Mre11 Is Essential for the Removal of Lethal Topoisomerase 2 Covalent Cleavage Complexes.

The Mre11/Rad50/Nbs1 complex initiates double-strand break repair by homologous recombination (HR). Loss of Mre11 or its nuclease activity in mouse cells is known to cause genome aberrations and cellular senescence, although the molecular basis for this phenotype is not clear. To identify the origin of these defects, we characterized Mre11-deficient (MRE11-/-) and nuclease-deficient Mre11 (MRE11-/H129N) chicken DT40 and human lymphoblast cell lines. These cells exhibit increased spontaneous chromosomal DSBs and extreme sensitivity to topoisomerase 2 poisons. The defects in Mre11 compromise the repair of etoposide-induced Top2-DNA covalent complexes, and MRE11-/- and MRE11-/H129N cells accumulate high levels of Top2 covalent conjugates even in the absence of exogenous damage. We demonstrate that both the genome instability and mortality of MRE11-/- and MRE11-/H129N cells are significantly reversed by overexpression of Tdp2, an enzyme that eliminates covalent Top2 conjugates; thus, the essential role of Mre11 nuclease activity is likely to remove these lesions.

Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence.

MCPH1 has been identified as the causal gene for primary microcephaly type 1, a neurodevelopmental disorder characterized by reduced brain size and delayed growth. As a multifunction protein, MCPH1 has been reported to repress the expression of TERT and interact with transcriptional regulator E2F1. However, it remains unclear whether MCPH1 regulates brain development through its transcriptional regulation function. This study showed that the knockout of Mcph1 in mice leads to delayed growth as early as the embryo stage E11.5. Transcriptome analysis (RNA-seq) revealed that the deletion of Mcph1 resulted in changes in the expression levels of a limited number of genes. Although the expression of some of E2F1 targets, such as Satb2 and Cdkn1c, was affected, the differentially expressed genes (DEGs) were not significantly enriched as E2F1 target genes. Further investigations showed that primary and immortalized Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibited cell cycle arrest and cellular senescence phenotype. Interestingly, the upregulation of p19ARF was detected in Mcph1 knockout MEFs, and silencing p19Arf restored the cell cycle and growth arrest to wild-type levels. Our findings suggested it is unlikely that MCPH1 regulates neurodevelopment through E2F1-mediated transcriptional regulation, and p19ARF-dependent cell cycle arrest and cellular senescence may contribute to the developmental abnormalities observed in primary microcephaly.

Discovery of a small-molecule inhibitor targeting the ovarian tumor domain of a novel Tamdy orthonairoviruse associated with human febrile illness.

Tick-borne orthonairoviruses have been characterized as a global health threat to humans and animals. Tacheng Tick virus 1 (TcTV-1) from this family was provided as evidence that is associated with the febrile illness syndrome. Here, we first identify and demonstrate that the ovarian tumor (OTU) domain of TcTV-1 has remarkable deubiquitinating activity both in vitro and in vivo. By solving the crystal structure of TcTV-1 OTU (tcOTU) domain and comparing it to that of human deubiquitinating enzymes, we found that overall structures of tcOTU and human OTU family are similar, but the residues involved in the catalytic pocket vary widely. Based on the tcOTU domain we screened 5090 bioactive compounds and found mecobalamin had a good effect on suppressing the deubiquitinating activity. The structural model of tcOTU and mecobalamin suggests that mecobalamin occupies the site of the substrate Ub, by blocking the substrate binding to the enzyme. Thus, our results showed OTU domain of TcTV-1 has a robust deubiquitinating activity and mecobalamin or its derivatives might be promising candidates for the treatment or prevention of disease caused by the TcTV-1 virus.

NBS1 interacts with Notch signaling in neuronal homeostasis.

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

Combination RSL3 Treatment Sensitizes Ferroptosis- and EGFR-Inhibition-Resistant HNSCCs to Cetuximab.

Head and neck squamous cell carcinomas (HNSCCs) are a type of cancer originating in the mucosal epithelium of the mouth, pharynx, and larynx, the sixth most common cancer in the world. However, there is no effective treatment for HNSCCs. More than 90% of HNSCCs overexpress epidermal growth factor receptors (EGFRs). Although small molecule inhibitors and monoclonal antibodies have been developed to target EGFRs, few EGFR-targeted therapeutics are approved for clinical use. Ferroptosis is a new kind of programmed death induced by the iron catalyzed excessive peroxidation of polyunsaturated fatty acids. A growing body of evidence suggests that ferroptosis plays a pivotal role in inhibiting the tumor process. However, whether and how ferroptosis-inducers (FINs) play roles in hindering HNSCCs are unclear. In this study, we analyzed the sensitivity of different HNSCCs to ferroptosis-inducers. We found that only tongue squamous cell carcinoma cells and laryngeal squamous cell carcinoma cells, but not nasopharyngeal carcinoma cells, actively respond to ferroptosis-inducers. The different sensitivities of HNSCC cells to ferroptosis induction may be attributed to the expression of KRAS and ferritin heavy chain (FTH1) since a high level of FTH1 is associated with the poor prognostic survival of HNSCCs, but knocked down FTH1 can promote HNSCC cell death. Excitingly, the ferroptosis-inducer RSL3 plays a synthetic role with EGFR monoclonal antibody Cetuximab to inhibit the survival of nasopharyngeal carcinoma cells (CNE-2), which are insensitive to both ferroptosis induction and EGFR inhibition due to a high level of FTH1 and a low level of EGFR, respectively. Our findings prove that FTH1 plays a vital role in ferroptosis resistance in HNSCCs and also provide clues to target HNSCCs resistant to ferroptosis induction and/or EGFR inhibition.

Aminoacyl-tRNA synthetases in Charcot-Marie-Tooth disease: A gain or a loss?

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurodegenerative disorders with an increasing number of CMT-associated variants identified as causative factors, however, there has been no effective therapy for CMT to date. Aminoacyl-tRNA synthetases (aaRS) are essential enzymes in translation by charging amino acids onto their cognate tRNAs during protein synthesis. Dominant monoallelic variants of aaRSs have been largely implicated in CMT. Some aaRSs variants affect enzymatic activity, demonstrating a loss-of-function property. In contrast, loss of aminoacylation activity is neither necessary nor sufficient for some aaRSs variants to cause CMT. Instead, accumulating evidence from CMT patient samples, animal genetic studies or protein conformational analysis has pinpointed toxic gain-of-function of aaRSs variants in CMT, suggesting complicated mechanisms underlying the pathogenesis of CMT. In this review, we summarize the latest advances in studies on CMT-linked aaRSs, with a particular focus on their functions. The current challenges, future direction and the promising candidates for potential treatment of CMT are also discussed.

The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1-ßTrCP2-Cdc25A-mediated mitotic entry to ensure neurogenesis.

Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase ßTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of ßTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1-deficient neuroprogenitors in vivo MCPH1 itself is degraded by APC/CCdh1, but not APC/CCdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1-knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin-proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development.

A Shortage of FTH Induces ROS and Sensitizes RAS-Proficient Neuroblastoma N2A Cells to Ferroptosis.

Ferroptosis, an iron-dependent form of programmed cell death, has excellent potential as an anti-cancer therapeutic strategy in different types of tumors, especially in RAS-mutated ones. However, the function of ferroptosis for inhibiting neuroblastoma, a common child malignant tumor with minimal treatment, is unclear. This study investigated the anti-cancer function of ferroptosis inducer Erastin or RSL3 in neuroblastoma N2A cells. Our results show that Erastin or RSL3 induces ROS level and cell death and, therefore, reduces the viability of RAS-proficient N2A cells. Importantly, inhibitors to ferroptosis, but not apoptosis, ameliorate the high ROS level and viability defect in Erastin- or RSL3-treated cells. In addition, our data also show that N2A cells are much more sensitive to ferroptosis inducers than primary mouse cortical neural stem cells (NSCs) or neurons. Moreover, a higher level of ROS and PARylation is evidenced in N2A, but not NSCs. Mechanically, ferritin heavy chain 1 (Fth), the ferroxidase function to oxidate redox-active Fe2+ to redox-inactive Fe3+, is likely responsible for the hypersensitivity of N2A to ferroptosis induction since its expression is lower in N2A compared to NSCs; ectopic expression of Fth reduces ROS levels and cell death, and induces expression of GPX4 and cell viability in N2A cells. Most importantly, neuroblastoma cell lines express a significantly low level of Fth than almost all other types of cancer cell lines. All these data suggest that Erastin or RSL3 induce ferroptosis cell death in neuroblastoma N2A cells, but not normal neural cells, regardless of RAS mutations, due to inadequate FTH. This study, therefore, provides new evidence that ferroptosis could be a promising therapeutic target for neuroblastoma.

A de novo mosaic mutation of PHEX in a boy with hypophosphatemic rickets.

X-linked dominant hypophosphatemic rickets (XLHR), is characterized mainly by renal phosphate wasting with hypophosphatemia, short stature and abnormal bone mineralization. PHEX, located at Xp22.1-p22.2, is the gene causing XLHR. We aim to characterize the pathogenesis of a Chinese boy who is apparently 'heterozygous' in PHEX gene. Direct sequencing showed two peaks: one was a wild-type 'G' and the other was one base substitution to 'A', though the patient was a male. TA clone assay clearly showed each sequences and the ratios. The mutation effect was predicted via bioinformatics and validated by exon-trapping assay. Real-time PCR was applied to determine the copy number of PHEX. TA clone assay showed the frequency of normal (G) to mutant allele (A) as 19:13. Normal karyotype and real-time PCR results indicate the normal copy number of PHEX. This splice site mutation leads to 4 bp of exon 18 skipping out causing frame shift p.Gly590Glufs*28 that ends up with a loss of active site and Zn(2+)-binding site of PHEX, which probably interfere with renal phosphate reabsorption and bone mineralization. In conclusion, mutation at conserved splice acceptor site resulted in aberrant splicing, ending up with a damaged protein product. This novel mutation is de novo in mosaic pattern that may be induced during early postzygotic period. Taking mosaic somatic mutation of PHEX into consideration is strongly suggested in genetic counseling and etiology research for XLHR.

The DNA damage response molecule MCPH1 in brain development and beyond.

Microcephalin (MCPH1) is identified as being responsible for the neurodevelopmental disorder primary microcephaly type 1, which is characterized by a smaller-than-normal brain size and mental retardation. MCPH1 has originally been identified as an important regulator of telomere integrity and of cell cycle control. Genetic and cellular studies show that MCPH1 controls neurogenesis by coordinating the cell cycle and the centrosome cycle and thereby regulating the division mode of neuroprogenitors to prevent the exhaustion of the progenitor pool and thereby microcephaly. In addition to its role in neurogenesis, MCPH1 plays a role in gonad development. MCPH1 also functions as a tumor suppressor in several human cancers as well as in mouse models. Here, we review the role of MCPH1 in DNA damage response, cell cycle control, chromosome condensation and chromatin remodeling. We also summarize the studies on the biological functions of MCPH1 in brain size determination and in pathologies, including infertility and cancer.

An essential function for the ATR-activation-domain (AAD) of TopBP1 in mouse development and cellular senescence.

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins - Dpb11(TopBP1), Ddc1(Rad9) and Dna2 - all interact with and activate Mec1(ATR). Each contains an ATR activation domain (ADD) that interacts directly with the Mec1(ATR):Ddc2(ATRIP) complex. Any of the Dpb11(TopBP1), Ddc1(Rad9) or Dna2 ADDs is sufficient to activate Mec1(ATR) in vitro. All three can also independently activate Mec1(ATR) in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.

Epigenetic and transcriptional landscapes during cerebral cortex development in a microcephaly mouse model.

The cerebral cortex is a pivotal structure integral to advanced brain functions within the mammalian central nervous system. DNA methylation and hydroxymethylation play important roles in regulating cerebral cortex development. However, it remains unclear whether abnormal cerebral cortex development, such as microcephaly, could rescale the epigenetic landscape, potentially contributing to dysregulated gene expression during brain development. In this study, we characterize and compare the DNA methylome/hydroxymethylome and transcriptome profiles of the cerebral cortex across several developmental stages in wild-type (WT) mice and Mcph1 knockout (Mcph1-del) mice with severe microcephaly. Intriguingly, we discover a global reduction of 5'-hydroxymethylcytosine (5hmC) level, primarily in TET1-binding regions, in Mcph1-del mice compared to WT mice during juvenile and adult stages. Notably, genes exhibiting diminished 5hmC levels and concurrently decreased expression are essential for neurodevelopment and brain functions. Additionally, genes displaying a delayed accumulation of 5hmC in Mcph1-del mice are significantly associated with the establishment and maintenance of the nervous system during the adult stage. These findings reveal that aberrant cerebral cortex development in the early stages profoundly alters the epigenetic regulation program, which provides unique insights into the molecular mechanisms underpinning diseases related to cerebral cortex development.

Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease.

The current COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an enormous threat to public health. The SARS-CoV-2 3C-like protease (3CLpro), which is critical for viral replication and transcription, has been recognized as an ideal drug target. Herein, it is identified that three herbal compounds, Salvianolic acid A (SAA), (-)-Epigallocatechin gallate (EGCG), and Oridonin, directly inhibit the activity of SARS-CoV-2 3CLpro. Further, blocking SARS-CoV-2 infectivity by Oridonin is confirmed in cell-based experiments. By solving the crystal structure of 3CLpro in complex with Oridonin and comparing it to that of other ligands with 3CLpro, it is identified that Oridonin binds at the 3CLpro catalytic site by forming a C-S covalent bond, which is confirmed by mass spectrometry and kinetic study, blocking substrate binding through a nonpeptidomimetic covalent binding mode. Thus, Oridonin is a novel candidate to develop a new antiviral treatment for COVID-19.

Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease.

Oridonin Inhibits SARS-CoV-2 Oridonin, a natural product extracted from Rabdosia rubescens, possesses a wide range of pharmacological properties, including anti-inflammatory, anti-cancer, anti-microbial, neuroprotection, immunoregulation, etc. In article number 2100124, Baisen Zhong, Litao Sun, and co-workers demonstrate that Oridonin targets the SARS-CoV-2 3CL protease by covalently binding to cysteine145 in its active pocket to exert an anti-SARS-CoV-2 effect, which provides a novel candidate for the treatment of COVID-19.

Post-Translational Modification of MRE11: Its Implication in DDR and Diseases.

Maintaining genomic stability is vital for cells as well as individual organisms. The meiotic recombination-related gene MRE11 (meiotic recombination 11) is essential for preserving genomic stability through its important roles in the resection of broken DNA ends, DNA damage response (DDR), DNA double-strand breaks (DSBs) repair, and telomere maintenance. The post-translational modifications (PTMs), such as phosphorylation, ubiquitination, and methylation, regulate directly the function of MRE11 and endow MRE11 with capabilities to respond to cellular processes in promptly, precisely, and with more diversified manners. Here in this paper, we focus primarily on the PTMs of MRE11 and their roles in DNA response and repair, maintenance of genomic stability, as well as their association with diseases such as cancer.

The Landscape of Aminoacyl-tRNA Synthetases Involved in Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in translation by linking amino acids onto their cognate tRNAs during protein synthesis. During evolution, aaRSs develop numerous non-canonical functions that expand the roles of aaRSs in eukaryotic organisms. Although aaRSs have been implicated in viral infection, the function of aaRSs during infections with coronaviruses (CoVs) remains unclear. Here, we analyzed the data from transcriptomic and proteomic database on human cytoplasmic (cyto) and mitochondrial (mt) aaRSs across infections with three highly pathogenic human CoVs, with a particular focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We found an overall downregulation of aaRSs at mRNA levels, while the protein levels of some mt-aaRSs and the phosphorylation of certain aaRSs were increased in response to SARS-CoV-2 infection. Strikingly, interaction network between SARS-CoV-2 and human aaRSs displayed a strong involvement of mt-aaRSs. Further co-immunoprecipitation (co-IP) experiments confirmed the physical interaction between SARS-CoV-2 M protein and TARS2. In addition, we identified the intermediate nodes and potential pathways involved in SARS-CoV-2 infection. This study provides an unbiased, overarching perspective on the correlation between aaRSs and SARS-CoV-2. More importantly, this work identifies TARS2, HARS2, and EARS2 as potential key factors involved in COVID-19.

ATR regulates neuronal activity by modulating presynaptic firing.

Ataxia Telangiectasia and Rad3-related (ATR) protein, as a key DNA damage response (DDR) regulator, plays an essential function in response to replication stress and controls cell viability. Hypomorphic mutations of ATR cause the human ATR-Seckel syndrome, characterized by microcephaly and intellectual disability, which however suggests a yet unknown role for ATR in non-dividing cells. Here we show that ATR deletion in postmitotic neurons does not compromise brain development and formation; rather it enhances intrinsic neuronal activity resulting in aberrant firing and an increased epileptiform activity, which increases the susceptibility of ataxia and epilepsy in mice. ATR deleted neurons exhibit hyper-excitability, associated with changes in action potential conformation and presynaptic vesicle accumulation, independent of DDR signaling. Mechanistically, ATR interacts with synaptotagmin 2 (SYT2) and, without ATR, SYT2 is highly upregulated and aberrantly translocated to excitatory neurons in the hippocampus, thereby conferring a hyper-excitability. This study identifies a physiological function of ATR, beyond its DDR role, in regulating neuronal activity.

The Essential DNA Damage Response Complex MRN Is Dispensable for the Survival and Function of Purkinje Neurons.

MRE11, RAD50, and NBS1 form the MRN complex in response to DNA damage to activate ATM, a gene responsible for Ataxia-Telangiectasia (A-T). Loss of any components of the MRN complex compromises cell life. Mutations in MRE11, RAD50, and NBS1 cause human genomic instability syndromes Ataxia-Telangiectasia-like disorder (A-TLD), NBS-like disorder (NBSLD), and Nijmegen Breakage Syndrome (NBS), respectively. Among other pathologies, neuronal deficits, including microcephaly, intellectual disabilities, and progressive cerebellar degeneration, are common in these disorders. Nbs1 deletion in neural stem cells of mouse models resulted in cerebellar atrophy and ataxia, mimicking the A-T syndrome suggesting an etiological function of MRN-mediated DDR in neuronal homeostasis and neuropathology. Here we show that deletion of Nbs1 or Mre11 specifically in Purkinje neurons of mouse models (Nbs1-PCΔ and Mre11-PCΔ, respectively) is compatible with cerebellar development. Deleting Nbs1 in Purkinje cells disrupts the cellular localization pattern of MRE11 or RAD50 without inducing apparent DNA damage, albeit impaired DNA damage response (judged by 53BP1 focus formation) to ionizing radiation (IR). However, neither survival nor morphology of Purkinje cells and thus locomotor capabilities is affected by Nbs1 deletion under physiological conditions. Similarly, deletion of Mre11 in Purkinje cells does not affect the numbers or morphology of Purkinje cells and causes no accumulation of DNA damage. Mre11-deleted Purkinje cells have regular intrinsic neuronal activity. Taken together, these data indicate that the MRN complex is not essential for the survival and functionality of postmitotic neurons such as Purkinje cells. Thus, cerebellar deficits in MRN defect-related disorders and mouse models are unlikely to be a direct consequence of loss of these factors compromising DDR in postmitotic neurons such as Purkinje cells.

ATR is essential for preservation of cell mechanics and nuclear integrity during interstitial migration.

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.