Glycogen synthase kinase‐3β inhibition decreases inflammation and relieves cancer induced bone pain via reducing Drp1‐mediated mitochondrial damage

Bone is the preferential site of metastasis for breast cancer. Invasion of cancer cells induces the destruction of bone tissue and damnification of peripheral nerves and consequently induced central sensitization which contributes to severe pain. Herein, cancer induced bone pain (CIBP) rats exhibited destruction of tibia, mechanical allodynia and spinal inflammation. Inflammatory response mainly mediated by astrocyte and microglia in central nervous system. Our immunofluorescence analysis revealed activation of spinal astrocytes and microglia in CIBP rats. Transmission electron microscopy (TEM) observations of mitochondrial outer membrane disruption and cristae damage in spinal mitochondria of CIBP rats. Proteomics analysis identified abnormal expression of proteins related to mitochondrial organization and function. Intrathecally, injection of GSK‐3β activity inhibitor TDZD‐8 significantly attenuated Drp1‐mediated mitochondrial fission and recovered mitochondrial function. Inhibition of GSK‐3β activity also suppressed NLRP3 inflammasome cascade and consequently decreased mechanical pain sensitivity of CIBP rats. For cell research, TDZD‐8 treatment significantly reversed TNF‐α induced mitochondrial membrane potential (MMP) deficiency and high mitochondrial reactive oxygen species level. Taken together, GSK‐3β inhibition by TDZD‐8 decreases spinal inflammation and relieves cancer induced bone pain via reducing Drp1‐mediated mitochondrial damage.     

Defective dimerization of FoF1‐ATP synthase secondary to glycation favors mitochondrial energy deficiency in cardiomyocytes during aging

Aged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1‐ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy‐dissipating channel involved in cell death. We investigated whether aging alters FoF1‐ATP synthase self‐assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1‐ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1‐ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes’ susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1‐ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1‐ATP synthase glycation in H9c2 myoblasts recapitulated the age‐related defective FoF1‐ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1‐ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.     

DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK^– osteosarcoma cells that had been depleted of mtDNA (ρ⁰) or not (wt). Despite the total absence of mtDNA in the ρ⁰ cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in ρ⁰ relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase γ activities were more affected, 65 and 45% lower, respectively, in ρ⁰ mitochondria. Overall BER activity in lysates was also about 65% reduced in ρ⁰ mitochondria. To identify the limiting deficiencies in BER of ρ⁰ mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase γ. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase γ. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in ρ⁰ mitochondria. While nuclear BER protein levels and activities were generally not altered in ρ⁰ cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in ρ⁰ cells, and not a general dysfunction of ρ⁰ cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.     

ATAD3B is a mitophagy receptor mediating clearance of oxidative stress‐induced damaged mitochondrial DNA

Mitochondrial DNA (mtDNA) encodes several key components of respiratory chain complexes that produce cellular energy through oxidative phosphorylation. mtDNA is vulnerable to damage under various physiological stresses, especially oxidative stress. mtDNA damage leads to mitochondrial dysfunction, and dysfunctional mitochondria can be removed by mitophagy, an essential process in cellular homeostasis. However, how damaged mtDNA is selectively cleared from the cell, and how damaged mtDNA triggers mitophagy, remain mostly unknown. Here, we identified a novel mitophagy receptor, ATAD3B, which is specifically expressed in primates. ATAD3B contains a LIR motif that binds to LC3 and promotes oxidative stress‐induced mitophagy in a PINK1‐independent manner, thus promoting the clearance of damaged mtDNA induced by oxidative stress. Under normal conditions, ATAD3B hetero‐oligomerizes with ATAD3A, thus promoting the targeting of the C‐terminal region of ATAD3B to the mitochondrial intermembrane space. Oxidative stress‐induced mtDNA damage or mtDNA depletion reduces ATAD3B‐ATAD3A hetero‐oligomerization and leads to exposure of the ATAD3B C‐terminus at the mitochondrial outer membrane and subsequent recruitment of LC3 for initiating mitophagy. Furthermore, ATAD3B is little expressed in m.3243A > G mutated cells and MELAS patient fibroblasts showing endogenous oxidative stress, and ATAD3B re‐expression promotes the clearance of m.3243A > G mutated mtDNA. Our findings uncover a new pathway to selectively remove damaged mtDNA and reveal that increasing ATAD3B activity is a potential therapeutic approach for mitochondrial diseases.     

Gain of PITRM1 peptidase in cortical neurons affords protection of mitochondrial and synaptic function in an advanced age mouse model of Alzheimer’s disease

Mitochondrial dysfunction is one of the early pathological features of Alzheimer''s disease (AD). Accumulation of cerebral and mitochondrial Aβ links to mitochondrial and synaptic toxicity. We have previously demonstrated the mechanism by which presequence peptidase (PITRM1)‐mediated clearance of mitochondrial Aβ contributes to mitochondrial and cerebral amyloid pathology and mitochondrial and synaptic stress in adult transgenic AD mice overexpressing Aβ up to 12 months old. Here, we investigate the effect of PITRM1 in an advanced age AD mouse model (up to 19–24 months) to address the fundamental unexplored question of whether restoration/gain of PITRM1 function protects against mitochondrial and synaptic dysfunction associated with Aβ accumulation and whether this protection is maintained even at later ages featuring profound amyloid pathology and synaptic failure. Using newly developed aged PITRM1/Aβ‐producing AD mice, we first uncovered reduction in PITRM1 expression in AD‐affected cortex of AD mice at 19–24 months of age. Increasing neuronal PITRM1 activity/expression re‐established mitochondrial respiration, suppressed reactive oxygen species, improved synaptic function, and reduced loss of synapses even at advanced ages (up to 19–24 months). Notably, loss of PITRM1 proteolytic activity resulted in Aβ accumulation and failure to rescue mitochondrial and synaptic function, suggesting that PITRM1 activity is required for the degradation and clearance of mitochondrial Aβ and Aβ deposition. These data indicate that augmenting PITRM1 function results in persistent life‐long protection against Aβ toxicity in an AD mouse model. Therefore, augmenting PITRM1 function may enhance Aβ clearance in mitochondria, thereby maintaining mitochondrial integrity and ultimately slowing the progression of AD.     

The C-terminal alphaO helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial beta-Ogg1 lacks this domain and does not have glycosylase activity

The human Ogg1 glycosylase is responsible for repairing 8-oxo-7,8-dihydroguanine (8-oxoG) in both nuclear and mitochondrial DNA. Two distinct Ogg1 isoforms are present; α-Ogg1, which mainly localizes to the nucleus and β-Ogg1, which localizes only to mitochondria. We recently showed that mitochondria from ρ⁰ cells, which lack mitochondrial DNA, have similar 8-oxoG DNA glycosylase activity to that of wild-type cells. Here, we show that β-Ogg1 protein levels are ~80% reduced in ρ⁰ cells, suggesting β-Ogg1 is not responsible for 8-oxoG incision in mitochondria. Thus, we characterized the biochemical properties of recombinant β-Ogg1. Surprisingly, recombinant β-Ogg1 did not show any significant 8-oxoG DNA glycosylase activity in vitro. Since β-Ogg1 lacks the C-terminal αO helix present in α-Ogg1, we generated mutant proteins with various amino acid substitutions in this domain. Of the seven amino acid positions substituted (317–323), we identified Val-317 as a novel critical residue for 8-oxoG binding and incision. Our results suggest that the αO helix is absolutely necessary for 8-oxoG DNA glycosylase activity, and thus its absence may explain why β-Ogg1 does not catalyze 8-oxoG incision in vitro. Western blot analysis revealed the presence of significant amounts of α-Ogg1 in human mitochondria. Together with previous localization studies in vivo, this suggests that α-Ogg1 protein may provide the 8-oxoG DNA glycosylase activity for the repair of these lesions in human mitochondrial DNA. β-Ogg1 may play a novel role in human mitochondria.     

SIAH proteins regulate the degradation and intra‐mitochondrial aggregation of PINK1: Implications for mitochondrial pathology in Parkinson's disease

Parkinson''s disease (PD) is characterized by degeneration of neurons, particularly dopaminergic neurons in the substantia nigra. PD brains show accumulation of α‐synuclein in Lewy bodies and accumulation of dysfunctional mitochondria. However, the mechanisms leading to mitochondrial pathology in sporadic PD are poorly understood. PINK1 is a key for mitophagy activation and recycling of unfit mitochondria. The activation of mitophagy depends on the accumulation of uncleaved PINK1 at the outer mitochondrial membrane and activation of a cascade of protein ubiquitination at the surface of the organelle. We have now found that SIAH3, a member of the SIAH proteins but lacking ubiquitin‐ligase activity, is increased in PD brains and cerebrospinal fluid and in neurons treated with α‐synuclein preformed fibrils (α‐SynPFF). We also observed that SIAH3 is aggregated together with PINK1 in the mitochondria of PD brains. SIAH3 directly interacts with PINK1, leading to their intra‐mitochondrial aggregation in cells and neurons and triggering a cascade of toxicity with PINK1 inactivation along with mitochondrial depolarization and neuronal death. We also found that SIAH1 interacts with PINK1 and promotes ubiquitination and proteasomal degradation of PINK1. Similar to the dimerization of SIAH1/SIAH2, SIAH3 interacts with SIAH1, promoting its translocation to mitochondria and preventing its ubiquitin‐ligase activity toward PINK1. Our results support the notion that the increase in SIAH3 and intra‐mitochondrial aggregation of SIAH3‐PINK1 may mediate α‐synuclein pathology by promoting proteotoxicity and preventing the elimination of dysfunctional mitochondria. We consider it possible that PINK1 activity is decreased in sporadic PD, which impedes proper mitochondrial renewal in the disease.     

Muscle mitochondrial energetics predicts mobility decline in well‐functioning older adults: The baltimore longitudinal study of aging

BackgroundMuscle mitochondrial dysfunction is associated with poor mobility in aging. Whether mitochondrial dysfunction predicts subsequent mobility decline is unknown.MethodsWe examined 380 cognitively normal participants aged 60 and older (53%women, 22%Black) who were well‐functioning (gait speed ≥ 1.0 m/s) and free of Parkinson''s disease and stroke at baseline and had data on baseline skeletal muscle oxidative capacity and one or more mobility assessments during an average 2.5 years. Muscle oxidative capacity was measured by phosphorus magnetic resonance spectroscopy as the post‐exercise recovery rate of phosphocreatine (k_PCr). Mobility was measured by four walking tests. Associations of baseline k_PCr with mobility changes were examined using linear mixed‐effects models, adjusted for covariates. In a subset, we examined whether changes in muscle strength and mass affected these associations by adjusting for longitudinal muscle strength, lean mass, and fat mass.ResultsLower baseline k_PCr was associated with greater decline in all four mobility measures (β, p‐value: (0.036, 0.020) 6‐m usual gait speed; (0.029, 0.038) 2.5‐min usual gait speed; (0.034, 0.011) 6‐m rapid gait speed; (−0.042, <0.001) 400‐m time). In the subset, further adjustment for longitudinal muscle strength, lean mass, and fat mass attenuated longitudinal associations with changes in mobility (Δβ reduced 26–63%).ConclusionAmong initially well‐functioning older adults, worse muscle mitochondrial function predicts mobility decline, and part of this longitudinal association is explained by decline in muscle strength and mass. Our findings suggest that worse mitochondrial function contributes to mobility decline with aging. These findings need to be verified in studies correlating longitudinal changes in mitochondrial function, muscle, and mobility performance.     

Effects of Fcj1-Mos1 and mitochondrial division on aggregation of mitochondrial DNA nucleoids and organelle morphology

Mitochondrial DNA (mtDNA) is packaged into DNA–protein complexes called nucleoids, which are distributed as many small foci in mitochondria. Nucleoids are crucial for the biogenesis and function of mtDNA. Here, using a yeast genetic screen for components that control nucleoid distribution and size, we identify Fcj1 and Mos1, two evolutionarily conserved mitochondrial proteins that maintain the connection between the cristae and boundary membranes. These two proteins are also important for establishing tubular morphology of mitochondria, as mitochondria lacking Fcj1 and Mos1 form lamellar sheets. We find that nucleoids aggregate, increase in size, and decrease in number in fcj1∆ and mos1∆ cells. In addition, Fcj1 form punctate structures and localized adjacent to nucleoids. Moreover, connecting mitochondria by deleting the DNM1 gene required for organelle division enhances aggregation of mtDNA nucleoids in fcj1∆ and mos1∆ cells, whereas single deletion of DNM1 does not affect nucleoids. Conversely, deleting F1Fo-ATP synthase dimerization factors generates concentric ring-like cristae, restores tubular mitochondrial morphology, and suppresses nucleoid aggregation in these mutants. Our findings suggest an unexpected role of Fcj1-Mos1 and organelle division in maintaining the distribution and size of mtDNA nucleoids.     

IFN‐β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource‐demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN‐β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN‐β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN‐β signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria–endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN‐β in the Ifnb ^–/– model of Parkinson disease (PD) disrupts STAT5‐PGAM5‐Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN‐β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN‐β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/^S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.     

WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures

The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long‐term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1‐SLX4 structure‐specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2‐MSH6), MutSβ (MSH2‐MSH3), and MutLα (MLH1‐PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic‐lethal interaction with promising applications in precision cancer therapy.     

Generation of ρ0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (ρ⁰ cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. ρ⁰ cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of ρ⁰ cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ⁰ cell lines within 3–5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK⁻ the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat ρ⁰ cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK⁻ ρ⁰ cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ⁰ mitochondria.     

Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability

Polymerase δ‐interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, “moonlighting” protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism and DNA replication and repair. How POLDIP2 interacts with many different proteins is unknown. Towards this goal, we present the crystal structure of POLDIP2 to 2.8 Å, which exhibited a compact two‐domain β‐strand‐rich globular structure, confirmed by circular dichroism and small angle X‐ray scattering approaches. POLDIP2 comprised canonical DUF525 and YccV domains, but with a conserved domain linker packed tightly, resulting in an “extended” YccV module. A central channel was observed, which we hypothesize could influence structural changes potentially mediated by redox conditions, following observation of a modified cysteine residue in the channel. Unstructured regions were rebuilt by ab initio modelling to generate a model of full‐length POLDIP2. Molecular dynamics simulations revealed a highly dynamic N‐terminal region tethered to the YccV‐domain by an extended linker, potentially facilitating interactions with distal binding partners. Models of POLDIP2 complexed with two of its partners, PrimPol and PCNA, indicated that dynamic flexibility of the POLDIP2 N‐terminus and loop regions likely mediate protein interactions.     

IFN‐γ+IL‐17+Th17 cells regulate fibrosis through secreting IL‐21 in systemic scleroderma

This study aimed to explore the function of IFN‐γ⁺IL‐17⁺Th17 cells on fibrosis in systemic scleroderma (SSc). Blood and skin samples were collected from 20 SSc cases and 10 healthy individuals. The percentage of IFN‐γ⁺IL‐17⁺Th17 cells was detected using flow cytometry. The in vitro induction of IFN‐γ⁺IL‐17⁺Th17 cells was performed adopting PHA and rIL‐12. Gene expression was detected via quantitative real‐time polymerase chain reaction (qRT‐PCR), whereas western blot analysis was adopted for protein analysis. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with SSc stages (P = .031), disease duration (P = .016), activity (P = .025) and skin scores (P < .001). In vitro, IFN‐γ⁺IL‐17⁺Th17 cells could promote the expressions of α‐SMA and COL1A1, revealing increased fibroblasts’ proliferation and enhanced collagen‐secreting capacity. In addition, IL‐21 expression was significantly increased in co‐culture medium of IFN‐γ⁺IL‐17⁺Th17 cells and fibroblasts (P < .001). IL‐21 neutralizer treatment resulted in the down‐regulation of α‐SMA and COL1A1. IL‐21 was confirmed as an effector of IFN‐γ⁺IL‐17⁺Th17 cells in fibrosis process. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with disease activity. IFN‐γ⁺IL‐17⁺Th17 cells could promote fibroblast proliferation and enhance collagen‐secreting ability via producing IL‐21, thus contributing to fibrosis in SSc.     

Decrease of MtDNA copy number affects mitochondrial function and involves in the pathological consequences of ischaemic stroke

The mtDNA copy number can affect the function of mitochondria and play an important role in the development of diseases. However, there are few studies on the mechanism of mtDNA copy number variation and its effects in IS. The specific mechanism of mtDNA copy number variation is still unclear. In this study, mtDNA copy number of 101 IS patients and 101 normal controls were detected by qRT‐PCR, the effect of D‐loop variation on mtDNA copy number of IS patients was explored. Then, a TFAM gene KD‐OE PC12 cell model was constructed to explore the effect of mtDNA copy number variation on mitochondrial function. The results showed that the mtDNA copy number level of the IS group was significantly lower than that of the normal control group (p < 0.05). The relative expression of TFAM gene mRNA in the cells of the OGD/R treatment group was significantly lower than that of the control group (p < 0.05). In addition, after TFAM gene knockdown and over‐expression plasmids were transfected into HEK 293T cells, mtDNA copy number and ATP production level of Sh‐TFAM transfection group was significantly decreased (p < 0.05), while mtDNA copy number and ATP production level of OE‐TFAM transfected group were significantly higher than that of blank control group and OE‐ctrl negative control group (p < 0.01). Our study demonstrated that mitochondrial D‐loop mutation and TFAM gene dysfunction can cause the decrease of mtDNA copy number, thus affecting the mitochondrial metabolism and function of nerve cells, participating in the pathological damage mechanism of IS.     

Structure of a human replisome shows the organisation and interactions of a DNA replication machine

The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45‐MCM‐GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading‐strand polymerase Pol ε, together with TIMELESS‐TIPIN, CLASPIN and AND‐1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Å cryo‐EM structure of a human replisome comprising CMG, Pol ε, TIMELESS‐TIPIN, CLASPIN and AND‐1 bound to replication fork DNA. The structure permits a detailed understanding of how AND‐1, TIMELESS‐TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS‐TIPIN with replication fork DNA suggests a mechanism for strand separation.     

CPT1α maintains phenotype of tubules via mitochondrial respiration during kidney injury and repair

Impaired energy metabolism in proximal tubular epithelial cells (PTECs) is strongly associated with various kidney diseases. Here, we characterized proximal tubular phenotype alternations during kidney injury and repair in a mouse model of folic acid nephropathy, in parallel, identified carnitine palmitoyltransferase 1α (CPT1α) as an energy stress response accompanied by renal tubular dedifferentiation. Genetic ablation of Cpt1α aggravated the tubular injury and interstitial fibrosis and hampered kidney repair indicate that CPT1α is vital for the preservation and recovery of tubular phenotype. Our data showed that the lipid accumulation and mitochondrial mass reduction induced by folic acid were persistent and became progressively more severe in PTECs without CPT1α. Interference of CPT1α reduced capacities of mitochondrial respiration and ATP production in PTECs, and further sensitized cells to folic acid-induced phenotypic changes. On the contrary, overexpression of CPT1α protected mitochondrial respiration and prevented against folic acid-induced tubular cell damage. These findings link CPT1α to intrinsic mechanisms regulating the mitochondrial respiration and phenotype of kidney tubules that may contribute to renal pathology during injury and repair.Subject terms: Mechanisms of disease, Chronic kidney disease, Pathogenesis