Highlights

Abstract

Graphical abstract

Abbreviations

Keywords

1. Introduction

2. De novo identification of mitochondrial proteins

2.1. In silico strategies

2.2. Experimental strategies

2.3. Integrative biology strategies

3. From proteins to functions: deorphanizing the unknown

3.1. In silico strategies

3.2. Experimental strategies

4. Perspectives

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgments

References

Abstract

Human mitochondria are complex and highly dynamic biological systems, comprised of over a thousand parts and evolved to fully integrate into the specialized intracellular signaling networks and metabolic requirements of each cell and organ. Over the last two decades, several complementary, top-down computational and experimental approaches have been developed to identify, characterize and modulate the human mitochondrial system, demonstrating the power of integrating classical reductionist and discovery-driven analyses in order to de-orphanize hitherto unknown molecular components of mitochondrial machineries and pathways. To this goal, systematic, multiomics-based surveys of proteome composition, protein networks, and phenotype-to-pathway associations at the tissue, cell and organellar level have been largely exploited to predict the full complement of mitochondrial proteins and their functional interactions, therefore catalyzing data-driven hypotheses. Collectively, these multidisciplinary and integrative research approaches hold the potential to propel our understanding of mitochondrial biology and provide a systems-level framework to unraveling mitochondria-mediated and disease-spanning pathomechanisms.

1. Introduction

Mitochondria are essential organelles for cellular and organismal life in virtually all eukaryotes (Fig. 1). Present-day human mitochondria originated from the integration of an endosymbiotic α-proteobacterium into a host cell, therefore exchanging their independence for a semi-autonomous life [1,2]. By the late 1990s, comparative genomics analyses of α-proteobacteria genomes and quantitative two-dimensional gels of highly purified mitochondria suggested that the mammalian mitochondrial proteome consists of ∼1,000–1,500 distinct proteins [3,4]. The majority of those proteins derive from the eukaryotic genome, whereas the prokaryotic genome was significantly reduced during the transition from endosymbiotic bacterium to organelle [2] (Fig. 2A). To date, only a handful of protein-coding genes – thirteen in mammals – are still retained in the mitochondrial DNA (mt-DNA) of almost all eukaryotes. Therefore, most of the mitochondrial proteome is encoded from the nuclear genome, translated in the cytosol, and then targeted and imported into the organelle.

Strikingly, only 1% of mammalian mitochondrial proteins are allocated to ATP synthesis, highlighting that the organelle’s functions reach far beyond energy production (Fig. 1). Indeed, mitochondria are at the core of multiple cellular pathways, including the biosynthesis of precursors for cholesterol, estrogen, testosterone and hemoglobin; the regulation of redox and ion homeostasis; the activation of antiviral responses and cell death. Adding an additional layer of complexity, mitochondrial functions are tied to the specialized tasks and physiology of different cell types, tissues, and organisms [5]. For instance, only between 40–70 % of the human mitochondrial proteome is conserved in commonly used model systems such as unicellular eukaryotes (e.g. S. cerevisiae) and invertebrates (e.g. C. elegans, D. melanogaster) (Fig. 2B). Furthermore, over 15 % of the mitochondrial system shows tissue-specificity [6] and profound differences even among cell types of the same tissue [6,7].

Background: Virtually all eukaryotic cells contain spatially distinct genomes, a single nuclear genome that harbours the vast majority of genes and much smaller genomes found in mitochondria present at thousands of copies per cell. To generate a coordinated gene response to various environmental cues, the genomes must communicate with each another. Much of this bi-directional crosstalk relies on epigenetic processes, including DNA, RNA, and histone modification pathways. Crucially, these pathways, in turn depend on many metabolites generated in specific pools throughout the cell, including the mitochondria. They also involve the transport of metabolites as well as the enzymes that catalyse these modifications between nuclear and mitochondrial genomes. Scope of review: This study examines some of the molecular mechanisms by which metabolites influence the activity of epigenetic enzymes, ultimately affecting gene regulation in response to metabolic cues. We particularly focus on the subcellular localisation of metabolite pools and the crosstalk between mitochondrial and nuclear proteins and RNAs. We consider aspects of mitochondrial-nuclear communication involving histone proteins, and potentially their epigenetic marks, and discuss how nuclear-encoded enzymes regulate mitochondrial function through epitranscriptomic pathways involving various classes of RNA molecules within mitochondria. Major conclusions: Epigenetic communication between nuclear and mitochondrial genomes occurs at multiple levels, ultimately ensuring a coordinated gene expression response between different genetic environments. Metabolic changes stimulated, for example, by environmental factors, such as diet or physical activity, alter the relative abundances of various metabolites, thereby directly affecting the epigenetic machinery. These pathways, coupled to regulated protein and RNA transport mechanisms, underpin the coordinated gene expression response. Their overall importance to the fitness of a cell is highlighted by the identification of many mutations in the pathways we discuss that have been linked to human disease including cancer.

Introduction

To fit within the confined space of a eukaryotic nucleus, a cell’s genome must be efficiently compacted, but in a highly ordered manner that maintains accessibility to the genetic information. This is achieved by DNA complexing with histone proteins to form nucleosome structures, which in turn further compact to form chromatin that ultimately packages entire chromosomes. Importantly, chromatin is not an inert packaging structure but rather an instructive scaffold capable of responding to various cues to regulate access of the DNA to different cellular machineries. This accessibility is fundamentally regulated by ATP-dependent chromatin remodelling activities and by covalent modification of both DNA and histone proteins. These modifications are commonly referred to as “epigenetic” modifications. Furthermore, in addition to epigenetics, the emerging field of epitranscriptomics is now adding numerous modifications of RNA molecules to the “modification repertoire,” thereby expanding our current view of epigenetics and how it regulates all DNA processes, in particular gene expression [1]. DNA and histone modifications regulate gene expression by modulating chromatin accessibility and/or providing binding sites for regulatory proteins. To date, more than 100 post-translational modifications (PTMs) have been detected on histones, including acetylation, methylation, ubiquitylation, phosphorylation, citrullination, and O-GlcNAcylation. Of these, acetylation and methylation are by far the best-studied and most characterised histone modifications (for review, see [2,3]).

Life emerges when biological structures are animated by energy. Energy is defined as a fundamental entity of nature that is transferred between parts of a system in the production of physical change within the system, and usually regarded as the capacity for doing work (Merriam-Webster, 2017). Without energy, there is no life – molecules alone do not interact in meaningful ways, and complex structures do not assemble nor replicate. A crucial factor that distinguishes the breathing-living organism from the inanimate body (i.e., cadaver) is the flow of energy. There are no intrinsic differences in the molecular composition of dead and living organisms. But it is a required quality of living organisms to experience a constant flow of energy through and between their different parts. This flow of energy sustains the movement, chemical reactions, and dynamic changes in the position and organization of molecules that is required to think, feel, move, and execute every element of the stress response. Without energy, stress adaptation is not possible, and the body dies. This review discusses the concept of stress as an energy-dependent and coordinated process, with an emphasis on the key role that mitochondria plays in these processes. With a broad view of energy, we take a multi-level approach exploring the role of mitochondria in glucocorticoid and catecholamine metabolism, and the effects of stress hormones on energy substrate distribution within the body. We also consider the reciprocal action of steroid hormones on mitochondria. The influence of stress on food/energy-seeking behavior and emerging evidence regarding the influence of mitochondrial functions on mental health, social behaviors, and physiological stress reactivity are also discussed. An energetic view of stress adaptation and pathophysiology opens new possibilities to rationalize and investigate the integrated adaptive and maladaptive effects of stress on the brain and body

The main function of mitochondria is the production of ATP in the process of oxidative phosphorylation. The main byproduct that is formed in the course of electron transport in the respiratory chain is superoxide anion radical. Superoxide emerges as a result of one-electron reduction of molecular oxygen in various parts of the respiratory chain, including Complex I (NADH: ubiquinone oxidoreductase), Complex II (succinate dehydrogenase), Complex III (ubiquinol: cytochrome oxidoreductase), and mitochondrial matrix dehydrogenases [Grivennikova and Vinogradov, 2013]. Subsequently, the superoxide gives rise to various reactive oxygen species (ROS). Normally, only 1-2% of the oxygen consumed by the cells are used for ROS formation in mitochondria (mtROS) [Turrens, 2003, Inoue et al., 2003]. Until recently, it was believed that mtROS predominantly play a destructive role causing cell damage and accelerating aging [Harman, 2003]. These views are exemplified by the ideas that are expressed in the work of Huang and co-workers [1997]. It was established that mice with a knockout of cytosolic CuZn-dependent superoxide dismutase gene are viable, while mice with a nonfunctional mitochondrial Mn-dependent superoxide dismutase gene do not survive. Recently, it has been established that mtROS formation is mandatory for the functioning of virtually all organs and tissues, being involved in numerous metabolic pathways and in the regulation of signaling processes [Dan Dunn et al., 2015]. Importantly, mtROS are essential for the functioning of the immune system. However, their influence may be either positive or negative, depending on the quantity of the mtROS involved. From the immunological viewpoint, the positive effects of mtROS are primarily based on their activating influence on antimicrobial immunity, while their negative effects, caused by their excessive production, result in the induction of autoinflammatory [van der Burgh and Boes, 2015] and autoimmune processes [Lee et al., 2016; Lood et al., 2016]. The formation of mtROS plays an important role in the development of the antiviral immunity including the production of proinflammatory cytokines by immune system cells [Kim et al., 2015]. This review is concerned with the analysis of the mtROS role in terms of antibacterial innate immunity.

The negative impact of obesity on reproductive success is well documented but the stages at which development of the conceptus is compromised and the mechanisms responsible for the developmental failure still remain unclear. Recent findings suggest that mitochondria may be a contributing factor. However to date no studies have directly addressed the consequences of maternal obesity on mitochondria in early embryogenesis. Using an established murine model of maternal diet induced obesity and a live cell dynamic fluorescence imaging techniques coupled with molecular biology we have investigated the underlying mechanisms of obesity-induced reduced fertility. Our study is the first to show that maternal obesity prior to conception is associated with altered mitochondria in mouse oocytes and zygotes. Specifically, maternal diet-induced obesity in mice led to an increase in mitochondrial potential, mitochondrial DNA content and biogenesis. Generation of reactive oxygen species (ROS) was raised while glutathione was depleted and the redox state became more oxidised, suggestive of oxidative stress. These altered mitochondrial properties were associated with significant developmental impairment as shown by the increased number of obese mothers who failed to support blastocyst formation compared to lean dams. We propose that compromised oocyte and early embryo mitochondrial metabolism, resulting from excessive nutrient exposure prior to and during conception, may underlie poor reproductive outcomes frequently reported in obese women.

ntroduction

Obesity and related metabolic disorders are a major health issue worldwide. With increasing prevalence in all populations and age groups, the proportion of women of reproductive age who are obese is rising [1]. Evidence is growing that excessive body fat has a detrimental effect on female fertility and pregnancy [2]. Obese women take longer to conceive and have a higher risk of miscarriage compared to lean women [3].Obesity also impairs the immediate outcome of assisted reproductive technologies suggesting that maternal body mass index (BMI) may influence the potential for fertilisation and viability of early embryos [4]. Since the earliest stages of embryo development are primarily controlled by the oocyte, it is likely that a sub-optimal environment within the ovary and/or oviduct accounts for these poor reproductive outcomes.

As recently reviewed [5], understanding of the effects of maternal obesity on the structure and metabolism in oocytes and pre-implantation embryos is very limited. It has been proposed that a high plane of nutrition might lead to excessive enrichment of the reproductive milieu [6]. This in turn may induce alterations in oocyte metabolism and impede embryonic development. Indeed several studies have shown that abnormally high or low rates of metabolism may compromise oocyte and embryo development [7,8]. Mitochondria are likely candidates for compromised metabolism in the embryo; these organelles are exclusively maternal in origin, and thus a deleterious influence of maternal BMI on mitochondria in the oocyte would strongly influence embryonic metabolism. Mitochondria also perform numerous regulatory functions during oocyte maturation [9], fertilization, initiation and progression of preimplantation embryos [10]. As energy producer, the central and most important function of mitochondria is the synthesis of adenosine triphosphate (ATP) by oxidative phosphorylation, a mechanism coupling the oxidation of nutrients and reducing equivalents (NAD(P)H, FADH2) with the phosphorylation of adenosine diphosphate. Both cytosolic and mitochondrial sources of NAD(P)H along with mitochondrial FADH2 stimulate the mitochondrial electron transport chain to pump H out of the mitochondrial matrix thereby hyperpolarising the inner mitochondrial membrane and generating the protonmotive force used to generate ATP. Electron donors NAD(P)H and FADH2 besides being used for energy production set the intracellular redox state. NADH oxidation in the mitochondria will produce ROS whereas NADPH oxidation (in the cytosol and mitochondria) serves to rejuvenate the antioxidant defence by reducing peroxiredoxins, thioredoxin and oxidised glutathione.