of promoter-reporter gene constructs showed that the two genes are expressed in complementary root regions: AtALMT1 is expressed in the root apices, while AtMATE is expressed in the mature portions of the roots. As citrate is a much more effective chelator of Al(3+) than malate, we used a promoter-swap strategy to test whether root tip-localized expression of the AtMATE coding region driven by the stronger AtALMT1 promoter (AtALMT1(P)::AtMATE) resulted in increased Arabidopsis Al resistance. Our results indicate that expression of AtALMT1(P)::AtMATE not only significantly increased Al resistance of the transgenic plants, but also enhanced carbon-use efficiency for Al resistance.     

The role of free fatty acids in mitochondrial energetic metabolism in winter wheat seedlings

The effect of fatty acids (FAs) (C12–C24) on the functioning of winter wheat (Triticum aestivum L.) mitochondria was studied. Such fatty acids as C12:0, C16:0, and C18:0 and unsaturated FAs, such as C18:1 (n-9 cis), C18:1 (n-12 cis), C18:2 (n-9, 12), (18:3, n-3), and C22:1 (n-9 cis) caused efficient uncoupling of oxidative phosphorylation in mitochondria, i.e., an increase in the nonphosphorylating respiration rate and a decrease in the respiratory control value. It was established that C16:0 had the strongest uncoupling effect among all saturated FAs, and C18:3, among unsaturated FAs. The uncoupling effect of saturated FAs is provided by the ADP/ATP-antiporter, while plant uncoupling proteins play an important role in the uncoupling effect of unsaturated FAs. In addition, unsaturated, as well as saturated FAs might serve as oxidative substrates for mitochondria. It was concluded that the role of FAs in energetic metabolism of winter wheat seedlings consisted of uncoupling of oxidative phosphorylation and of serving as substrates for oxidation.     

Malate plays a central role in plant nutrition

Malate occupies a central role in plant metabolism. Its importance in plant mineral nutrition is reflected by the role it plays in symbiotic nitrogen fixation, phosphorus acquisition, and aluminum tolerance. In nitrogen-fixing root nodules, malate is the primary substrate for bacteroid respiration, thus fueling nitrogenase. Malate also provides the carbon skeletons for assimilation of fixed nitrogen into amino acids. During phosphorus deficiency, malate is frequently secreted from roots to release unavailable forms of phosphorus. Malate is also involved with plant adaptation to aluminum toxicity. To define the genetic and biochemical regulation of malate formation in plant nutrition we have isolated and characterized genes involved in malate metabolism from nitrogen-fixing root nodules of alfalfa and those involved in organic acid excretion from phosphorus-deficient proteoid roots of white lupin. Moreover, we have overexpressed malate dehydrogenase in alfalfa in attempts to improve nutrient acquisition. This report is an overview of our efforts to understand and modify malate metabolism, particularly in the legumes alfalfa and white lupin.     

Predictive role of mitochondrial genome in the stress resistance of insects and nematodes

Certain insects (e.g., moths and butterflies; order Lepidoptera) and nematodes are considered as excellent experimental models to study the cellular stress signaling mechanisms since these organisms are far more stress-resistant as compared to mammalian system. Multiple factors have been implicated in this unusual response, including the oxidative stress response mechanisms. Radiation or chemical-induced mitochondrial oxidative stress occurs through damage caused to the components of electron transport chain (ETC) leading to leakage of electrons and generation of superoxide radicals. This may be countered through quick replacement of damaged mitochondrial proteins by upregulated expression. Since the ETC comprises of various proteins coded by mitochondrial DNA, variation in the composition, expressivity and regulation of mitochondrial genome could greatly influence mitochondrial role under oxidative stress conditions. Therefore, we carried out in silico analysis of mitochondrial DNA in these organisms and compared it with that of the stress-sensitive humans/mammals. Parameters such as mitochondrial genome organization, codon bias, gene expressivity and GC₃ content were studied. Gene arrangement and Shine-Dalgarno (SD) sequence patterns indicating translational regulation were distinct in insect and nematodes as compared to humans. A higher codon bias (ENC≫35) and lower GC₃ content (≫0.20) were observed in mitochondrial genes of insect and nematodes as compared to humans (ENC>42; GC3>0.20), coupled with low codon adaptation index among insects. These features indeed favour higher expressivity of mitochondrial proteins and might help maintain the mitochondrial physiology under stress conditions. Therefore, our study indicates that mitochondrial genome organization may influence stress-resistance of insects and nematodes.     

The mitochondrial contact site complex, a determinant of mitochondrial architecture

Mitochondria are organelles with a complex architecture. They are bounded by an envelope consisting of the outer membrane and the inner boundary membrane (IBM). Narrow crista junctions (CJs) link the IBM to the cristae. OMs and IBMs are firmly connected by contact sites (CS). The molecular nature of the CS remained unknown. Using quantitative high-resolution mass spectrometry we identified a novel complex, the mitochondrial contact site (MICOS) complex, formed by a set of mitochondrial membrane proteins that is essential for the formation of CS. MICOS is preferentially located at the CJs. Upon loss of one of the MICOS subunits, CJs disappear completely or are impaired, showing that CJs require the presence of CS to form a superstructure that links the IBM to the cristae. Loss of MICOS subunits results in loss of respiratory competence and altered inheritance of mitochondrial DNA.     

植物适应酸铝胁迫机理的研究进展

酸铝胁迫是限制植物正常生长发育的重要非生物胁迫因子,严重制约了我国酸性土壤地区的农业生产水平。植物抵御酸铝胁迫的形式复杂多样,如分泌有机酸、提高根际pH、分泌黏液、细胞壁对Al³⁺的固定、有机酸对细胞溶质中Al³⁺的螯合与液泡区隔化等。现有研究多集中于常规生理特征分析,缺乏深入的分子生物学解析。基于此,本文对国内外植物适应酸铝胁迫机理的相关研究进行了归纳和总结,从酸铝胁迫对植物生长与生理代谢的影响、植物适应酸铝胁迫最主要的两种生理机制(Al排除机制、Al耐受机制)以及分子水平上调控相关耐铝基因进行了综述。最后针对现有研究的不足提出了展望,以期为深入揭示植物适应酸铝胁迫的机理以及挖掘适于酸土生长的优质作物资源提供理论依据。     

线粒体动力学改变在神经变性疾病中的地位

线粒体是一种处于高度运动状态的细胞器,频繁地出现分裂和融合,线粒体分裂和融合的动态过程被称为线粒体动力学。对于神经元来说,线粒体的动力学过程具有十分重要的生物学意义。已知线粒体融合介导蛋白的功能缺失性突变可以导致常染色体显性遗传性视神经萎缩和Charcot-Marie-Tooth病等神经变性疾病。近来发现,在迟发性神经变性疾病中,线粒体动力学的改变也具有重要地位。本文将在线粒体动力学的分子调控以及与细胞死亡的关系、在神经变性疾病中的地位等方面综述这一领域的最新进展。     

The real‐time quantification of autofluorescence spectrum shape for the monitoring of mitochondrial metabolism

The cellular proportion of free and protein‐bound NADH complexes is increasingly recognized as a metabolic indicator and biomarker. Because free and bound forms exhibit different fluorescence spectra, we consider whether autofluorescence shape sufficiently correlates with mitochondrial metabolism to be useful for monitoring in cellular suspensions. Several computational approaches for rapidly quantifying spectrum shape are used to detect Saccharomyces cereviseae response to oxygenation, and to the addition of mitochondrial functional modifiers and metabolic substrates. Observed changes appear consistent with previous studies probing free/protein‐bound proportions, making this a potentially useful approach for the real‐time monitoring of metabolism. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Mechanisms of metal resistance in plants: aluminum and heavy metals

Plants have evolved sophisticated mechanisms to deal with toxic levels of metals in the soil. In this paper, an overview of recent progress with regards to understanding fundamental molecular and physiological mechanisms underlying plant resistance to both aluminum (Al) and heavy metals is presented. The discussion of plant Al resistance will focus on recent advances in our understanding of a mechanism based on Al exclusion from the root apex, which is facilitated by Al-activated exudation of organic acid anions. The consideration of heavy metal resistance will focus on research into a metal hyperaccumulating plant species, the Zn/Cd hyperaccumulator, Thlaspi caerulescens, as an example for plant heavy metal research. Based on the specific cases considered in this paper, it appears that quite different strategies are used for Al and heavy metal resistance. For Al, our current understanding of a resistance mechanism based on excluding soil-borne Al from the root apex is presented. For heavy metals, a totally different strategy based on extreme tolerance and metal hyperaccumulation is described for a hyperaccumulator plant species that has evolved on naturally metalliferous soils. The reason these two strategies are the focus of this paper is that, currently, they are the best understood mechanisms of metal resistance in terrestrial plants. However, it is likely that other mechanisms of Al and/or heavy metal resistance are also operating in certain plant species, and there may be common features shared for dealing with Al and heavy resistance. Future research may uncover a number of novel metal resistance mechanisms in plants. Certainly the complex genetics of Al resistance in some crop plant species, such as rice and maize, suggests that a number of presently unidentified mechanisms are part of an overall strategy of metal resistance in crop plants.     

Oxidative stress triggered by aluminum in plant roots

Aluminum (Al) is a major growth-limiting factor for plants in acid soils. The primary site of Al accumulation and toxicity is the root meristem, and the inhibition of root elongation is the most sensitive response to Al. Al cannot catalyze redox reactions but triggers lipid peroxidation and reactive oxygen species (ROS) production in roots. Furthermore, Al causes respiration inhibition and ATP depletion. Comparative studies of Al toxicity in roots with that in cultured plant cells suggest that Al causes dysfunction and ROS production in mitochondria, and that ROS production, but not lipid peroxidation, seems to be a determining factor of root-elongation inhibition by Al.     

Defects in the mitochondrial energy metabolism outside the respiratory chain and the pyruvate dehydrogenase complex

Disturbances in substrate oxidations in muscle mitochondria from patients with a suspicion of a mitochondrial myopathy may arise from a deficiency of one or more of the complexes of the respiratory chain or of the pyruvate dehydrogenase complex. However, we found no clear-cut defect in a substantial part of such patients. In this report we discuss some of the other possibilities which could account for the disturbed substrate oxidation rates. Special attention will be paid to defects which are localized outside the respiratory chain, such as defects in post-respiratory chain enzymes, defects in transport mechanisms of the mitochondrial inner or outer membrane, deficiency of cofactors and deficiency of heat-shock protein. (Mol Cell Biochem 174: 243–247, 1997)     

植物核编码线粒体蛋白转动的研究进展

植物核编码蛋白向线粒体内的转运对于线粒体正常功能的发挥有着不可忽视的作用。目前在诸如前序列、前体蛋白加工、分子伴侣及线粒体与质体（或叶绿体）之间分选等几方面开展了许多研究。本综述以上方面的研究进展。     

泛素化在植物抗病中的作用

泛素化作为植物体内一种广泛存在的调控细胞反应的机制,参与调控植物抗病反应。本文综述了泛素化系统在植物抗病反应中的功能及作用机制,重点介绍了CRLs型E3泛素连接酶和RING/U-box型E3泛素连接酶如何参与调控植物抗病信号途径,以及病原物通过效应蛋白和毒性因子调控植物抗病性的分子机理,为阐明植物抗病机理和植物病害防治方法提供参考。     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

In vivo and in vitro effects of aluminum treatment on rat liver mitochondrial function

This study examines the effect on mitochondrial respiration and permeability of in vivo and in vitro aluminium (Al) exposure. Rats were treated intraperitoneally with AlCl₃ to achieve serum and liver Al concentrations comparable to those seen in Al-related disorders. Mitochondria isolated from Al-treated rats had higher (p<0.01) Al concentration, lower (p<0.05) state 3 respiration, respiratory control (RCR), and ADP/O ratio (succinate substrate), and greater passive swelling in 100 mM KCl or 200 mM NH₄NO₃ than controls. The in vitro addition of Al (0–180 μM) to mitochondria from normal rats also decreased (p<0.01) state 3 respiration, RCR, and ADP/O and stimulated passive swelling in KCl and NH₄NO₃ at 42–180 μM Al. These studies show that Al depresses mitochondrial energy metabolism and increases membrane permeability. The toxicity associated with Al may be related to its effect on mitochondria.     

The mitochondrial complexome of Arabidopsis thaliana

Mitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein–protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot‐gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re‐annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource ( https://complexomemap.de/at_mito_leaves ) to deposit the data and to allow straightforward access and custom data analyses.     

Modulation of mitochondrial permeability transition pore affects multidrug resistance in human hepatocellular carcinoma cells

Multidrug resistance (MDR) is a critical problem in the chemotherapy of cancers. Human hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to its potent MDR. Chemotherapeutic drugs primarily act by inducing apoptosis of cancer cells, and defects in apoptosis may result in MDR. Mitochondrial permeability transition (mPT) is implicated as an important event in the control of cell death or survival and mPT represents a target for the development of cytotoxic drugs. This study aimed to investigate the effects of selective opener (Atractyloside glycoside, ATR) and inhibitor (Cyclosporine A, CsA) of mitochondrial permeability transition pore (mPTP) on a CDDP-resistant HCC cell line (SK-Hep1 cells). In this study, a stable MDR phenotype characterization of SK-Hep1 cell line (SK-Hep1/CDDP cells) was established and used to investigate the role of mPTP in MDR. Results suggested that ATR accelerated the decrease of mitochondrial membrane potential (ΔΨm), reduced the Bax activity, and increased the apoptosis of SK-Hep1/CDDP cells; while CsA inhibited mPTP opening, reduced and delayed the decline of mitochondrial membrane potential, and increased the Bax activity, leading to increased tolerance of SK-Hep1/CDDP cells to apoptosis induction. However, mPTP activity had no effect on the expression of MDR1 in cells,meanwhile the P-gp translocation to mitochondria was increased, and functionally activated. In conclusion, selective modulation of mPTP can affect MDR in human HCC cells. Therefore, activation of mPTP may provide a new strategy to sensitize cancer cells to chemotherapeutic drugs and to reverse the MDR in cancer cells.