Sirt3 Regulates Proliferation and Progesterone Production in Leydig Cells via Suppression of Reactive Oxygen Species.

Sirt3 is a mitochondrial protein deacetylase functioning in energy metabolism, regulation of intracellular reactive oxygen species (ROS) levels, and aging. Although Sirt3 loss has negative effects on fertility of oocytes during in vitro fertilization and on progesterone production in granulosa cells, Sirt3's function in Leydig cells remains unclear. Therefore, we investigated Sirt3 activity in Leydig cells, focusing on androgen production. To do so, we performed immunohistochemistry to confirm Sirt3 localization in gonads and observed strong Sirt3 immunostaining in Leydig cells of human testes and of Sirt3+/+ and Sirt3+/- mouse testes, while Sirt3-/- mouse testis tissue was negative. In human ovary, hilus cells were strongly Sirt3-positive, theca cells showed weak positivity, and granulosa cells showed very weak or almost no immunostaining. Next, we used the murine Leydig tumor cell line MA-10 as a model. We overexpressed Sirt3 but observed no changes in proliferation, expression of Star, Cyp11a1 (p450scc gene), and Hsd3b, or progesterone production in MA-10 cells. Sirt3 knockdown significantly reduced proliferation, suppressed expressions of steroidogenic enzymes and of transcription factors Ad4bp (Sf-1 gene) and Gata4, and decreased progesterone production. Sirt3 knockdown in MA-10 cells also increased intracellular ROS levels based on CM-H2DCFDA fluorescence dye analysis and increased the proportion of both early and late apoptotic (necrotic) cells based on Annexin V/7AAD assays. These results indicate that Sirt3 has a potential function in androgen production in Leydig cells by regulating intracellular ROS levels.

A three-dimensional model with two-body interactions for endothelial cells in angiogenesis.

We introduce a three-dimensional mathematical model for the dynamics of vascular endothelial cells during sprouting angiogenesis. Angiogenesis is the biological process by which new blood vessels form from existing ones. It has been the subject of numerous theoretical models. These models have successfully replicated various aspects of angiogenesis. Recent studies using particle-based models have highlighted the significant influence of cell shape on network formation, with elongated cells contributing to the formation of branching structures. While most mathematical models are two-dimensional, we aim to investigate whether ellipsoids also form branch-like structures and how their shape affects the pattern. In our model, the shape of a vascular endothelial cell is represented as a spheroid, and a discrete dynamical system is constructed based on the simple assumption of two-body interactions. Numerical simulations demonstrate that our model reproduces the patterns of elongation and branching observed in the early stages of angiogenesis. We show that the pattern formation of the cell population is strongly dependent on the cell shape. Finally, we demonstrate that our current mathematical model reproduces the cell behaviours, specifically cell-mixing, observed in sprouts.

Hey2 enhancer activity defines unipotent progenitors for left ventricular cardiomyocytes in juxta-cardiac field of early mouse embryo.

The cardiac crescent is the first structure of the heart and contains progenitor cells of the first heart field, which primarily differentiate into left ventricular cardiomyocytes. The interface between the forming cardiac crescent and extraembryonic tissue is known as the juxta-cardiac field (JCF), and progenitor cells in this heart field contribute to the myocardium of the left ventricle and atrioventricular canal as well as the epicardium. However, it is unclear whether there are progenitor cells that differentiate specifically into left ventricular cardiomyocytes. We have previously demonstrated that an enhancer of the gene encoding the Hey2 bHLH transcriptional repressor is activated in the ventricular myocardium during mouse embryonic development. In this study, we aimed to investigate the characteristics of cardiomyocyte progenitor cells and their cell lineages by analyzing Hey2 enhancer activity at the earliest stages of heart formation. We found that the Hey2 enhancer initiated its activity prior to cardiomyocyte differentiation within the JCF. Hey2 enhancer-active cells were present rostrally to the Tbx5-expressing region at the early phase of cardiac crescent formation and differentiated exclusively into left ventricular cardiomyocytes in a lineage distinct from the Tbx5-positive lineage. By the late phase of cardiac crescent formation, Hey2 enhancer activity became significantly overlapped with Tbx5 expression in cells that contribute to the left ventricular myocardium. Our study reveals that a population of unipotent progenitor cells for left ventricular cardiomyocytes emerge in the JCF, providing further insight into the mode of cell type diversification during early cardiac development.

Notch and retinoic acid signals regulate macrophage formation from endocardium downstream of Nkx2-5.

Hematopoietic progenitors are enriched in the endocardial cushion and contribute, in a Nkx2-5-dependent manner, to tissue macrophages required for the remodeling of cardiac valves and septa. However, little is known about the molecular mechanism of endocardial-hematopoietic transition. In the current study, we identified the regulatory network of endocardial hematopoiesis. Signal network analysis from scRNA-seq datasets revealed that genes in Notch and retinoic acid (RA) signaling are significantly downregulated in Nkx2-5-null endocardial cells. In vivo and ex vivo analyses validate that the Nkx2-5-Notch axis is essential for the generation of both hemogenic and cushion endocardial cells, and the suppression of RA signaling via Dhrs3 expression plays important roles in further differentiation into macrophages. Genetic ablation study revealed that these macrophages are essential in cardiac valve remodeling. In summary, the study demonstrates that the Nkx2-5/Notch/RA signaling plays a pivotal role in macrophage differentiation from hematopoietic progenitors.

Biallelic truncating variants in VGLL2 cause syngnathia in humans.

BACKGROUND: Syngnathia is an ultrarare craniofacial malformation characterised by an inability to open the mouth due to congenital fusion of the upper and lower jaws. The genetic causes of isolated bony syngnathia are unknown. METHODS: We used whole exome and Sanger sequencing and microsatellite analysis in six patients (from four families) presenting with syngnathia. We used CRISPR/Cas9 genome editing to generate vgll2a and vgll4l germline mutant zebrafish, and performed craniofacial cartilage analysis in homozygous mutants. RESULTS: We identified homozygous truncating variants in vestigial-like family member 2 (VGLL2) in all six patients. Two alleles were identified: one in families of Turkish origin and the other in families of Moroccan origin, suggesting a founder effect for each. A shared haplotype was confirmed for the Turkish patients. The VGLL family of genes encode cofactors of TEAD transcriptional regulators. Vgll2 is regionally expressed in the pharyngeal arches of model vertebrate embryos, and morpholino-based knockdown of vgll2a in zebrafish has been reported to cause defects in development of pharyngeal arch cartilages. However, we did not observe craniofacial anomalies in vgll2a or vgll4l homozygous mutant zebrafish nor in fish with double knockout of vgll2a and vgll4l. In Vgll2 -/- mice, which are known to present a skeletal muscle phenotype, we did not identify defects of the craniofacial skeleton. CONCLUSION: Our results suggest that although loss of VGLL2 leads to a striking jaw phenotype in humans, other vertebrates may have the capacity to compensate for its absence during craniofacial development.

Coronary artery established through amniote evolution.

Coronary arteries are a critical part of the vascular system and provide nourishment to the heart. In humans, even minor defects in coronary arteries can be lethal, emphasizing their importance for survival. However, some teleosts survive without coronary arteries, suggesting that there may have been some evolutionary changes in the morphology and function of coronary arteries in the tetrapod lineage. Here, we propose that the true ventricular coronary arteries were newly established during amniote evolution through remodeling of the ancestral coronary vasculature. In mouse (Mus musculus) and Japanese quail (Coturnix japonica) embryos, the coronary arteries unique to amniotes are established by the reconstitution of transient vascular plexuses: aortic subepicardial vessels (ASVs) in the outflow tract and the primitive coronary plexus on the ventricle. In contrast, amphibians (Hyla japonica, Lithobates catesbeianus, Xenopus laevis, and Cynops pyrrhogaster) retain the ASV-like vasculature as truncal coronary arteries throughout their lives and have no primitive coronary plexus. The anatomy and development of zebrafish (Danio rerio) and chondrichthyans suggest that their hypobranchial arteries are ASV-like structures serving as the root of the coronary vasculature throughout their lives. Thus, the ventricular coronary artery of adult amniotes is a novel structure that has acquired a new remodeling process, while the ASVs, which occur transiently during embryonic development, are remnants of the ancestral coronary vessels. This evolutionary change may be related to the modification of branchial arteries, indicating considerable morphological changes underlying the physiological transition during amniote evolution.

Coronary arteries are tasked with supplying the heart with oxygenated blood and nutrients. Any blockage or developmental problem in these blood vessels can have severe and sometimes lethal consequences. Due to their importance for health, researchers have extensively studied how coronary arteries form in humans and mice; a more limited range of studies have also looked at their equivalent in zebrafish. However, little is known about these structures develop in animals such as birds, amphibians, or other groups of fish. This makes it difficult to retrace the evolutionary processes that have given rise to the coronary arteries we are familiar with in mammals. To address this knowledge gap, Mizukami et al. set out to compare blood vessel development around the heart of mammals, birds, amphibians, and fish. To do this, they performed detailed anatomical studies of blood vessel structure at different stages of development in mice as well as quail, frogs and newts, zebrafish and sharks. In both mice and quail, small arterial subepicardial vessels (or ASVs) emerged early in development around the heart; these subsequently reorganised and remodelled themselves to give rise to the 'true' coronary arteries characteristic of the mature heart. Frogs and newts also developed similar ASV-like structures; however, unlike their mammalian and bird equivalents, these vessels did not reorganise, instead being retained into adulthood. In fish, blood vessel development resembled that of amphibians, suggesting that the coronary artery-like structures seen in some fish are an 'ancestral' form of ASVs, rather than the equivalent of the mature coronary arteries in mammals and birds. This work sheds light on the evolutionary processes shaping essential structures in the heart. In the future, Mizukami et al. hope that this knowledge will help develop a greater range of experimental animal models for studying heart disease and potential treatments.

Persistent homological cell tracking technology.

In this paper, we develop a cell tracking method based on persistent homological figure detection technology. We apply our tracking method to 9 different time-series cell images and extract several kinds of cell movements. Being able to analyze various images with a single method allows researchers to systematically understand and compare different tracking data. Persistent homological cell tracking technology's 9 parameters all have clear meanings. Thus, researchers can decide the parameters not by black box trial-and-error but by the purpose of their analysis. We use model data with ground truth to see our method's performance. We compare persistent homological figure detection and cell tracking technology with Image-Pro, sure-foreground in watershed method, and cell detection methods in previous studies. We see that there are some cases where Image-Pro's tracking stops and requires manual plots, while our method does not require manual plots. We show that our technology includes sure-foreground and has more information, and can be applied to different types of data that previously needed different methods. We also show that our technology is powerful as a detection technology by applying the technology to 5 different types of cell images.

Coordinated linear and rotational movements of endothelial cells compartmentalized by VE-cadherin drive angiogenic sprouting.

Angiogenesis is a sequential process to extend new blood vessels from preexisting ones by sprouting and branching. During angiogenesis, endothelial cells (ECs) exhibit inhomogeneous multicellular behaviors referred to as "cell mixing," in which ECs repetitively exchange their relative positions, but the underlying mechanism remains elusive. Here we identified the coordinated linear and rotational movements potentiated by cell-cell contact as drivers of sprouting angiogenesis using in vitro and in silico approaches. VE-cadherin confers the coordinated linear motility that facilitated forward sprout elongation, although it is dispensable for rotational movement, which was synchronous without VE-cadherin. Mathematical modeling recapitulated the EC motility in the two-cell state and angiogenic morphogenesis with the effects of VE-cadherin-knockout. Finally, we found that VE-cadherin-dependent EC compartmentalization potentiated branch elongations, and confirmed this by mathematical simulation. Collectively, we propose a way to understand angiogenesis, based on unique EC behavioral properties that are partially dependent on VE-cadherin function.

Increased PDGFRB and NF-κB signaling caused by highly prevalent somatic mutations in intracranial aneurysms.

Intracranial aneurysms (IAs) are a high-risk factor for life-threatening subarachnoid hemorrhage. Their etiology, however, remains mostly unknown at present. We conducted screening for sporadic somatic mutations in 65 IA tissues (54 saccular and 11 fusiform aneurysms) and paired blood samples by whole-exome and targeted deep sequencing. We identified sporadic mutations in multiple signaling genes and examined their impact on downstream signaling pathways and gene expression in vitro and an arterial dilatation model in mice in vivo. We identified 16 genes that were mutated in at least one IA case and found that these mutations were highly prevalent (92%: 60 of 65 IAs) among all IA cases examined. In particular, mutations in six genes (PDGFRB, AHNAK, OBSCN, RBM10, CACNA1E, and OR5P3), many of which are linked to NF-κB signaling, were found in both fusiform and saccular IAs at a high prevalence (43% of all IA cases examined). We found that mutant PDGFRBs constitutively activated ERK and NF-κB signaling, enhanced cell motility, and induced inflammation-related gene expression in vitro. Spatial transcriptomics also detected similar changes in vessels from patients with IA. Furthermore, virus-mediated overexpression of a mutant PDGFRB induced a fusiform-like dilatation of the basilar artery in mice, which was blocked by systemic administration of the tyrosine kinase inhibitor sunitinib. Collectively, this study reveals a high prevalence of somatic mutations in NF-κB signaling pathway-related genes in both fusiform and saccular IAs and opens a new avenue of research for developing pharmacological interventions.

Mandibulofacial dysostosis with alopecia results from ETAR gain-of-function mutations via allosteric effects on ligand binding.

Mutations of G protein-coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here, we establish p.E303K in the gene encoding the endothelin receptor type A (ETAR/EDNRA) as a recurrent mutation causing mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibited a partial maxillary-to-mandibular transformation, which was rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ETAR mutations as gain of function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increased flexibility of this portion of the helix, thus favoring G protein binding and leading to G protein-mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduced the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, which may provide the basis for drug design targeting GPCRs.

Evolution of the therian face through complete loss of the premaxilla.

The anatomical framework of the jawbones is highly conserved among most of the Osteichthyes, including the tetrapods. However, our recent study suggested that the premaxilla, the rostralmost upper jaw bone, was rearranged during the evolution of therian mammals, being replaced by the septomaxilla at least in the lateral part. In the present study, to understand more about the process of evolution from the ancestral upper jaw to the therian face, we re-examined the development of the therian premaxilla (incisive bone). By comparing mouse, bat, goat, and cattle fetuses, we confirmed that the therian premaxilla has dual developmental origins, the lateral body and the palatine process. This dual development is widely conserved among the therian mammals. Cell-lineage-tracing experiments using Dlx1-CreERT2 mice revealed that the palatine process arises in the ventral part of the premandibular domain, where the nasopalatine nerve distributes, whereas the lateral body develops from the maxillary prominence in the domain of the maxillary nerve. Through comparative analysis using various tetrapods, we concluded that the palatine process should not be considered part of the ancestral premaxilla. It rather corresponds to the anterior region of the vomerine bone of nonmammalian tetrapods. Thus, the present findings indicate that the true premaxilla was completely lost during the evolution of the therian mammals, resulting in the establishment of the unique therian face as an evolutionary novelty. Reconsideration of the homological framework of the cranial skeleton based on the topographical relationships of the ossification center during embryonic development is warranted.

Pattern formation of elliptic particles by two-body interactions: A model for dynamics of endothelial cells in angiogenesis.

A two-dimensional mathematical model for dynamics of endothelial cells in angiogenesis is investigated. Angiogenesis is a morphogenic process in which new blood vessels emerge from an existing vascular network. Recently a one-dimensional discrete dynamical model has been proposed to reproduce elongation, bifurcation, and cell motility such as cell-mixing during angiogenesis on the assumption of a simple two-body interaction between endothelial cells. The present model is its two-dimensional extension, where endothelial cells are represented as the ellipses with the two-body interactions: repulsive interaction due to excluded volume effect, attractive interaction through pseudopodia and rotation by contact. We show that the oblateness of ellipses and the magnitude of contact rotation significantly affect the shape of created vascular patterns and elongation of branches.

The cardiopharyngeal mesoderm contributes to lymphatic vessel development in mouse.

Lymphatic vessels are crucial for tissue homeostasis and immune responses in vertebrates. Recent studies have demonstrated that lymphatic endothelial cells (LECs) arise from both venous sprouting (lymphangiogenesis) and de novo production from non-venous origins (lymphvasculogenesis), which is similar to blood vessel formation through angiogenesis and vasculogenesis. However, the contribution of LECs from non-venous origins to lymphatic networks is considered to be relatively small. Here, we identify the Islet1 (Isl1)-expressing cardiopharyngeal mesoderm (CPM) as a non-venous origin of craniofacial and cardiac LECs. Genetic lineage tracing with Isl1Cre/+ and Isl1CreERT2/+ mice suggested that a subset of CPM cells gives rise to LECs. These CPM-derived LECs are distinct from venous-derived LECs in terms of their developmental processes and anatomical locations. Later, they form the craniofacial and cardiac lymphatic vascular networks in collaboration with venous-derived LECs. Collectively, our results demonstrate that there are two major sources of LECs, the cardinal vein and the CPM. As the CPM is evolutionarily conserved, these findings may improve our understanding of the evolution of lymphatic vessel development across species. Most importantly, our findings may provide clues to the pathogenesis of lymphatic malformations, which most often develop in the craniofacial and mediastinal regions.

Diverse contribution of amniogenic somatopleural cells to cardiovascular development: With special reference to thyroid vasculature.

BACKGROUND: The somatopleure serves as the primordium of the amnion, an extraembryonic membrane surrounding the embryo. Recently, we have reported that amniogenic somatopleural cells (ASCs) not only form the amnion but also migrate into the embryo and differentiate into cardiomyocytes and vascular endothelial cells. However, detailed differentiation processes and final distributions of these intra-embryonic ASCs (hereafter referred to as iASCs) remain largely unknown. RESULTS: By quail-chick chimera analysis, we here show that iASCs differentiate into various cell types including cardiomyocytes, smooth muscle cells, cardiac interstitial cells, and vascular endothelial cells. In the pharyngeal region, they distribute selectively into the thyroid gland and differentiate into vascular endothelial cells to form intra-thyroid vasculature. Explant culture experiments indicated sequential requirement of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling for endothelial differentiation of iASCs. Single-cell transcriptome analysis further revealed heterogeneity and the presence of hemangioblast-like cell population within ASCs, with a switch from FGF to VEGF receptor gene expression. CONCLUSION: The present study demonstrates novel roles of ASCss especially in heart and thyroid development. It will provide a novel clue for understanding the cardiovascular development of amniotes from embryological and evolutionary perspectives.

Dlx5/6 Expression Levels in Mouse GABAergic Neurons Regulate Adult Parvalbumin Neuronal Density and Anxiety/Compulsive Behaviours.

Neuronal circuits integrating Parvalbumin-positive GABAergic inhibitory interneurons (PV) are essential for normal brain function and are often altered in psychiatric conditions. During development, Dlx5 and Dlx6 (Dlx5/6) genes are involved in the differentiation of PV-interneurons. In the adult, Dlx5/6 continue to be expressed at low levels in most telencephalic GABAergic neurons, but their importance in determining the number and distribution of adult PV-interneurons is unknown. Previously, we have shown that targeted deletion of Dlx5/6 in mouse GABAergic neurons (Dlx5/6VgatCre mice) results in altered behavioural and metabolic profiles. Here we evaluate the consequences of targeted Dlx5/6 gene dosage alterations in adult GABAergic neurons. We compare the effects on normal brain of homozygous and heterozygous (Dlx5/6VgatCre and Dlx5/6VgatCre/+ mice) Dlx5/6 deletions to those of Dlx5 targeted overexpression (GABAergicDlx5/+ mice). We find a linear correlation between Dlx5/6 allelic dosage and the density of PV-positive neurons in the adult prelimbic cortex and in the hippocampus. In parallel, we observe that Dlx5/6 expression levels in GABAergic neurons are also linearly associated with the intensity of anxiety and compulsivity-like behaviours. Our findings reinforce the notion that regulation of Dlx5/6 expression is involved in individual cognitive variability and, possibly, in the genesis of certain neuropsychiatric conditions.

Mammalian face as an evolutionary novelty.

The anterior end of the mammalian face is characteristically composed of a semimotile nose, not the upper jaw as in other tetrapods. Thus, the therian nose is covered ventrolaterally by the "premaxilla," and the osteocranium possesses only a single nasal aperture because of the absence of medial bony elements. This stands in contrast to those in other tetrapods in whom the premaxilla covers the rostral terminus of the snout, providing a key to understanding the evolution of the mammalian face. Here, we show that the premaxilla in therian mammals (placentals and marsupials) is not entirely homologous to those in other amniotes; the therian premaxilla is a composite of the septomaxilla and the palatine remnant of the premaxilla of nontherian amniotes (including monotremes). By comparing topographical relationships of craniofacial primordia and nerve supplies in various tetrapod embryos, we found that the therian premaxilla is predominantly of the maxillary prominence origin and associated with mandibular arch. The rostral-most part of the upper jaw in nonmammalian tetrapods corresponds to the motile nose in therian mammals. During development, experimental inhibition of primordial growth demonstrated that the entire mammalian upper jaw mostly originates from the maxillary prominence, unlike other amniotes. Consistently, cell lineage tracing in transgenic mice revealed a mammalian-specific rostral growth of the maxillary prominence. We conclude that the mammalian-specific face, the muzzle, is an evolutionary novelty obtained by overriding ancestral developmental constraints to establish a novel topographical framework in craniofacial mesenchyme.

Surgical protocol for permanent ligation of the left anterior descending coronary artery in mice to generate a model of myocardial infarction.

Myocardial infarction (MI) is one of the most common causes of death worldwide. Animal models for MI are useful for studying the pathophysiology and developing therapies. Here, we describe a surgical protocol for permanent ligation of the left anterior descending coronary artery in mice, which mimics human acute coronary syndrome. This protocol includes descriptive step-by-step surgical procedures and high-quality surgical videos, which are useful for performing stable and highly reproducible operations. For complete details on the use and execution of this protocol, please refer to Maruyama et al. (2021).

Semaphorin3E-PlexinD1 signaling in coronary artery and lymphatic vessel development with clinical implications in myocardial recovery.

Blood and lymphatic vessels surrounding the heart develop through orchestrated processes from cells of different origins. In particular, cells around the outflow tract which constitute a primordial transient vasculature, referred to as aortic subepicardial vessels, are crucial for the establishment of coronary artery stems and cardiac lymphatic vessels. Here, we revealed that the epicardium and pericardium-derived Semaphorin 3E (Sema3E) and its receptor, PlexinD1, play a role in the development of the coronary stem, as well as cardiac lymphatic vessels. In vitro analyses demonstrated that Sema3E may demarcate areas to repel PlexinD1-expressing lymphatic endothelial cells, resulting in proper coronary and lymphatic vessel formation. Furthermore, inactivation of Sema3E-PlexinD1 signaling improved the recovery of cardiac function by increasing reactive lymphangiogenesis in an adult mouse model of myocardial infarction. These findings may lead to therapeutic strategies that target Sema3E-PlexinD1 signaling in coronary artery diseases.

Murine neonatal ketogenesis preserves mitochondrial energetics by preventing protein hyperacetylation.

Ketone bodies are generated in the liver and allow for the maintenance of systemic caloric and energy homeostasis during fasting and caloric restriction. It has previously been demonstrated that neonatal ketogenesis is activated independently of starvation. However, the role of ketogenesis during the perinatal period remains unclear. Here, we show that neonatal ketogenesis plays a protective role in mitochondrial function. We generated a mouse model of insufficient ketogenesis by disrupting the rate-limiting hydroxymethylglutaryl-CoA synthase 2 enzyme gene (Hmgcs2). Hmgcs2 knockout (KO) neonates develop microvesicular steatosis within a few days of birth. Electron microscopic analysis and metabolite profiling indicate a restricted energy production capacity and accumulation of acetyl-CoA in Hmgcs2 KO mice. Furthermore, acetylome analysis of Hmgcs2 KO cells revealed enhanced acetylation of mitochondrial proteins. These findings suggest that neonatal ketogenesis protects the energy-producing capacity of mitochondria by preventing the hyperacetylation of mitochondrial proteins.