Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the ...catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.
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•DNA-PKcs inhibits ATM activity through phosphorylation at multiple sites•DNA-PKcs phosphorylation of ATM impairs ATM signaling upon DNA damage•DNA-PKcs phosphorylation of ATM does not affect ATM function upon oxidative stress•Loss of DNA-PKcs regulation of ATM affects normal cell growth
Zhou et al. show that DNA-PKcs phosphorylates ATM at multiple sites in response to DNA damage, resulting in inhibition of ATM activity. DNA-PKcs phosphorylation of ATM regulates its activity in DNA repair and cell-cycle checkpoint activation, and also reduces apoptosis during normal cell growth.
The pathogenesis of post-stroke depression (PSD) remains largely unknown. There is growing evidence indicating that gut microbiota participates in the development of brain diseases through the ...gut-brain axis. Here, we aim to determine whether and how microbial composition and function altered among control, stroke and PSD rats.
After the PSD rat model was successfully established, gut microbiome combined with fecal metabolome approach were performed to identify potentially PSD-related gut microbes and their functional metabolites. Then, correlations between behavior indices and altered gut microbes, as well as correlations between altered gut microbial operational taxonomic units (OTUs) with differential metabolites in PSD rats were explored. Enrichment analysis was also conducted to uncover the crucial metabolic pathways related to PSD.
Although there were some alterations in the microbiome and metabolism of the control and stroke rats, we found that the microbial and metabolic phenotypes of PSD rats were significantly different. The microbial composition of PSD showed a decreased species richness indices, characterized by 22 depleted OTUs mainly belonging to phylum
, genus
and
. In addition, PSD was associated with disturbances of fecal metabolomics, among them Glutamate, Maleic acid, 5-Methyluridine, Gallocatechin, 1,5-Anhydroglucitol, L-Kynurenine, Daidzein, Cyanoalanine, Acetyl Alanine and 5-Methoxytryptamine were significantly related to disturbed gut microbiome (P ≤ 0.01). Disordered fecal metabolomics in PSD rats mainly assigned to lipid, amino acid, carbohydrate and nucleotide metabolism. The steroid biosynthesis was particularly enriched in PSD.
Our findings suggest that gut microbiome may participate in the development of PSD, the mechanism may be related to the regulation of lipid metabolism.
Brain inflammation induced by ischemic stroke is an important cause of secondary brain injury. The nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and NLRP3 inflammasome ...signaling are believed to drive the progression of brain inflammation. Spermatogenesis-associated protein2 (SPATA2) functions as a partner protein that recruits CYLD, a negative regulator of NF-κB signaling, to signaling complexes. However, the role of SPATA2 in the central nervous system remains unclear and whether it is involved in regulating inflammatory responses remains controversial. Rats were subjected to transient middle cerebral artery occlusion followed by reperfusion (tMCAO/R) surgery. The expression and localization of SPATA2 in the brain were investigated. The lentivirus-mediated shRNA was employed to inhibit SPATA2 expression. The inflammatory responses and outcomes of
Spata2
knockdown were investigated. SPATA2 was co-localized with CYLD in neurons. SPATA2 expression was reduced in tMCAO/R rats.
Spata2
knockdown resulted in increased microglia, increased expression of
Tnfa
,
Il-1β,
and
Il-18
, decreased Garcia score, and increased infarct volume.
Spata2
knockdown resulted in the activation of P38MAPK and NLRP3 inflammasome and the increased activation of NF-κB signaling. These results suggest that SPATA2 plays a protective role against brain inflammation induced by ischemia/reperfusion injury. Therefore, SPATA2 could be a potential therapeutic target for treating ischemic stroke.
Identifying traffic congestion accurately is crucial for improving the expressway service level. Because the distributions of microscopic traffic quantities are highly sensitive to slight changes, ...the traffic congestion measurement is affected by many factors. As an essential part of the expressway, service areas should be considered when measuring the traffic state. Although existing studies pay increasing attention to service areas, the impact caused by service areas is hard to measure for evaluating traffic congestion events. By merging ETC transaction datasets and service area entrance data, this work proposes a traffic congestion measurement with the influence of expressway service areas. In this model, the traffic congestion with the influence of service areas is corrected by three modules: 1) the pause rate prediction module; 2) the fitting module for the relationship between effect and pause rate; 3) the measurement module with correction terms. Extensive experiments were conducted on the real dataset of the Fujian Expressway, and the results show that the proposed method can be applied to measure the effect caused by service areas in the absence of service area entry data. The model can also provide references for other traffic indicator measurements under the effect of the service area.
Nonhomologous end-joining (NHEJ) is a major DNA double-strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin ...opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate-specific NHEJ factor that can be autophosphorylated or transphosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated transphosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strict autophosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcsKD/KD mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations.
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•DNA-PKcs protein inhibits direct end-ligation at the DNA ends•Intermolecular autophosphorylation of DNA-PKcs is required for end-ligation•DNA-PKcs protein, but not its kinase activity, is required for end-processing•ATM phosphorylates DNA-PKcs to recruit Artemis and promote end-processing
Differential phosphorylation of DNA-PKcs at DNA ends is the molecular switch between end-processing and end-ligation. ATM-mediated phosphorylation of DNA-PKcs is necessary for Artemis recruitment and end-processing, while intermolecular autophosphorylation of DNA-PKcs relieves the physical block on end-ligation imposed by the DNA-PKcs protein at DNA ends and is required for direct end-ligation.
The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor ...suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here, we compare mice with mutations that ablate BRCT phospho-recognition by Bard1 (Bard1S563F and Bard1K607A) or Brca1 (Brca1S1598F). Brca1S1598F abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1 mutant tumors. Although not affecting HDR, the Bard1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, Bard1S563F/S563F and Bard1K607A/K607A mice, unlike Brca1S1598F/S1598F mice, are not tumor prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, because SFP, unlike HDR, is also impaired in heterozygous Brca1/Bard1 mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.
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•The BRCT domain of BARD1 recruits BRCA1/BARD1 to stalled replication forks•Stalled fork protection (SFP), but not HDR, requires BARD1 BRCT phospho-recognition•SFP and HDR both require BRCA1 BRCT phospho-recognition•HDR is sufficient for tumor suppression in the absence of SFP
Billing et al. describe the mechanism by which the BRCA1/BARD1 tumor suppressor complex is recruited to stalled DNA replication forks and identify molecular features of the complex required for maintenance of genome integrity though homology-directed repair (HDR) and stalled fork protection (SFP).
The ATR kinase, which coordinates cellular responses to DNA replication stress, is also essential for the proliferation of normal unstressed cells. Although its role in the replication stress ...response is well defined, the mechanisms by which ATR supports normal cell proliferation remain elusive. Here, we show that ATR is dispensable for the viability of G0-arrested naïve B cells. However, upon cytokine-induced proliferation, Atr-deficient B cells initiate DNA replication efficiently, but by mid-S phase they display dNTP depletion, fork stalling, and replication failure. Nonetheless, productive DNA replication and dNTP levels can be restored in Atr-deficient cells by suppressing origin firing, such as partial inhibition of CDC7 and CDK1 kinase activities. Together, these findings indicate that ATR supports the proliferation of normal unstressed cells by tempering the pace of origin firing during the early S phase to avoid exhaustion of dNTPs and importantly also other replication factors.
mRNA vaccines have proven to be pivotal in the fight against COVID-19. A recommended booster, given 3 to 4 weeks post the initial vaccination, can substantially amplify protective antibody levels. ...Here, we show that, compared to contralateral boost, ipsilateral boost of the SARS-CoV-2 mRNA vaccine induces more germinal center B cells (GCBCs) specific to the receptor binding domain (RBD) and generates more bone marrow plasma cells. Ipsilateral boost can more rapidly generate high-affinity RBD-specific antibodies with improved cross-reactivity to the Omicron variant. Mechanistically, the ipsilateral boost promotes the positive selection and plasma cell differentiation of pre-existing GCBCs from the prior vaccination, associated with the expansion of T follicular helper cells. Furthermore, we show that ipsilateral immunization with an unrelated antigen after a prior mRNA vaccination enhances the germinal center and antibody responses to the new antigen compared to contralateral immunization. These findings propose feasible approaches to optimize vaccine effectiveness.
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•Ipsilateral boost of mRNA vaccine induces superior GC and plasma cell responses•Ipsilateral boost more rapidly induces high-affinity antibodies with improved breadth•Ipsilateral boost directly activates the ongoing GCs from the prior vaccination•Ipsilateral new antigen immunization post mRNA vaccination improves humoral responses
COVID-19 mRNA vaccines require a prime-boost approach to stimulate robust levels of protective antibodies. Jiang et al. demonstrate that receiving the booster shot or a different vaccine on the same side following a prior mRNA vaccination may result in more optimal B cell and antibody responses.
Vascular dementia (VD) is the second most common cause of dementia following Alzheimer’s disease (AD). The major symptoms of VD including memory loss, language deficits and impairment of executive ...functions. Its specific etiology and pathogenesis remain unknown. Currently, treatment options of VD are still limited. The therapeutic strategies aim to control the vascular risk factors and improve the cognitive function. In recent years, cell therapy for neurodegenerative diseases has attracted a great deal of attention. Evidence suggested that stem cell transplantation could improve the symptoms of cerebral infarction and AD. Therefore, it may serve as a potential therapy for VD. We summarized the latest research results both in vitro and in vivo. Further, the clinical trial of stem cell transplantation in VD patients was also reviewed. Finally, the limitations and future directions of cell therapy in VD treatment were discussed.
ATR kinase is activated by RPA-coated single-stranded DNA (ssDNA) to orchestrate DNA damage responses. Here we show that ATR inhibition differs from ATR loss. Mouse model expressing kinase-dead ATR ...(Atr
), but not loss of ATR (Atr
), displays ssDNA-dependent defects at the non-homologous region of X-Y chromosomes during male meiosis leading to sterility, and at telomeres, rDNA, and fragile sites during mitosis leading to lymphocytopenia. Mechanistically, we find that ATR kinase activity is necessary for the rapid exchange of ATR at DNA-damage-sites, which in turn promotes CHK1-phosphorylation. ATR-KD, but not loss of ATR, traps a subset of ATR and RPA on chromatin, where RPA is hyper-phosphorylated by ATM/DNA-PKcs and prevents downstream repair. Consequently, Atr
cells have shorter inter-origin distances and are vulnerable to induced fork collapses, genome instability and mitotic catastrophe. These results reveal mechanistic differences between ATR inhibition and ATR loss, with implications for ATR signaling and cancer therapy.
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