Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.

Genetic changes in the FH gene cause vagal paraganglioma.

Vagal paraganglioma (VPGL) is a rare neuroendocrine tumor that originates from the paraganglion associated with the vagus nerve. VPGLs present challenges in terms of diagnostics and treatment. VPGL can occur as a hereditary tumor and, like other head and neck paragangliomas, is most frequently associated with mutations in the SDHx genes. However, data regarding the genetics of VPGL are limited. Herein, we report a rare case of a 41-year-old woman with VPGL carrying a germline variant in the FH gene. Using whole-exome sequencing, a variant, FH p.S249R, was identified; no variants were found in other PPGL susceptibility and candidate genes. Loss of heterozygosity analysis revealed the loss of the wild-type allele of the FH gene in the tumor. The pathogenic effect of the p.S249R variant on FH activity was confirmed by immunohistochemistry for S-(2-succino)cysteine (2SC). Potentially deleterious somatic variants were found in three genes, SLC7A7, ZNF225, and MED23. The latter two encode transcriptional regulators that can impact gene expression deregulation and are involved in tumor development and progression. Moreover, FH-mutated VPGL was characterized by a molecular phenotype different from SDHx-mutated PPGLs. In conclusion, the association of genetic changes in the FH gene with the development of VPGL was demonstrated. The germline variant FH: p.S249R and somatic deletion of the second allele can lead to biallelic gene damage that promotes tumor initiation. These results expand the clinical and mutation spectra of FH-related disorders and improve our understanding of the molecular genetic mechanisms underlying the pathogenesis of VPGL.

Ppx1 putative exopolyphosphatase is essential for polyphosphate accumulation in Lacticaseibacillus paracasei.

The linear polymer polyphosphate (poly-P) is present across all three domains of life and serves diverse physiological functions. The enzyme polyphosphate kinase (Ppk) is responsible for poly-P synthesis, whereas poly-P degradation is carried out by the enzyme exopolyphosphatase (Ppx). In many Lactobacillaceae, the Ppk-encoding gene (ppk) is found clustered together with two genes encoding putative exopolyphosphatases (ppx1 and ppx2) each having different domain compositions, with the gene order ppx1-ppk-ppx2. However, the specific function of these ppx genes remains unexplored. An in-frame deletion of ppx1 in Lacticaseibacillus paracasei BL23 resulted in bacteria unable to accumulate poly-P, whereas the disruption of ppx2 did not affect poly-P synthesis. The expression of ppk was not altered in the Δppx1 strain, and poly-P synthesis in this strain was only restored by expressing ppx1 in trans. Moreover, no poly-P synthesis was observed when ppk was expressed from a plasmid in the Δppx1 strain. Purified Ppx2 exhibited in vitro exopolyphosphatase activity, whereas no in vitro enzymatic activity could be demonstrated for Ppx1. This observation corresponds with the absence in Ppx1 of conserved motifs essential for catalysis found in characterized exopolyphosphatases. Furthermore, assays with purified Ppk and Ppx1 evidenced that Ppx1 enhanced Ppk activity. These results demonstrate that Ppx1 is essential for poly-P synthesis in Lc. paracasei and have unveiled, for the first time, an unexpected role of Ppx1 exopolyphosphatase in poly-P synthesis.IMPORTANCEPoly-P is a pivotal molecular player in bacteria, participating in a diverse array of processes ranging from stress resilience to pathogenesis while also serving as a functional component in probiotic bacteria. The synthesis of poly-P is tightly regulated, but the underlying mechanisms remain incompletely elucidated. Our study sheds light on the distinctive role played by the two exopolyphosphatases (Ppx) found in the Lactobacillaceae bacterial group, of relevance in food and health. This particular group is noteworthy for possessing two Ppx enzymes, supposedly involved in poly-P degradation. Remarkably, our investigation uncovers an unprecedented function of Ppx1 in Lacticaseibacillus paracasei, where its absence leads to the total cessation of poly-P synthesis, paralleling the impact observed upon eliminating the poly-P forming enzyme, poly-P kinase. Unlike the anticipated role as a conventional exopolyphosphatase, Ppx1 demonstrates an unexpected function. Our results added a layer of complexity to our understanding of poly-P dynamics in bacteria.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Structural Evolution of Bacterial Polyphosphate Degradation Enzyme for Phosphorus Cycling.

Living organisms ranging from bacteria to animals have developed their own ways to accumulate and store phosphate during evolution, in particular as the polyphosphate (polyP) granules in bacteria. Degradation of polyP into phosphate is involved in phosphorus cycling, and exopolyphosphatase (PPX) is the key enzyme for polyP degradation in bacteria. Thus, understanding the structure basis of PPX is crucial to reveal the polyP degradation mechanism. Here, it is found that PPX structure varies in the length of ɑ-helical interdomain linker (ɑ-linker) across various bacteria, which is negatively correlated with their enzymatic activity and thermostability - those with shorter ɑ-linkers demonstrate higher polyP degradation ability. Moreover, the artificial DrPPX mutants with shorter ɑ-linker tend to have more compact pockets for polyP binding and stronger subunit interactions, as well as higher enzymatic efficiency (kcat/Km) than that of DrPPX wild type. In Deinococcus-Thermus, the PPXs from thermophilic species possess a shorter ɑ-linker and retain their catalytic ability at high temperatures (70 °C), which may facilitate the thermophilic species to utilize polyP in high-temperature environments. These findings provide insights into the interdomain linker length-dependent evolution of PPXs, which shed light on enzymatic adaption for phosphorus cycling during natural evolution and rational design of enzyme.

Arabidopsis Fhit-like tumor suppressor resumes early terminated constitutive triple response1-10 mRNA translation.

The Arabidopsis (Arabidopsis thaliana) constitutive triple response1-10 (ctr1-10) mutant produces a reduced level of CTR1 protein and exhibits a weak ctr1 mutant phenotype. Sequence analysis revealed highly active translation of the upstream open reading frame (uORF) at the extended 5'-UTR of the ctr1-10 mRNA, resulting from T-DNA insertion. Enhancer screening for ctr1-10 isolated the fragile histidine triad-1 (fhit-1) mutation. The fhit-1 ctr1-10 mutant phenotypically resembled strong ctr1 mutants and barely produced CTR1, and the fhit-1 mutation reduced the translation efficiency of ctr1-10 but not that of CTR1 mRNA. The human (Homo sapiens) Fhit that involves tumorigenesis and genome instability has the in vitro dinucleotide 5',5'″-P1, P3-triphosphate hydrolase activity, and expression of the human HsFHIT or the hydrolase-defective HsFHITH96N transgene reversed the fhit-1 ctr1-10 mutant phenotype and restored CTR1 levels. Genetic editing that in situ disrupts individual upstream ATG codons proximal to the ctr1-10 mORF elevated CTR1 levels in ctr1-10 plants independent of FHIT. EUKARYOTIC INITIATION FACTOR3G (eIF3G), which is involved in translation and reinitiation, interacted with FHIT, and both were associated with the polysome. We propose that FHIT resumes early terminated ctr1-10 mORF translation in the face of active and complex uORF translation. Our study unveils a niche that may lead to investigations on the molecular mechanism of Fhit-like proteins in translation reinitiation. The biological significance of FHIT-regulated translation is discussed.

Structure of the exopolyphosphatase (PPX) from Zymomonas mobilis reveals a two-magnesium-ions PPX.

Rapid adaptation of metabolic capabilities is crucial for bacterial survival in habitats with fluctuating nutrient availability. In such conditions, the bacterial stringent response is a central regulatory mechanism activated by nutrient starvation or other stressors. This response is primarily controlled by exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes. To gain further insight into these enzymes, the high-resolution crystal structure of PPX from Zymomonas mobilis (ZmPPX) was determined at 1.8 Å. The phosphatase activity of PPX was strictly dependent on the presence of divalent metal cations. Notably, the structure of ZmPPX revealed the presence of two magnesium ions in the active site center, which is atypical compared to other PPX structures where only one divalent ion is observed. ZmPPX exists as a dimer in solution and belongs to the "long" PPX group consisting of four domains. Remarkably, the dimer configuration exhibits a substantial and deep aqueduct with positive potential along its interface. This aqueduct appears to extend towards the active site region, suggesting that this positively charged aqueduct could potentially serve as a binding site for polyP.

Integrated analysis of FHIT gene alterations in cancer.

The Fragile Histidine Triad Diadenosine Triphosphatase (FHIT) gene is located in the Common Fragile Site FRA3B and encodes an enzyme that hydrolyzes the dinucleotide Ap3A. Although FHIT loss is one of the most frequent copy number alterations in cancer, its relevance for cancer initiation and progression remains unclear. FHIT is frequently lost in cancers from the digestive tract, which is compatible with being a cancer driver event in these tissues. However, FHIT loss could also be a passenger event due to the inherent fragility of the FRA3B locus. Moreover, the physiological relevance of FHIT enzymatic activity and the levels of Ap3A is largely unclear. We have conducted here a systematic pan-cancer analysis of FHIT status in connection with other mutations and phenotypic alterations, and we have critically discussed our findings in connection with the literature to provide an overall view of FHIT implications in cancer.

PRUNE1 and NME/NDPK family proteins influence energy metabolism and signaling in cancer metastases.

We describe here the molecular basis of the complex formation of PRUNE1 with the tumor metastasis suppressors NME1 and NME2, two isoforms appertaining to the nucleoside diphosphate kinase (NDPK) enzyme family, and how this complex regulates signaling the immune system and energy metabolism, thereby shaping the tumor microenvironment (TME). Disrupting the interaction between NME1/2 and PRUNE1, as suggested, holds the potential to be an excellent therapeutic target for the treatment of cancer and the inhibition of metastasis dissemination. Furthermore, we postulate an interaction and regulation of the other Class I NME proteins, NME3 and NME4 proteins, with PRUNE1 and discuss potential functions. Class I NME1-4 proteins are NTP/NDP transphosphorylases required for balancing the intracellular pools of nucleotide diphosphates and triphosphates. They regulate different cellular functions by interacting with a large variety of other proteins, and in cancer and metastasis processes, they can exert pro- and anti-oncogenic properties depending on the cellular context. In this review, we therefore additionally discuss general aspects of class1 NME and PRUNE1 molecular structures as well as their posttranslational modifications and subcellular localization. The current knowledge on the contributions of PRUNE1 as well as NME proteins to signaling cascades is summarized with a special regard to cancer and metastasis.

Pathological Roles of INPP5D in Alzheimer's Disease.

Current hypothesis of Alzheimer's disease (AD) postulates that amyloid ß (Aß) deposition in the brain causes tau inclusion in neurons and leads to cognitive decline. The discovery of the genetic association between triggering receptor expressed on myeloid cells 2 (TREM2) with increased AD risk points to a causal link between microglia and AD pathogenesis, and revealed a crucial role of TREM2-dependent clustering of microglia around amyloid plaques that prevents Aß toxicity to facilitate tau deposition near the plaques. Here we review the physiological and pathological roles of another AD risk gene expressed in microglia, inositol polyphosphate-5-polyphosphatase D (INPP5D), which encodes a phosphoinositide phosphatase. Evidence suggests that its risk polymorphisms alter the expression level and/or function of INPP5D, while concomitantly affecting tau levels in cerebrospinal fluids. In ß-amyloidosis mice, INPP5D was upregulated upon Aß deposition and negatively regulated the microglial clustering toward amyloid plaques. INPP5D seems to exert its function by acting antagonistically at downstream of the TREM2 signaling pathway, suggesting that it is a novel regulator of the protective barrier by microglia. Further studies to elucidate INPP5D's role in AD may help in developing new therapeutic targets for AD treatment.

Importance of Germline and Somatic Alterations in Human MRE11, RAD50, and NBN Genes Coding for MRN Complex.

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.

RBBP4 regulates the expression of the Mre11-Rad50-NBS1 (MRN) complex and promotes DNA double-strand break repair to mediate glioblastoma chemoradiotherapy resistance.

For treatment of glioblastoma (GBM), temozolomide (TMZ) and radiotherapy (RT) exert antitumor effects by inducing DNA double-strand breaks (DSBs), mainly via futile DNA mismatch repair (MMR) and inducing apoptosis. Here, we provide evidence that RBBP4 modulates glioblastoma resistance to chemotherapy and radiotherapy by recruiting transcription factors and epigenetic regulators that bind to their promoters to regulate the expression of the Mre11-Rad50-NBS1(MRN) complex and the level of DNA-DSB repair, which are closely associated with recovery from TMZ- and radiotherapy-induced DNA damage in U87MG and LN229 glioblastoma cells, which have negative MGMT expression. Disruption of RBBP4 induced GBM cell DNA damage and apoptosis in response to TMZ and radiotherapy and enhanced radiotherapy and chemotherapy sensitivity by the independent pathway of MGMT. These results displayed a possible chemo-radioresistant mechanism in MGMT negative GBM. In addition, the RBBP4-MRN complex regulation axis may provide an interesting target for developing therapy-sensitizing strategies for GBM.

Systematic Review and Meta-Analysis of the Influence of Genetic Variation on Ototoxicity in Platinum-Based Chemotherapy.

OBJECTIVE: The objective of this meta-analysis is to evaluate the impact of genetic polymorphisms on platinum-based chemotherapy (PBC)-induced ototoxicity. DATA SOURCES: Systematic searches of PubMed, Embase, Cochrane, and Web of Science were conducted from the inception of the databases to May 31, 2022. Abstracts and presentations from conferences were also reviewed. REVIEW METHODS: Four investigators independently extracted data in adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Differences in the prevalence of PBC-induced ototoxicity between reference and variant (i) genotypes and (ii) alleles were analyzed. The overall effect size was presented using the random-effects model as an odds ratio (OR) with a 95% confidence interval (CI). RESULTS: From 32 included articles, 59 single nucleotide polymorphisms on 28 genes were identified, with 4406 total unique participants. For allele frequency analysis, the A allele in ACYP2 rs1872328 was positively associated with ototoxicity (OR: 2.61; 95% CI: 1.06-6.43; n = 2518). Upon limiting to cisplatin use only, the T allele of COMT rs4646316 and COMT rs9332377 revealed significant results. For genotype frequency analysis, the CT/TT genotype in ERCC2 rs1799793 demonstrated an otoprotective effect (OR: 0.50; 95% CI: 0.27-0.94; n = 176). Excluding studies using carboplatin or concomitant radiotherapy revealed significant effects with COMT rs4646316, GSTP1 rs1965, and XPC rs2228001. Major sources of variations between studies include differences in patient demographics, ototoxicity grading systems, and treatment protocols. CONCLUSION: Our meta-analysis presents polymorphisms that exert ototoxic or otoprotective effects in patients undergoing PBC. Importantly, several of these alleles are observed at high frequencies globally, highlighting the potential for polygenic screening and cumulative risk evaluation for personalized care.

The Rosetta Stone Hypothesis-Based Interaction of the Tumor Suppressor Proteins Nit1 and Fhit.

In previous studies, we have identified the tumor suppressor proteins Fhit (fragile histidine triad) and Nit1 (Nitrilase1) as interaction partners of ß-catenin both acting as repressors of the canonical Wnt pathway. Interestingly, in D. melanogaster and C. elegans these proteins are expressed as NitFhit fusion proteins. According to the Rosetta Stone hypothesis, if proteins are expressed as fusion proteins in one organism and as single proteins in others, the latter should interact physically and show common signaling function. Here, we tested this hypothesis and provide the first biochemical evidence for a direct association between Nit1 and Fhit. In addition, size exclusion chromatography of purified recombinant human Nit1 showed a tetrameric structure as also previously observed for the NitFhit Rosetta Stone fusion protein Nft-1 in C. elegans. Finally, in line with the Rosetta Stone hypothesis we identified Hsp60 and Ubc9 as other common interaction partners of Nit1 and Fhit. The interaction of Nit1 and Fhit may affect their enzymatic activities as well as interaction with other binding partners.

Advanced ovarian clear cell carcinoma with RAD50 mutation treated by PARP inhibitor pamiparib combined with anti-angiogenesis therapy: a case report.

Ovarian clear cell carcinoma (OCCC) is a relatively uncommon epithelial ovarian malignancy with unique clinical, histopathologic and genetic characteristics. Patients with advanced OCCC have poor outcomes and are resistant to standard chemotherapy. Targeted therapy offers a novel approach for treating OCCC. We report the case of a 45-year-old female patient with advanced OCCC who experienced relapse after standard treatment. Further, a frameshift mutation in the homologous recombination repair-related gene RAD50 (RAD50-p.I371Ffs*8) was identified by genetic testing. Next, the patient had received targeted combination therapy with poly (ADP-ribose) polymerase (PARP) inhibitor pamiparib and bevacizumab, achieving partial remission. Patient's symptoms improved significantly compared to before. To date, the patient has been followed up for more than half a year with favorable survival and high quality of life. The case report suggested that parmiparib-targeted therapy is a viable treatment option for advanced OCCC patients with RAD50 mutation.

The emerging landscape of eukaryotic polyphosphatases.

Polyphosphate (polyP) is a conserved polymer of inorganic phosphate residues that can reach thousands of moieties in length. PolyP has been implicated in cellular functions ranging from energy and phosphate homeostasis to cell signalling in eukaryotes from yeast to humans. Despite the interest in the role of polyP as a signalling molecule, the spatiotemporal regulation of polyP itself remains poorly understood. This knowledge gap limits our ability to understand how polyP impacts the physiology of normal and diseased cells and how this might be exploited in a therapeutic context. Polyphosphatases, enzymes that degrade polyP to generate shorter chains and free inorganic phosphate are ideally positioned to mediate polyP dynamics. However, little is known about how the activities of these enzymes are linked to specific cellular functions and how they might be regulated. Here, we provide an in-depth overview of polyphosphatase enzymes in budding yeast, which has served as a workhorse for polyP research, and in mammalian cells where the enzymes that make and degrade polyP have remained elusive. We identify critical open questions in both systems and propose strategies to guide future work.

TRIM24 is critical for the cellular response to DNA double-strand breaks through regulating the recruitment of MRN complex.

The MRE11-RAD50-NBS1 (MRN) complex plays a crucial role in DNA double-strand breaks (DSBs) sensing and initiation of signaling cascades. However, the precise mechanisms by which the recruitment of MRN complex is regulated has yet to be elucidated. Here, we identified TRIpartite motif-containing protein 24 (TRIM24), a protein considered as an oncogene overexpressed in cancers, as a novel signaling molecule in response to DSBs. TRIM24 is essential for DSBs-induced recruitment of MRN complex and activation of downstream signaling. In the absence of TRIM24, MRN mediated DSBs repair is remarkably diminished. Mechanistically, TRIM24 is phosphorylated by ataxia-telangiectasia mutated (ATM) and then recruited to DSBs sites, facilitating the accumulation of the MRN components to chromatin. Depletion of TRIM24 sensitizes human hepatocellular carcinoma cells to cancer therapy agent-induced apoptosis and retards the tumor growth in a subcutaneous xenograft tumor mouse model. Together, our data reveal a novel function of TRIM24 in response to DSBs through regulating the MRN complex, which suggests that TRIM24 may be a potential therapeutic molecular target for tumor treatment.

Deubiquitinase USP2 stabilizes the MRE11-RAD50-NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1.

The MRE11-RAD50-NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.

Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway.

Cisplatin and other platinum-based anticancer agents are among the most widely used chemotherapy drugs in the treatment of different types of cancer. However, it is common to find patients who respond well to treatment at first but later relapse due to the appearance of resistance to cisplatin. Among the mechanisms responsible for this phenomenon is the increase in DNA damage repair. Here, we elucidate the effect of cisplatin on the MRN (MRE11-RAD50-NBS1) DNA damage sensor complex. We found that the tumor suppressor FBXW7 is a key factor in controlling the turnover of the MRN complex by inducing its degradation through lysosomes. Inhibition of lysosomal enzymes allowed the detection of the association of FBXW7-dependent ubiquitylated MRN with LC3 and the autophagy adaptor p62/SQSTM1 and the localization of MRN in lysosomes. Furthermore, cisplatin-induced cell death increased MRN degradation, suggesting that this complex is one of the targets that favor cell death. These findings open the possibility of using the induction of the degradation of the MRN complex after genotoxic damage as a potential therapeutic strategy to eliminate tumor cells.

Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions.

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer. MRN has two DNA-binding modes, one ATP-dependent mode for loading onto DNA ends and one ATP-independent mode through Mre11's C terminus, suggesting how it may interact with DSBs and intact DNA. MRNs two 60-nm-long coiled-coil domains form a linear rod structure, the apex of which is assembled by the two joined zinc-hook motifs. Apices from two MRN complexes can further dimerize, forming 120-nm spanning MRN-MRN structures. Our results illustrate the architecture of MRN and suggest how it mechanistically integrates catalytic and tethering functions.