Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

Introduction

Subcellular compartmentalization enables eukaryotic cells to carry out different reactions at the same time, resulting in different metabolite pools in the subcellular compartments. Thus, mutations affecting the mitochondrial energy metabolism could cause different metabolic alterations in mitochondria compared to the cytoplasm. Given that the metabolite pool in the cytosol is larger than that of other subcellular compartments, metabolic profiling of total cells could miss these compartment-specific metabolic alterations.

Objectives

To reveal compartment-specific metabolic differences, mitochondria and the cytoplasmic fraction of baker’s yeast Saccharomyces cerevisiae were isolated and subjected to metabolic profiling.

Methods

Mitochondria were isolated through differential centrifugation and were analyzed together with the remaining cytoplasm by gas chromatography–mass spectrometry (GC–MS) based metabolic profiling.

Results

Seventy-two metabolites were identified, of which eight were found exclusively in mitochondria and sixteen exclusively in the cytoplasm. Based on the metabolic signature of mitochondria and of the cytoplasm, mutants of the succinate dehydrogenase (respiratory chain complex II) and of the F_OF₁-ATP-synthase (complex V) can be discriminated in both compartments by principal component analysis from wild-type and each other. These mitochondrial oxidative phosphorylation machinery mutants altered not only citric acid cycle related metabolites but also amino acids, fatty acids, purine and pyrimidine intermediates and others.

Conclusion

By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations affecting the mitochondrial metabolism.

     

Mitochondria-cytoskeleton associations in mammalian cytokinesis

Background

The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown.

Methods

We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells.

Results

Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division.

Conclusions

Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.

     

Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

Background

Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid.

Results

Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclx_s, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclx_s oligomerization, accumulation of Ca²⁺, and increased oxygen consumption.

Conclusion

Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions.

     

Evolution of mitochondrial TAT translocases illustrates the loss of bacterial protein transport machines in mitochondria

Background

Bacteria and mitochondria contain translocases that function to transport proteins across or insert proteins into their inner and outer membranes. Extant mitochondria retain some bacterial-derived translocases but have lost others. While BamA and YidC were integrated into general mitochondrial protein transport pathways (as Sam50 and Oxa1), the inner membrane TAT translocase, which uniquely transports folded proteins across the membrane, was retained sporadically across the eukaryote tree.

Results

We have identified mitochondrial TAT machinery in diverse eukaryotic lineages and define three different types of eukaryote-encoded TatABC-derived machineries (TatAC, TatBC and TatC-only). Here, we investigate TatAC and TatC-only machineries, which have not been studied previously. We show that mitochondria-encoded TatAC of the jakobid Andalucia godoyi represent the minimal functional pathway capable of substituting for the Escherichia coli TatABC complex and can transport at least one substrate. However, selected TatC-only machineries, from multiple eukaryotic lineages, were not capable of supporting the translocation of this substrate across the bacterial membrane. Despite the multiple losses of the TatC gene from the mitochondrial genome, the gene was never transferred to the cell nucleus. Although the major constraint preventing nuclear transfer of mitochondrial TatC is likely its high hydrophobicity, we show that in chloroplasts, such transfer of TatC was made possible due to modifications of the first transmembrane domain.

Conclusions

At its origin, mitochondria inherited three inner membrane translocases Sec, TAT and Oxa1 (YidC) from its bacterial ancestor. Our work shows for the first time that mitochondrial TAT has likely retained its unique function of transporting folded proteins at least in those few eukaryotes with TatA and TatC subunits encoded in the mitochondrial genome. However, mitochondria, in contrast to chloroplasts, abandoned the machinery multiple times in evolution. The overall lower hydrophobicity of the Oxa1 protein was likely the main reason why this translocase was nearly universally retained in mitochondrial biogenesis pathways.

     

Impaired mitochondrial calcium uptake caused by tacrolimus underlies beta-cell failure

Background

One of the most common side effects of the immunosuppressive drug tacrolimus (FK506) is the increased risk of new-onset diabetes mellitus. However, the molecular mechanisms underlying this association have not been fully clarified.

Methods

We studied the effects of the therapeutic dose of tacrolimus on mitochondrial fitness in beta-cells.

Results

We demonstrate that tacrolimus impairs glucose-stimulated insulin secretion (GSIS) in beta-cells through a previously unidentified mechanism. Indeed, tacrolimus causes a decrease in mitochondrial Ca²⁺ uptake, accompanied by altered mitochondrial respiration and reduced ATP production, eventually leading to impaired GSIS.

Conclusion

Our observations individuate a new fundamental mechanism responsible for the augmented incidence of diabetes following tacrolimus treatment. Indeed, this drug alters Ca²⁺ fluxes in mitochondria, thereby compromising metabolism-secretion coupling in beta-cells.

     

Intracellular organelle networks: Understanding their organization and communication through systems-level modeling and analysis

Background

Membrane-bound intracellular organelles are biochemically distinct compartments used by eukaryotic cells for serving specialized physiological functions and organizing their internal environment. Recent studies revealed surprisingly extensive communication between these organelles and highlighted the network nature of their organization and communication. Since organization and communication of the organelles are carried out at the systems level through their networks, systems-level studies are essential for understanding the underlying mechanisms.

Methods

We reviewed recent studies that used systems-level quantitative modeling and analysis to understand organization and communication of intracellular organelle networks.

Results

We first review modeling and analysis studies on how fusion/fission and degradation/biogenesis, two essential and closely related classes of activities of individual organelles, collectively mediate the dynamic organization of their networks. We then turn to another important aspect of the dynamic organization of the organelle networks, namely how organelles are physically connected within their networks, a property referred to as the topology of the networks in mathematics, and summarize some of their distinct properties. Lastly, we briefly review modeling and analysis studies that aim to understand communication between different organelle networks, focusing on cellular calcium homeostasis as an example. We conclude with a brief discussion of future directions for research in this area.

Conclusion

Together, the reviewed studies provide critical insights into how diverse activities of individual organelles collectively mediate the organization and communication of their networks. They demonstrate the essential role of systemslevel modeling and analysis in understanding complex behavior of such networks.

     

Proteomic profiling of neuronal mitochondria reveals modulators of synaptic architecture

Background

Neurons are highly polarized cells consisting of three distinct functional domains: the cell body (and associated dendrites), the axon and the synapse. Previously, it was believed that the clinical phenotypes of neurodegenerative diseases were caused by the loss of entire neurons, however it has recently become apparent that these neuronal sub-compartments can degenerate independently, with synapses being particularly vulnerable to a broad range of stimuli. Whilst the properties governing the differential degenerative mechanisms remain unknown, mitochondria consistently appear in the literature, suggesting these somewhat promiscuous organelles may play a role in affecting synaptic stability. Synaptic and non-synaptic mitochondrial subpools are known to have different enzymatic properties (first demonstrated by Lai et al., 1977). However, the molecular basis underpinning these alterations, and their effects on morphology, has not been well documented.

Methods

The current study has employed electron microscopy, label-free proteomics and in silico analyses to characterize the morphological and biochemical properties of discrete sub-populations of mitochondria. The physiological relevance of these findings was confirmed in-vivo using a molecular genetic approach at the Drosophila neuromuscular junction.

Results

Here, we demonstrate that mitochondria at the synaptic terminal are indeed morphologically different to non-synaptic mitochondria, in both rodents and human patients. Furthermore, generation of proteomic profiles reveals distinct molecular fingerprints – highlighting that the properties of complex I may represent an important specialisation of synaptic mitochondria. Evidence also suggests that at least 30% of the mitochondrial enzymatic activity differences previously reported can be accounted for by protein abundance. Finally, we demonstrate that the molecular differences between discrete mitochondrial sub-populations are capable of selectively influencing synaptic morphology in-vivo. We offer several novel mitochondrial candidates that have the propensity to significantly alter the synaptic architecture in-vivo.

Conclusions

Our study demonstrates discrete proteomic profiles exist dependent upon mitochondrial subcellular localization and selective alteration of intrinsic mitochondrial proteins alters synaptic morphology in-vivo.

     

Mitofusin 1 is degraded at G<Subscript>2</Subscript>/M phase through ubiquitylation by MARCH5

Background

Mitochondria exhibit a dynamic morphology in cells and their biogenesis and function are integrated with the nuclear cell cycle. In mitotic cells, the filamentous network structure of mitochondria takes on a fragmented form. To date, however, whether mitochondrial fusion activity is regulated in mitosis has yet to be elucidated.

Findings

Here, we report that mitochondria were found to be fragmented in G₂ phase prior to mitotic entry. Mitofusin 1 (Mfn1), a mitochondrial fusion protein, interacted with cyclin B1, and their interactions became stronger in G₂/M phase. In addition, MARCH5, a mitochondrial E3 ubiquitin ligase, reduced Mfn1 levels and the MARCH5-mediated Mfn1 ubiquitylation were enhanced in G₂/M phase.

Conclusions

Mfn1 is degraded through the MARCH5-mediated ubiquitylation in G₂/M phase and the cell cycle-dependent degradation of Mfn1 could be facilitated by interaction with cyclin B1/Cdk1 complexes.

     

Esub8: A novel tool to predict protein subcellular localizations in eukaryotic organisms

Background

Subcellular localization of a new protein sequence is very important and fruitful for understanding its function. As the number of new genomes has dramatically increased over recent years, a reliable and efficient system to predict protein subcellular location is urgently needed.

Results

Esub8 was developed to predict protein subcellular localizations for eukaryotic proteins based on amino acid composition. In this research, the proteins are classified into the following eight groups: chloroplast, cytoplasm, extracellular, Golgi apparatus, lysosome, mitochondria, nucleus and peroxisome. We know subcellular localization is a typical classification problem; consequently, a one-against-one (1-v-1) multi-class support vector machine was introduced to construct the classifier. Unlike previous methods, ours considers the order information of protein sequences by a different method. Our method is tested in three subcellular localization predictions for prokaryotic proteins and four subcellular localization predictions for eukaryotic proteins on Reinhardt's dataset. The results are then compared to several other methods. The total prediction accuracies of two tests are both 100% by a self-consistency test, and are 92.9% and 84.14% by the jackknife test, respectively. Esub8 also provides excellent results: the total prediction accuracies are 100% by a self-consistency test and 87% by the jackknife test.

Conclusions

Our method represents a different approach for predicting protein subcellular localization and achieved a satisfactory result; furthermore, we believe Esub8 will be a useful tool for predicting protein subcellular localizations in eukaryotic organisms.

     

The <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> homologue of Twinkle,Twm1, is a mitochondrial DNA helicase,an active primase and promotes mitochondrial DNA replication

Background

DNA replication requires contributions from various proteins, such as DNA helicases; in mitochondria Twinkle is important for maintaining and replicating mitochondrial DNA. Twinkle helicases are predicted to also possess primase activity, as has been shown in plants; however this activity appears to have been lost in metazoans. Given this, the study of Twinkle in other organisms is required to better understand the evolution of this family and the roles it performs within mitochondria.

Results

Here we describe the characterization of a Twinkle homologue, Twm1, in the amoeba Dictyostelium discoideum, a model organism for mitochondrial genetics and disease. We show that Twm1 is important for mitochondrial function as it maintains mitochondrial DNA copy number in vivo. Twm1 is a helicase which unwinds DNA resembling open forks, although it can act upon substrates with a single 3′ overhang, albeit less efficiently. Furthermore, unlike human Twinkle, Twm1 has primase activity in vitro. Finally, using a novel in bacterio approach, we demonstrated that Twm1 promotes DNA replication.

Conclusions

We conclude that Twm1 is a replicative mitochondrial DNA helicase which is capable of priming DNA for replication. Our results also suggest that non-metazoan Twinkle could function in the initiation of mitochondrial DNA replication. While further work is required, this study has illuminated several alternative processes of mitochondrial DNA maintenance which might also be performed by the Twinkle family of helicases.

     

Succinate dehydrogenase in Parkinson’s disease

Background

The prevalence of neurodegenerative disorders such as Parkinson’s disease (PD) is increased by age. Alleviation of their symptoms and protection of normal neurons against degeneration are the main aspects of the researches to establish novel therapeutic strategies. Many studies have shown that mitochondria as the most important organelles in the brain which show impairment in PD models. Succinate dehydrogenase (SDH) as a component of the oxidative phosphorylation system in mitochondria connects Krebs cycle to the electron transport chain. Dysfunction or inhibition of the SDH can trigger mitochondrial impairment and disruption in ATP generation. Excessive in lipid synthesis and induction of the excitotoxicity as inducers in PD are controlled by SDH activity directly and indirectly. On the other hand, mutation in subunits of the SDH correlates with the onset of neurodegenerative disorders. Therefore, SDH could behave as one of the main regulators in neuroprotection.

Objective

In this review we will consider contribution of the SDH and its related mechanisms in PD.

Methods

Pubmed search engine was used to find published studies from 1977 to 2016. “Succinate dehydrogenase”, “lipid and brain”, “mitochondria and Parkinson’s disease” were the main keywords for searching in the engine.

Results

Wide ranges of studies (59 articles) in neurodegenerative disorders especially Parkinson’s disease like genetics of the Parkinson’s disease, effects of the mutant SDH on cell activity and physiology and lipid alteration in neurodegenerative disorders have been used in this review.

Conclusion

Mitochondria as key organelles in the energy generation plays crucial roles in PD. ETC complex in this organelle consists four complexes which alteration in their activities cause ROS generation and ATP depletion. Most of complexes are encoded by mtDNA while complex II is the only part of the ETC which is encoded by nuclear genome. So, focusing on the SDH and related pathways which have important role in neuronal survival and SDH has a potential to further studies as a novel neuroprotective agent.

     

Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells

Introduction

Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.

Objectives

The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.

Methods

To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.

Results

Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.

Conclusion

Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.

     

Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in <Emphasis Type="Italic">Drosophila</Emphasis>

Background

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure.

Results

The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits.

Conclusions

We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

     

Association of mitochondrial dysfunction and lipid metabolism with type 2 diabetes mellitus: A review of literature

Background

Diabetes mellitus (DM) is one of the most prevalent chronic diseases, and its prevalence continues to increase globally. The impact of mitochondrial dysfunction and lipid metabolism on diabetes mellitus and insulin resistance (IR) has been implicated in several previous reports; however, the results of studies are confusing despite four decades of study.

Methods/Results

This review has evaluated updated understanding of the role of mitochondrial dysfunction and lipid metabolism on type 2 diabetes, and found that mitochondrial dysfunction and lipid metabolism disorder induce the dysregulation of liver and pancreatic beta cells, insulin resistance, and type 2 diabetes.

Conclusion

Mitochondrial dysfunction and lipid metabolism induce metabolic dysregulation and finally increasing the possibility of diabetes.

     

A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells

Background

In previous research, we found that cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells, but the protein with cytocidal activity was unidentified.

Methods

A novel lamprey immune protein (LIP) as defense molecule was first purified and identified in jawless vertebrates (cyclostomes) using hydroxyapatite column and Q Sepharose Fast Flow column. After LIP stimulation, morphological changes of tumor cells were analysed and measured whether in vivo or in vitro.

Results

LIP induces remarkable morphological changes in tumor cells, including cell blebbing, cytoskeletal alterations, mitochondrial fragmentation and endoplasmic reticulum vacuolation, and most of the cytoplasmic and organelle proteins are released following treatment with LIP. LIP evokes an elevation of intracellular calcium and inflammatory molecule levels. Our analysis of the cytotoxic mechanism suggests that LIP can upregulate the expression of caspase 1, RIPK1, RIP3 to trigger pyroptosis and necroptosis. To examine the effect of LIP in vivo, tumor xenograft experiments were performed, and the results indicated that LIP inhibits tumor growth without damage to mice. In addition, the cytotoxic action of LIP depended on the phosphatidylserine (PS) content of the cell membrane.

Conclusions

These observations suggest that LIP plays a crucial role in tumor cell survival and growth. The findings will also help to elucidate the mechanisms of host defense in lamprey.

     

Deferiprone targets aconitase: Implication for Friedreich's ataxia treatment

Background

Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly affected. As a mean to counteract disease progression, it has been suggested to chelate free mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of availability of deferiprone, a chelator preferentially targeting mitochondrial iron.

Method

Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a particular attention paid to iron-sensitive aconitase activity.

Results

We found that a direct consequence of chelating mitochondrial free iron in various cell systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity was shown to precede decreased cell proliferation.

Conclusion

We conclude that, if chelating excessive mitochondrial iron may be beneficial at some stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in order to avoid undesirable consequences.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

Eg5 orchestrates porcine oocyte maturational progression by maintaining meiotic organelle arrangement

Background

Kinesin superfamily proteins are microtubule-based molecular motors essential for the intracellular transport of various cargos, including organelles, proteins, and RNAs. However, their exact roles during mammalian oocyte meiosis have not been fully clarified.

Results

Herein, we investigated the critical events during porcine oocyte meiotic maturation with the treatment of Eg5-specific inhibitor monastrol. We found that Eg5 inhibition resulted in oocyte meiotic failure by displaying the poor expansion of cumulus cells and reduced rate of polar body extrusion. In the meantime, the spindle assembly and chromosome alignment were compromised, accompanied by the decreased level of acetylated α-tubulin, indicative of less stable microtubules. Impaired actin dynamics and mitochondria integrity were also observed in Eg5-inhibited oocytes. Additionally, inhibition of Eg5 caused the abnormal distribution of cortical granules and ovastacin, a cortical granule component, potentially leading to the fertilization failure.

Conclusions

Our findings reveal that Eg5 possesses an important function in porcine oocyte meiotic progression by regulating the organelle dynamics and arrangement.