The ends of linear eukaryotic chromosomes are protected by telomeres, which serve to ensure proper chromosome replication and to prevent spurious recombination at chromosome ends. In this study, we ...show by single cell analysis that in the absence of telomerase, a single short telomere is sufficient to induce the recruitment of checkpoint and recombination proteins. Notably, a DNA damage response at eroded telomeres starts many generations before senescence and is characterized by the recruitment of Cdc13 (cell division cycle 13), replication protein A, DNA damage checkpoint proteins and the DNA repair protein Rad52 into a single focus. Moreover, we show that eroded telomeres, although remaining at the nuclear periphery, move to the nuclear pore complex. Our results link the DNA damage response at eroded telomeres to changes in subnuclear localization and suggest the existence of collapsed replication forks at eroded telomeres.
The vacuolar protein sorting 33B (VPS33B) was rarely reported in malignant tumors. In this research, we demonstrated that overexpression of VPS33B inhibited proliferation and chemoresistance to ...fluorouracil (5-FU) in nasopharyngeal carcinoma (NPC) in vivo and in vitro. Mechanistic analysis confirmed that overexpression of VPS33B modulated EGFR/PI3K/AKT/c-Myc/P53 signaling to arrest the cell cycle at G1/S phase. In addition, miR-133a-3p, a tumor-suppressive miRNA, was induced by P53 and directly targeted the EGFR/PI3K/AKT/c-Myc/P53 signaling and thus formed a negative feedback loop. Furthermore, another tumor suppressor, NESG1, interacted with VPS33B by colocalizing in the cytoplasm. The knockdown of NESG1 reversed the inhibitory effects of the overexpression of VPS33B in NPC cells by downregulating the PI3K/AKT/c-Jun-mediated transcription repression. Surprisingly, VPS33B was downregulated in the nicotine-treated and LMP-1-overexpressing NPC cells by targeting PI3K/AKT/c-Jun-mediated signaling. In addition, patients with higher VPS33B expression had a longer overall survival. Our study is the first to demonstrate that VPS33B is negatively regulated by LMP-1 and nicotine and thus suppresses the proliferation of NPC cells by interacting with NESG1 to regulate EGFR/PI3K/AKT/c-Myc/P53/miR-133a-3p signaling in NPC cells.
Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work ...was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer.
Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively.
The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells.
Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.
The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the ...heterotetrameric (ε-β4-μ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as "AP-4 deficiency syndrome". In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the μ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.
The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better ...understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).
Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 ...(LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (Scl
N93A
), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that Scl
N93A
competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of Scl
N93A
fused to the fragment crystallizable (Fc) domain of immunoglobulin (Scl
N93A
Fc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl–LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl–LRP4 interactions offer a potential strategy to promote anabolic bone functions.
Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to ...heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.
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•DSBs in G1 are repaired by resection-independent or resection-dependent c-NHEJ•Resection-dependent c-NHEJ is induced by Plk3-dependent CtIP interaction with Brca1•G1 resection is executed from DSB ends by Mre11 exo, EXD2, and Exo1, but not Mre11 endo•Artemis completes resection in G1, forming deletions and, potentially, translocations
Biehs et al. identify and characterize a resection-dependent canonical non-homologous end joining process that operates in G1 phase cells. Resection in G2 leads to homologous recombination. Resection in G1 is distinct from that because the same toolbox of resection factors is orchestrated in a way that is compatible with an end-joining process.
TolR is a 15-kDa inner membrane protein subunit of the Tol-Pal complex in Gram-negative bacteria, and its function is poorly understood. Tol-Pal is recruited to cell division sites where it is ...involved in maintaining the integrity of the outer membrane. TolR is related to MotB, the peptidoglycan (PG)-binding stator protein from the flagellum, suggesting it might serve a similar role in Tol-Pal. The only structure thus far reported for TolR is of the periplasmic domain from Haemophilus influenzae in which N- and C-terminal residues had been deleted (TolR(62–133), Escherichia coli numbering). H. influenzae TolR(62–133) is a symmetrical dimer with a large deep cleft at the dimer interface. Here, we present the 1.7-Å crystal structure of the intact periplasmic domain of E. coli TolR (TolR(36–142)). E. coli TolR(36–142) is also dimeric, but the architecture of the dimer is radically different from that of TolR(62–133) due to the intertwining of its N and C termini. TolR monomers are rotated ∼180° relative to each other as a result of this strand swapping, obliterating the putative PG-binding groove seen in TolR(62–133). We found that removal of the strand-swapped regions (TolR(60–133)) exposes cryptic PG binding activity that is absent in the full-length domain. We conclude that to function as a stator in the Tol-Pal complex dimeric TolR must undergo large scale structural remodeling reminiscent of that proposed for MotB, where the N- and C-terminal sequences unfold in order for the protein to both reach and bind the PG layer ∼90 Å away from the inner membrane.
Background: TolR forms part of the proton-motive force (pmf)-linked stator of the Tol-Pal complex in bacteria.
Results: Structural and biophysical analysis shows the periplasmic domain of TolR to be a strand-swapped dimer.
Conclusion: Strand swapping blocks peptidoglycan binding by TolR.
Significance: Pmf-linked stator proteins must disassemble strand-swapped dimer structures to traverse the periplasm and bind peptidoglycan.
Glioblastoma (GBM) is the most common primary brain tumor, with poor prognosis and a lack of effective therapeutic options. The aberrant expression of transcription factor REST (repressor element ...1-silencing transcription factor) had been reported in different kinds of tumors. However, the function of REST and its mechanisms in GBM remain elusive. Here, REST expression was inhibited by siRNA silencing in U-87 and U-251 GBM cells. Then CCK-8 assay showed significantly decreased cell proliferation, and the inhibition of migration was verified by scratch wound healing assay and transwell assay. Using cell cycle analysis and Annexin V/PI straining assay, G1 phase cell cycle arrest was found to be a reason for the suppression of cell proliferation and migration upon REST silencing, while apoptosis was not affected by REST silencing. Further, the detection of REST-downstream genes involved in cytostasis and migration inhibition demonstrated that CCND1 and CCNE1 were reduced; CDK5R1, BBC3, EGR1, SLC25A4, PDCD7, MAPK11, MAPK12, FADD and DAXX were enhanced, among which BBC3 and DAXX were direct targets of REST, as verified by ChIP (chromatin immunoprecipitation) and Western blotting. These data suggested that REST is a master regulator that maintains GBM cells proliferation and migration, partly through regulating cell cycle by repressing downstream genes, which might represent a potential target for GBM therapy.
The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT ...(Tel2, Tti1, Tti2) cochaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks), of which IP6K2 has been implicated in p53-associated cell death. In the present study we report an apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex, thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine 15 to activate the cell death program in human cancer cells and in murine B cells.
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•IP7 binds with high affinity to CK2•IP7 binding enhances CK2-mediated phosphorylation of Tel2/Tti1•IP6K2 binds Tti1 to facilitate stabilization of DNA-PKcs/ATM•The IP7/CK2/TTT cascade activates the DNA-PKcs/ATM-p53 apoptotic program
Human IP6K2 mediates p53-dependent cell death. Rao et al. show that IP6K2 functions by promoting p53 phosphorylation by DNA-PKcs/ATM. IP6K2 acts on DNA-PKcs/ATM indirectly by generating an inositol pyrophosphate (IP7) that binds to CK2 and mediates CK2-dependent phosphorylation of a chaperone complex (Tel2/Tti1/Tti2) known to control the stability and activity of DNA-PKcs/ATM.
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