Mitochondrial Outer Membrane Permeability Change and Hypersensitivity to Digitonin Early in Staurosporine-induced Apoptosis

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK- cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK- osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK- cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage.

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes

Role of Pirh2 in Mediating the Regulation of p53 and c-Myc

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2−/− mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops

Cloning and characterization of an Arabidopsis thaliana vacuolar Na§+/H§+ antiport-AtNHX3

grantor: University of TorontoIn 'Arabidopsis', overexpression of vacuolar Na+/H+ antiport, AtNHX 1, increases salt tolerance of the plant. Genomic DNA database search indicates that there are four other genes that are homologous to 'AtNHX1'. In this thesis, the cloning and characterization of 'AtNHX3' is reported. The ORF of 'AtNHX3' is about 1.5 kb, coding for 521 amino acids. AtNHX3 protein shares about 35% identical sequence with AtNHX1 and AtNHX2. Western blotting shows that AtNHX3 is located on tonoplast. Northern-blotting and semi-quantitative RT-PCR suggest that AtNHX3 mRNA is in root, stem, leaf and flower, and there is no significant difference for mRNA levels between young seedlings and adult plants. Furthermore, AtNHX3 mRNA levels do not change in response to salt treatment. AtNHX3 can complement a yeast salt sensitive mutant, [Delta]nhx1, and overexpression of AtNHX3 in 'Arabidopsis' greatly increases salt tolerance of the plant. Yeast complementation studies suggest that C-terminus of AtNHX3 is necessary for the full function of this antiport.M.Sc

Cbx2 targets PRC1 to constitutive heterochromatin in mouse zygotes in a parent-of-origin-dependent manner

Polycomb repressive complexes PRC1 and PRC2 regulate expression of genes involved in proliferation and development. In mouse early embryos, however, canonical PRC1 localizes to paternal pericentric heterochromatin (pat-PCH), where it represses transcription of major satellite repeats. In contrast, maternal PCH (mat-PCH) is enriched for H3 lysine 9 tri-methylation (H3K9me3) and Hp1β. How PRC1 is targeted to pat-PCH, yet excluded from mat-PCH, has remained elusive. Here, we identify a PRC1 targeting mechanism that relies on Cbx2 and Hp1β. Cbx2 directs catalytically active PRC1 to PCH via its chromodomain (CDCbx2) and neighboring AT-hook (ATCbx2) binding to H3K27me3 and AT-rich major satellites, respectively. CDCbx2 prevents ATCbx2 from interacting with DNA at PCH marked by H3K9me3 and Hp1β. Loss-of-function studies show that Hp1β and not H3K9me3 prevents PRC1 targeting to mat-PCH. Our findings indicate that CDCbx2 and ATCbx2 separated by a short linker function together to integrate H3K9me3/HP1 and H3K27me3 states

Cryo-EM HTT Q23 (+/- DNA) sample generation (2017/06/05)

Huntingtin structure-function open lab notebook projec

Huntingtin Q23 with FLAG antibody Grafix in 0-1 percent Glutaraldehyde – 28th November 2017

Huntingtin structure function open lab noteboo