GSTpi Reduces DNA Damage And Cell Death By Regulating The Ubiquitination And Nuclear Translocation of NBS1

Glutathione S-transferase pi (GSTpi) is an important phase Ⅱ detoxifying enzyme that participates in various physiological processes, such as antioxidant, detoxification, and signal transduction. The high expression level of GSTpi has been reported to be related to drug-resistant and anti-inflammatory and it functioned via its non-catalytic ligandin. However, the previous protection mechanism of GSTpi in DNA damage has not been addressed so far. Nijmegen breakage syndrome 1 (NBS1) is one of the most important sensor proteins to detect damaged DNA. Here, we investigated the interaction between GSTpi and NBS1 in HEK-293T cells and human breast adenocarcinoma cells during DNA damage. Our results showed that overexpression of GSTpi in cells by transfecting DNA vector decreased the DNA damage level after Methyl methanesulfonate (MMS) or Adriamycin (ADR) treatment. We found that cytosolic GSTpi could increase NBS1 ubiquitin-mediated degradation in unstimulated cells, which suggested that GSTpi could maintain the basal level of NBS1 during normal conditions. In response to DNA damage, GSTpi can be phosphorylated in Ser184 and inhibit the ubiquitination degradation of NBS1 mediated by Skp2 to recover NBS1 protein level. Phosphorylated GSTpi can further enhance NBS1 nuclear translocation to activate the ATM-Chk2-p53 signaling pathway. Finally, GSTpi blocked the cell cycle in the G2/M phase to gain more time for cells to repair. Thus, our finding revealed the novel mechanism of GSTpi via its Ser184 phosphorylation to protect cells from cell death during DNA damage and it enriches the function of GSTpi in drug resistance.

The Thousand Polish Genomes Project - a national database of Polish variant allele frequencies

Although Slavic populations account for over 3.5% of world inhabitants, no centralized, open source reference database of genetic variation of any Slavic population exists to date. Such data are crucial for either biomedical research and genetic counseling and are essential for archeological and historical studies. Polish population, homogenous and sedentary in its nature but influenced by many migrations of the past, is unique and could serve as a good genetic reference for middle European Slavic nations. The aim of the present study was to describe first results of analyses of a newly created national database of Polish genomic variant allele frequencies. Never before has any study on the whole genomes of Polish population been conducted on such a large number of individuals (1,079). A wide spectrum of genomic variation was identified and genotyped, such as small and structural variants, runs of homozygosity, mitochondrial haplogroups and Mendelian inconsistencies. The allele frequencies were calculated for 943 unrelated individuals and released publicly as The Thousand Polish Genomes database. A precise detection and characterisation of rare variants enriched in the Polish population allowed to confirm the allele frequencies for known pathogenic variants in diseases, such as Smith-Lemli-Opitz syndrome (SLOS) or Nijmegen breakage syndrome (NBS). Additionally, the analysis of OMIM AR genes led to the identification of 22 genes with significantly different cumulative allele frequencies in the Polish (POL) vs European NFE population. We hope that The Thousand Polish Genomes database will contribute to the worldwide genomic data resources for researchers and clinicians.

Geographical Distribution, Incidence, Malignancies, and Outcome of 136 Eastern Slavic Patients With Nijmegen Breakage Syndrome and NBN Founder Variant c.657_661del5

Nijmegen breakage syndrome (NBS) is a DNA repair disorder characterized by combined immunodeficiency and a high predisposition to lymphoid malignancies. The majority of NBS patients are identified with a homozygous five base pair deletion in the Nibrin (NBN) gene (c.657_661del5, p.K219fsX19) with a founder effect observed in Caucasian European populations, especially of Slavic origin. We present here an analysis of a cohort of 136 NBS patients of Eastern Slav origin across Belarus, Ukraine, Russia, and Latvia with a focus on understanding the geographic distribution, incidence of malignancy, and treatment outcomes of this cohort. Our analysis shows that Belarus had the highest prevalence of NBS (2.3 per 1,000,000), followed by Ukraine (1.3 per 1,000,000), and Russia (0.7 per 1,000,000). Of note, the highest concentration of NBS cases was observed in the western regions of Belarus and Ukraine, where NBS prevalence exceeds 20 cases per 1,000,000 people, suggesting the presence of an “Eastern Slavic NBS hot spot.” The median age at diagnosis of this cohort ranged from 4 to 5 years, and delay in diagnosis was more pervasive in smaller cities and rural regions. A total of 62 (45%) patients developed malignancies, more commonly in males than females (55.2 vs. 34.2%; p=0.017). In 27 patients, NBS was diagnosed following the onset of malignancies (mean age: 8 years). Malignancies were mostly of lymphoid origin and predominantly non-Hodgkin lymphoma (NHL) (n=42, 68%); 38% of patients had diffuse large B-cell lymphoma. The 20-year overall survival rate of patients with malignancy was 24%. However, females with cancer experienced poorer event-free survival rates than males (16.6% vs. 46.8%, p=0.036). Of 136 NBS patients, 13 underwent hematopoietic stem cell transplantation (HSCT) with an overall survival of 3.5 years following treatment (range: 1 to 14 years). Indications for HSCT included malignancy (n=7) and immunodeficiency (n=6). Overall, 9% of patients in this cohort reached adulthood. Adult survivors reported diminished quality of life with significant physical and cognitive impairments. Our study highlights the need to improve timely diagnosis and clinical management of NBS among Eastern Slavs. Genetic counseling and screening should be offered to individuals with a family history of NBS, especially in hot spot regions.

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.

Impaired p53-mediated DNA damage response contributes to microcephaly in Nijmegen Breakage Syndrome patient-derived cerebral organoids

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within NBN, a DNA-damage repair protein. Hallmarks of NBS include several clinical manifestations such growth retardation, chromosomal instability, immunodeficiency and progressive microcephaly. However, the etiology of microcephaly in NBS patients remains elusive. Here, we employed induced pluripotent stem cell-derived brain organoids from two NBS patients to analyze the underlying mechanisms of microcephaly. We show that NBS-organoids carrying the homozygous 647del5 NBN mutation are significantly smaller in size with disrupted cyto-architecture Patient-derived organoids exhibit premature differentiation together with neuronatin (NNAT) overexpression and key pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. Moreover, we show that after exposure to bleomycin, NBS organoids undergo a delayed p53-mediated DNA damage response and aberrant trans-synaptic signalling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders.

NBS1-CtIP–Mediated DNA End Resection Regulates cGAS Binding to Micronuclei

Cyclic GMP-AMP synthase (cGAS), an important component of immune signaling, is hyperactivated in cells defective for DNA damage response (DDR) signaling. However, a direct role for DDR factors in the regulation of cGAS functions is mostly unknown. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor, in coordination with ATM, a protein kinase, and CtBP-interacting protein (CtIP), a DNA end resection factor, functions as an upstream regulator of cGAS binding to micronuclei. Upon NBS1 binding to micronuclei via its fork-head–associated domain, it recruits ATM and CtIP via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1’s interaction with micronuclei, and CtIP converts DSB ends into single-strand DNA ends, and these two key events preclude cGAS from binding to micronuclei. Notably, we show that purified cGAS cannot form a complex with DNA substrates that mimic resected DNA ends in vitro. Thus, NBS1 together with its binding partners modify the chromatin architecture of the micronuclei and that plays a critical role in cGAS’s binding to micronuclei.