The clinical presentation and genotype of protein C deficiency with double mutations of the protein C gene

BackgroundSevere protein C (PC) deficiency is a rare heritable thrombophilia leading to thromboembolic events during the neonatal period. It remains unclear how individuals with complete PC gene (PROC) defects develop or escape neonatal stroke or purpura fulminans (PF).ProcedureWe studied the onset of disease and the genotype of 22 PCâ deficient patients with double mutations in PROC based on our cohort (n = 12) and the previous reports (n = 10) in Japan.ResultsTwentyâ two patients in 20 unrelated families had 4 homozygous and 18 compound heterozygous mutations. Sixteen newborns presented with PF (n = 11, 69%), intracranial thromboembolism and hemorrhage (n = 13, 81%), or both (n = 8, 50%), with most showing a plasma PC activity of <10%. Six others first developed overt thromboembolism when they were over 15 years of age, showing a median PC activity of 31% (range: 19â 52%). Fifteen of the 22 patients (68%) had the five major mutations (G423VfsX82, V339M, R211W, M406I, and F181V) or two others (E68K and K193del) that have been reported in Japan. Three of the six lateâ onset cases, but none of the 16 neonatal cases, had the K193del mutation, which has been reported to be the most common variant of Chinese thrombophilia. A novel mutation of A309V was determined in a family of two patients with late onset.ConclusionsThe genotype of doubleâ PROC mutants might show less diversity than heterozygous mutants in terms of the timing of the onset of thrombophilia (newborn onset or late onset).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137364/1/pbc26404_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137364/2/pbc26404.pd

Crystal structure of the anion exchanger domain of human erythrocyte band 3

Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients

Synopsis Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype

Overexpression of TFAM or Twinkle Increases mtDNA Copy Number and Facilitates Cardioprotection Associated with Limited Mitochondrial Oxidative Stress

Background Mitochondrial DNA (mtDNA) copy number decreases in animal and human heart failure (HF), yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM) or Twinkle helicase in volume overload (VO)-induced HF. Methods and Results Two strains of transgenic (TG) mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT) mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP) activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS) upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM) overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice. Conclusions The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF.Peer reviewe

ERAL1 is associated with mitochondrial ribosome and elimination of ERAL1 leads to mitochondrial dysfunction and growth retardation

ERAL1, a homologue of Era protein in Escherichia coli, is a member of conserved GTP-binding proteins with RNA-binding activity. Depletion of prokaryotic Era inhibits cell division without affecting chromosome segregation. Previously, we isolated ERAL1 protein as one of proteins which were associated with mitochondrial transcription factor A by using immunoprecipitation. In this study, we analysed the localization and function of ERAL1 in mammalian cells. ERAL1 was localized in mitochondrial matrix and associated with mitoribosomal proteins including the 12S rRNA. siRNA knockdown of ERAL1 decreased mitochondrial translation, caused redistribution of ribosomal small subunits and reduced 12S rRNA. The knockdown of ERAL1 in human HeLa cells elevated mitochondrial superoxide production and slightly decreased mitochondrial membrane potential. The knockdown inhibited the growth of HeLa cells with an accumulation of apoptotic cells. These results suggest that ERAL1 is localized in a small subunit of the mitochondrial ribosome, plays an important role in the small ribosomal constitution, and is also involved in cell viability

Suicide and Microglia: Recent Findings and Future Perspectives Based on Human Studies

Suicide is one of the most disastrous outcomes for psychiatric disorders. Recent advances in biological psychiatry have suggested a positive relationship between some specific brain abnormalities and specific symptoms in psychiatric disorders whose organic bases were previously completely unknown. Microglia, immune cells in the brain, are regarded to play crucial roles in brain inflammation by releasing inflammatory mediators and are suggested to contribute to various psychiatric disorders such as depression and schizophrenia. Recently, activated microglia have been suggested to be one of the possible contributing cells to suicide and suicidal behaviors via various mechanisms especially including the tryptophan-kynurenine pathway. Animal model research focusing on psychiatric disorders has a long history, however, there are only limited animal models that can properly express psychiatric symptoms. In particular, to our knowledge, animal models of human suicidal behaviors have not been established. Suicide is believed to be limited to humans, therefore human subjects should be the targets of research despite various ethical and technical limitations. From this perspective, we introduce human biological studies focusing on suicide and microglia. We first present neuropathological studies using the human postmortem brain of suicide victims. Second, we show recent findings based on positron emission tomography (PET) imaging and peripheral blood biomarker analysis on living subjects with suicidal ideation and/or suicide-related behaviors especially focusing on the tryptophan-kynurenine pathway. Finally, we propose future perspectives and tasks to clarify the role of microglia in suicide using multi-dimensional analytical methods focusing on human subjects with suicidal ideation, suicide-related behaviors and suicide victims

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals’ samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp−/− mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp−/− MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia

Maintenance of Mitochondrial DNA in Somatic Cell : Diseases and Aging

ヒトのミトコンドリアゲノムは13の電子伝達系サブユニットと22のtRNAと2つのrRNAをコードしており, 細胞あたり数十から数千コピー存在している. コードしている遺伝子の数は限られているが, 全ての遺伝子はミトコンドリアにおける酸化的リン酸化による好気的ATP合成に必須である. ミトコンドリアは細胞におけるATP合成の80％以上を占めていることから, ミトコンドリアゲノム異常は細胞の生存と機能に重大な支障を来す. 培養細胞系ではミトコンドリアDNAを失ったいわゆるrho0細胞も特殊な環境下で生存可能であるが, 個体としては, ミトコンドリアDNAの維持は種々の機能を正常に保ち生存していくために必須である. 実際これまで, ミトコンドリアゲノムの変異に起因する先天的なミトコンドリア脳筋症が数多く報告されており, 新たな変異の報告は現在もさらに増えつづけている. ミトコンドリアゲノムは細胞あたり複数コピーで存在し, そのコピー数は細胞のエネルギー需要とおおよそ平行している. つまり細胞が要求するレベルのミトコンドリア機能を保持するのには, 一定のコピー数のミトコンドリアゲノムが維持されることが必要である. このため, ミトコンドリアゲノムの維持という場合, 遺伝情報そのものの維持に加え, 遺伝情報量の維持も考える必要がある. 最近は, 心不全, がん, 糖尿病などのより一般的な疾患においてミトコンドリアゲノム異常関与が解明され, 先天性のミトコンドリアゲノム異常に加え, 加齢に伴う体細胞性のミトコンドリアゲノムの変異とその維持の重要性が認められつつある

ミトコンドリアの機能維持と疾患

Mitochondria are responsible for about 90% of ATP synthesis in most aerobic cells, which inevitably accompanies production of huge reactive oxygen species. Therefore, mitochondrial genome (mtDNA) is under far more oxidative stress than nuclear genome. Resultantly, mtDNA as well as the organelle itself suffers higher damage over age. As mitochondria are a central hub for many cellular metabolisms including sugars, lipids, proteins, and nucleic acids in addition to energy production, the damage of mitochondria seriously affects the cellular overall processes leading to various common diseases. We have found Mitochondrial transcription factor A (TFAM) is a main component forming mtDNA higher structure, so called nucleoid. We also have shown TFAM is essential for its stability in mitochondrial matrix, in other words, protects mtDNA in vivo. Accordingly, overexpression of TFAM increases mtDNA and has beneficial effects for many pathologic situations such as heart failure, aging, neurodegenerative disease, diabetes, and so on. We found p32 protein among the TFAM-binding proteins. p32 is already reported by us to be in mitochondrial matrix and critical for oxidative phosphorylation. Through several p32 conditional knockout mice, we have shown p32 plays critical roles in efficient mitochondrial translation, regulation of IL6 production, antigen presentation, and differentiation to erythrocyte and B lymphocyte