Knockout MEFs Research Articles

ARTS and small-molecule ARTS mimetics upregulate p53 levels by promoting the degradation of XIAP

Mutations resulting in decreased activity of p53 tumor suppressor protein promote tumorigenesis. P53 protein levels are tightly regulated through the Ubiquitin Proteasome System (UPS). Several E3 ligases were shown to regulate p53 stability, including MDM2. Here we report that the ubiquitin E3 ligase XIAP (X-linked Inhibitors of Apoptosis) is a direct ligase for p53 and describe a novel approach for modulating the levels of p53 by targeting the XIAP pathway. Using in vivo (live-cell) and in vitro (cell-free reconstituted system) ubiquitylation assays, we show that the XIAP-antagonist ARTS regulates the levels of p53 by promoting the degradation of XIAP. XIAP directly binds and ubiquitylates p53. In apoptotic cells, ARTS inhibits the ubiquitylation of p53 by antagonizing XIAP. XIAP knockout MEFs express higher p53 protein levels compared to wild-type MEFs. Computational screen for small molecules with high affinity to the ARTS-binding site within XIAP identified a small-molecule ARTS-mimetic, B3. This compound stimulates apoptosis in a wide range of cancer cells but not normal PBMC (Peripheral Blood Mononuclear Cells). Like ARTS, the B3 compound binds to XIAP and promotes its degradation via the UPS. B3 binding to XIAP stabilizes p53 by disrupting its interaction with XIAP. These results reveal a novel mechanism by which ARTS and p53 regulate each other through an amplification loop to promote apoptosis. Finally, these data suggest that targeting the ARTS binding pocket in XIAP can be used to increase p53 levels as a new strategy for developing anti-cancer therapeutics.

Bcl-xL regulates radiation-induced ferroptosis through chaperone-mediated autophagy of GPX4 in tumor cells

ObjectiveTo investigate the role and the molecular mechanisms of apoptotic signaling in ferroptosis to regulate tumor radiosensitivity. MethodsReactive oxygen species (ROS) and lipid peroxide levels were detected in Mouse embryonic fibroblasts(MEFs) with Bcl-xL or Mcl-1 deficiency induced by erastin. Colony formation, ROS, lipid peroxidation and the transcription/translation levels of PTGS2 were measured in Bcl-xL knockdown tumor cells induced by 5 ​Gy γ-rays or co-treated with ferrostatin-1 (Ferr-1). The protein levels of LPCAT3, ACSL4 and PEBP1 in Bcl-xL knockout MEF cells were evaluated in Bcl-xL knockout MEF cells post-radiation. Moreover, the interaction of heat shock protein 90 (HSP90) with Bcl-xL, GPX4, or LAMP2A was detected by protein mass spectrometry and immunoprecipitation assays. ResultsManipulating Bcl-xL levels facilitated radiation-induced ferroptosis by augmenting the enzymatic oxidation of polyunsaturated fatty acids (PUFAs) and enhancing chaperone-mediated autophagy (CMA) of glutathione peroxidase 4 (GPX4) (MEF cell line: t=4.540, P＜0.01; A549 ​cell line: t=56.16, P＜0.0001; t=4.885, P＜0.01; HCT116 ​cell line: t=14.75, P＜0.01; t=7.363, P＜0.05). Downregulating Bcl-xL expression promoted the activity of acyl-CoA synthetase long-chain family member 4 (ACSL4), thus increasing the enzymatic oxidation of PUFAs (t=4.258, P＜0.01). Moreover, depletion of Bcl-xL expedited the CMA process targeting GPX4 by facilitating the association of GPX4 with heat shock protein 90 (HSP90) and LAMP2A following radiation exposure. Subsequent degradation of GPX4 led to the accumulation of lipid peroxides, ultimately triggering ferroptosis. ConclusionsOur study provides initial insights into the regulatory role of Bcl-xL in ferroptosis and underscores the potential of targeting Bcl-xL as a promising therapeutic strategy for cancer by modulating both apoptotic and ferroptotic pathways.

IF1 Promotes Cellular Proliferation and Inhibits Oxidative Phosphorylation in Mouse Embryonic Fibroblasts under Normoxia and Hypoxia.

ATP synthase inhibitory factor subunit 1 (IF1) is an inhibitory subunit of mitochondrial ATP synthase, playing a crucial role in regulating mitochondrial respiration and energetics. It is well-established that IF1 interacts with the F1 sector of ATP synthase to inhibit the reversal rotation and, thus, ATP hydrolysis. Recent evidence supports that IF1 also inhibits forward rotation or the ATP synthesis activity. Adding to the complexity, IF1 may also facilitate mitophagy and cristae formation. The implications of these complex actions of IF1 for cellular function remain obscure. In the present study, we found that IF1 expression was markedly upregulated in hypoxic MEFs relative to normoxic MEFs. We investigate how IF1 affects cellular growth and function in cultured mouse embryonic fibroblasts derived from mouse lines with systemic IF1 overexpression and knockout under normoxia and hypoxia. Cell survival and proliferation analyses revealed that IF1 overexpression exerted limited effects on cellular viability but substantially increased proliferation under normoxia, whereas it facilitated both cellular viability and proliferation under hypoxia. The absence of IF1 may have a pro-survival effect but not a proliferative one in both normoxia and hypoxia. Cellular bioenergetic analyses revealed that IF1 suppressed cellular respiration when subjected to normoxia and was even more pronounced when subjected to hypoxia with increased mitochondrial ATP production. In contrast, IF1 knockout MEFs showed markedly increased cellular respiration under both normoxia and hypoxia with little change in mitochondrial ATP. Glycolytic stress assay revealed that IF1 overexpression modestly increased glycolysis in normoxia and hypoxia. Interestingly, the absence of IF1 in MEFs led to substantial increases in glycolysis. Therefore, we conclude that IF1 mainly inhibits cellular respiration and enhances cellular glycolysis to preserve mitochondrial ATP. On the other hand, IF1 deletion can significantly facilitate cellular respiration and glycolysis without leading to mitochondrial ATP deficit.

Yeast Bxi1/Ybh3 mediates conserved mitophagy and apoptosis in yeast and mammalian cells: convergence in Bcl-2 family.

The process of degrading unwanted or damaged mitochondria by autophagy, called mitophagy, is essential for mitochondrial quality control together with mitochondrial apoptosis. In mammalian cells, pan-Bcl-2 family members including conical Bcl-2 members and non-conical ones are involved in and govern the two processes. We have illustrated recently the BH3 receptor Hsp70 interacts with Bim to mediate both apoptosis and mitophagy. However, whether similar pathways exist in lower eukaryotes where conical Bcl-2 members are absent remained unclear. Here, a specific inhibitor of the Hsp70-Bim PPI, S1g-10 and its analogs were used as chemical tools to explore the role of yeast Bxi1/Ybh3 in regulating mitophagy and apoptosis. Using Om45-GFP processing assay, we illustrated that yeast Ybh3 mediates a ubiquitin-related mitophagy pathway in both yeast and mammalian cells through association with Hsp70, which is in the same manner with Bim. Moreover, by using Bax/Bak double knockout MEF cells, Ybh3 was identified to induce apoptosis through forming oligomerization to trigger mitochondrial outer membrane permeabilization (MOMP) like Bax. We not only illustrated a conserved ubiquitin-related mitophagy pathway in yeast but also revealed the multi-function of Ybh3 which combines the function of BH3-only protein and multi-domain Bax protein as one.

Deletion of exons 2 and 3 from Actb and cell immortalization lead to widespread, β-actin independent alterations in gene expression associated with cell cycle control

The cytoplasmic actin proteins, β- and γ-actin, are 99% identical but thought to perform non-redundant functions. The nucleotide coding regions of cytoplasmic actin genes, Actb and Actg1, are 89% identical. Knockout (KO) of Actb by Cre-mediated deletion of first coding exons 2 and 3 in mice is embryonic lethal and fibroblasts derived from KO embryos (MEFs) fail to proliferate. In contrast, Actg1 KO MEFs display with a much milder defect in cell proliferation and Actg1 KO mice are viable, but present with increased perinatal lethality. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the β-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry reveal largescale changes to the transcriptome, proteome, and phosphoproteome in cells lacking Actb but not those only lacking β-actin protein. Pathway analysis of genes and proteins differentially expressed upon Actb KO suggest widespread dysregulation of genes involved in the cell cycle that may explain the severe defect in proliferation.

HDAC6 preserves BNIP3 expression and mitochondrial integrity by deacetylating p53 at lysine 320

HDAC6 has been reported as a deacetylase of p53 at multiple lysine residues, associated with the canonical functions of p53, such as apoptosis and tumor suppression. We have previously reported that p53 acetylation at the lysine 320 site accumulates due to the genetic ablation of HDAC6 in mice liver. However, the biological processes affected by K320 acetylation of p53 are yet to be elucidated. In this study, we demonstrate that K320 acetylation of p53 is regulated by HDAC6 deacetylase activity. HDAC6 knockout mouse brains exhibit a significant accumulation of K320 acetylated p53 compared to other tissues. The level of K320 acetylation of p53 inversely correlates with the level of BNIP3, a direct target of p53 and essential for mitophagy. Notably, overexpressing the deacetylation mimic K320R mutant p53 restored BNIP3 expression in HDAC6 knockout MEFs. Furthermore, we observed that neurons are particularly susceptible to the genetic ablation of HDAC6, impacting BNIP3 expression, which inversely correlates with the accumulation of abnormal mitochondria characterized by swollen cristae. Our findings suggest that HDAC6 plays a crucial role in maintaining BNIP3 expression by deacetylating p53 at the K320 site, which is linked to the structural integrity of mitochondria.

Circadian control of cisplatin-DNA adduct repair and apoptosis in culture cells.

Cisplatin, a widely prescribed chemotherapeutic agent for treating solid tumors, induces DNA adducts and activates cellular defense mechanisms, including DNA repair, cell cycle checkpoint control, and apoptosis. Considering the circadian rhythmicity displayed by most chemotherapeutic agents and their varying therapeutic efficacy based on treatment timing, our study aimed to investigate whether the circadian clock system influences the DNA damage responses triggered by cisplatin in synchronized cells. We examined the DNA damage responses in circadian-synchronized wild-type mouse embryonic fibroblasts (WT-MEF; clock-proficient cells), cryptochrome1 and 2 double knock-out MEF (CRYDKO; clock-deficient cells), and mouse hepatocarcinoma Hepa1c1c7 cells. Varying the treatment time resulted in a significant difference in the rate of platinum-DNA adduct removal specifically in circadian-synchronized WT-MEF, while CRYDKO did not exhibit such variation. Moreover, diurnal variation in other DNA damage responses, such as cell cycle checkpoint activity indicated by p53 phosphorylation status and apoptosis measured by DNA break frequency, was observed only in circadian-synchronized WT-MEF, not in CRYDKO or mouse hepatocarcinoma Hepa1c1c7 cells. These findings highlight that the DNA damage responses triggered by cisplatin are indeed governed by circadian control exclusively in clock-proficient cells. This outcome bears potential implications for enhancing or devising chronotherapy approaches for cancer patients.

MKRN2 knockout causes male infertility through decreasing STAT1, SIX4, and TNC expression.

Makorin-2 (Mkrn2) is an evolutionarily conserved gene whose biological functions are not fully known. Although recent studies have shed insights on the potential causes of male infertility, its underlining mechanisms still remain to be elucidated. We developed a Mrkn2 knockout mice model to study this gene and found that deletion of Mkrn2 in mice led to male infertility. Interestingly, the expression level of signal transducer and activator of the transcription (STAT)1 was significantly decreased in MKRN2 knockout testis and MEF cells. Co-IP assay showed an interaction between MKRN2 and STAT1. Moreover, our results further indicated that MKRN2 regulated the expression level of SIX4 and tenascin C (TNC) via the EBF transcription factor 2 (EBF2) in mice. The results of our study will provide insights into a new mechanism of male infertility.

Opposite roles of Kindlin orthologs in cell survival and proliferation.

ObjectiveIt is unclear why adhesion‐dependent cells such as epithelium undergo anoikis without anchorage, while adhesion‐independent blood cells thrive in suspension. The adhesive machinery of these cells is similar, with the exception of Kindlin orthologs, Kindlin 2 (K2) and Kindlin 3 (K3). Here we address how Kindlins control cell survival and proliferation in anchorage‐dependent and independent cells.Material and MethodsTo demonstrate the opposite roles of Kindlin's in cell survival we utilized in vivo and in vitro models and K3 and K2 knockdown and knockin cells. We used human lymphocytes from the K3 deficient patients in tumour model, K3 knockout and knockin macrophages and K2 knockout and knockin MEF cells for experiments in under conditions of adhesion and in suspension.ResultsDepletion of K3 promotes cell proliferation and survival of anchorage‐independent cells regardless of cell attachment. In contrast, the absence of K2 in anchorage‐dependent cells accelerates apoptosis and limits proliferation. K3 deficiency promotes human lymphoma growth and survival in vivo. Kindlins' interaction with paxillin, is critical for their differential roles in cell anchorage. While disruption of K2‐paxillin binding leads to increased apoptosis, the lack of K3‐paxillin binding has an opposite effect in adhesion‐independent cells.ConclusionKindlin ortologs and their interaction to cytoskeletal protein paxillin define the mechanisms of anchorage dependence. Our study identifies the key elements of the cell adhesion machinery in cell survival and tumour metastasis, proposing possible targets for tumour treatment.

Curcumin activates Nrf2 through PKC\u03b4-mediated p62 phosphorylation at Ser351

Curcumin, a phytochemical extracted from Curcuma longa rhizomes, is known to be protective in neurons via activation of Nrf2, a master regulator of endogenous defense against oxidative stress in cells. However, the exact mechanism by which curcumin activates Nrf2 remains controversial. Here, we observed that curcumin induced the expression of genes downstream of Nrf2 such as HO-1, NQO1, and GST-mu1 in neuronal cells, and increased the level of Nrf2 protein. Notably, the level of p62 phosphorylation at S351 (S349 in human) was significantly increased in cells treated with curcumin. Additionally, curcumin-induced Nrf2 activation was abrogated in p62 knockout (−/−) MEFs, indicating that p62 phosphorylation at S351 played a crucial role in curcumin-induced Nrf2 activation. Among the kinases involved in p62 phosphorylation at S351, PKCδ was activated in curcumin-treated cells. The phosphorylation of p62 at S351 was enhanced by transfection of PKCδ expression plasmid; in contrast, it was inhibited in cells treated with PKCδ-specific siRNA. Together, these results suggest that PKCδ is mainly involved in curcumin-induced p62 phosphorylation and Nrf2 activation. Accordingly, we demonstrate for the first time that curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at S351.

Loss of Function of the Gene Encoding the Histone Methyltransferase KMT2D Leads to Deregulation of Mitochondrial Respiration

KMT2D encodes a methyltransferase responsible for histone 3 lysine 4 (H3K4) mono-/di-methylation, an epigenetic mark correlated with active transcription. Here, we tested the hypothesis that KMT2D pathogenic loss-of-function variants, which causes the Kabuki syndrome type 1, could affect the mitochondrial metabolic profile. By using Seahorse technology, we showed a significant reduction of the mitochondrial oxygen consumption rate as well as a reduction of the glycolytic flux in both Kmt2d knockout MEFs and skin fibroblasts of Kabuki patients harboring heterozygous KMT2D pathogenic variants. Mass-spectrometry analysis of intermediate metabolites confirmed alterations in the glycolytic and TCA cycle pathways. The observed metabolic phenotype was accompanied by a significant increase in the production of reactive oxygen species. Measurements of the specific activities of the mitochondrial respiratory chain complexes revealed significant inhibition of CI (NADH dehydrogenase) and CIV (cytochrome c oxidase); this result was further supported by a decrease in the protein content of both complexes. Finally, we unveiled an impaired oxidation of glucose and larger reliance on long-chain fatty acids oxidation. Altogether, our findings clearly indicate a rewiring of the mitochondrial metabolic phenotype in the KMT2D-null or loss-of-function context that might contribute to the development of Kabuki disease, and represents metabolic reprogramming as a potential new therapeutic approach.

The induction of host cell autophagy triggers defense mechanisms against Trypanosoma cruzi infection in vitro

Trypanosoma cruzi causes Chagas disease, a neglected illness that affects millions of people worldwide, especially in Latin America. The balance between biochemical pathways triggered by the parasite and host cells response will ultimately define the progression of a life-threatening disease, justifying the efforts to understand cellular mechanisms for infection restrain. In this interaction, parasite and host cells are affected by different physiological responses as autophagy modulation, which could be under intense cellular stress, such as nutrient deprivation, hormone depletion, or infection. Autophagy is a constitutive pathway that leads to degradation of macromolecules and cellular structures and may induce cell death. In Trypanosoma cruzi infection, the relevance of host autophagy is controversial regarding in vitro parasite intracellular life cycle. In the present study, we evaluated host cell autophagy during T. cruzi infection in phagocytic and non-professional phagocytic cells. We described that the presence of the parasite increased the number of LC3 puncta, a marker for autophagy, in cardiac cells and peritoneal macrophages in vitro. The induction of host autophagy decreased infection in macrophages in early and late time-periods. We suggest that starved phagocytic cells reduced internalization, also confirmed by inert particles and dead trypomastigotes. Whereas, in cardiac cells, starvation-induced autophagy decreased lipid droplets and infection in later time-point, by reducing parasite differentiation/proliferation. In ATG5 knockout MEF cells, we confirmed our hypothesis of autophagy machinery activation during parasite internalization, increasing infection. Our data suggest that host autophagy downregulates T. cruzi infection through impairing parasite intracellular life cycle, reducing the infection in primary culture cells.

MITOCHONDRIAL-DERIVED PEPTIDE, SHLP2, A NOVEL PROTECTIVE FACTOR IN PARKINSON’S DISEASE

Mitochondrial DNA (mtDNA) variants are associated with a wide range of diseases of aging, from diabetes to Alzheimer’s, as well as with longevity itself. However, to date, little work has thoroughly examined the functional roles of mtDNA variants in such age-related diseases or the therapeutic potential of mitochondrial-derived peptides (MDPs) in these conditions. Our lab hypothesizes that mtDNA SNPs could affect MDPs, and we recently showed that a mtDNA SNP is associated with reduced circulating levels of an MDP called humanin and with cognitive decline. How other mtDNA SNPs affect MDPs and disease risk has yet to be analyzed. Remarkably, a recent paper showed a mtDNA SNP (m.2158 T>C) reduces the risk of Parkinson’s disease (PD). Of note, this SNP changes lysine (K) 4 to arginine (R) of a MDP called SHLP2, which is encoded by the 16S rRNA region of the mtDNA. SHLP2 acts as a neuroprotective factor and as a metabolic regulator. We hypothesized that K4R SHLP2 – produced by individuals who carry mtDNA m.2158 T>C – is a protective factor for Parkinson’s disease. Cycloheximide-treated pulse-chase experiments additionally showed that K4R SHLP2 is more stable than WT SHLP2. WT SHLP2 has a polyubiquitination whereas K4R SHLP2 diminish the polyubiquitination. K4R SHLP2 more potently inhibits PD toxin (MPP+) induced apoptosis in neuronal cells. K4R SHLP2 reverse the mitochondrial membrane potential loss and mitochondria respiration defect in TFAM heterozygous knockout MEFs. Altogether, SHLP2 has the therapeutic potential as a precision medicine in PD.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

P11 Loss-of-Function is Associated with Decreased Cell Proliferation and Neurobehavioral Disorders in Mice.

Although depression is associated with anxiety and memory deficit in humans, the molecular mechanisms of the complication remain largely unknown. In this study, we generated P11 knockout mice using CRISPR/Cas9 technology, as well as P11 knockout MEF cell lines, and confirmed depression-like phenotype. We observed that knockout of P11 in MEFs led to a decreased cell proliferation compared with P11+/+ MEFs. Moreover, P11 knockout resulted in a larger cell size, which resulted probably from accumulated F-actin stress fibers. The number of proliferating cells was decreased in the hippocampus of P11 KO mice. We observed anxiety-like disorder in addition to depression phenotype in the knockout mice. In addition, knockout of P11 led to memory deficit in female mice, but not in males. These data indicated that P11 is involved in regulating cell proliferation and cell size. The molecular associations of depression behavior with anxiety and memory deficit suggested a potential approach to improve therapeutic intervention through P11 in these disorders.

Abstract 1332: Autophagic clearance of protein aggregates is impaired in cancer cells with dysfunctional mitochondria

Abstract Mitophagy and aggrephagy are selective types of autophagy that degrade damaged mitochondria and protein aggregates, respectively. Dysfunctional mitochondria are known to undergo mitophagy and cause protein aggregation. How a cell can simultaneously coordinate the removal of dysfunctional mitochondria and protein aggregates via autophagy is unknown. Here, two agents, Mitoquinone (MitoQ) and MitoApocynin (MitoApo), known to induce mitochondrial dysfunction and mitophagy in MDA-MB-231 cells were used to investigate the accumulation of protein aggregates and the mechanisms that contribute to selective autophagy. Using a protein aggregate dye and a poly-ubiquitinated antibody, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), MitoQ, and MitoApo were identified as agents that caused an accumulation of poly-ubiquitinated protein aggregates in cancer cells by FACS analysis and confocal imaging. Microflow imaging of the cellular lysates confirmed a relative accumulation of large protein (>460 kDa) complexes. To establish the presence of aggrephagy, the aggregates were colocalized with LC3. Wild type and ATG7 knockout MEF cells were used to test whether protein aggregates accumulated in an autophagy-dependent manner. Our data currently demonstrates the kinetics of autophagic clearance of drug-induced protein aggregation and dysfunctional mitochondria. Citation Format: Thomas Biel, Ashutosh Rao. Autophagic clearance of protein aggregates is impaired in cancer cells with dysfunctional mitochondria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1332.

­­­Proteomic analysis of H3K36me3 and PSIP1/p75 (LEDGF) complexes reveal their wider role in DNA repair

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses a H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). Furthermore, we performed stable isotope labelling with amino acids in cell culture (SILAC) for a longer isoform of PSIP1 (p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts (MEFS). Results: Proteomic analysis of H3K36me3 chromatin show association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP1/p75. We validated the association of PSIP1/p75 with gamma H2A.X, an early marker of DNA damage and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP1/p75 in promoting HDR in mammals, our data supports the wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting several DNA repair proteins to transcribed gene bodies.

­­­Proteomic analysis of H3K36me3 and PSIP1/p75 (LEDGF) complexes reveal their wider role in DNA repair

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses a H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). Furthermore, we performed stable isotope labelling with amino acids in cell culture (SILAC) for a longer isoform of PSIP1 (p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts (MEFS). Results: Proteomic analysis of H3K36me3 chromatin show association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP1/p75. We validated the association of PSIP1/p75 with gamma H2A.X, an early marker of DNA damage and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP1/p75 in promoting HDR in mammals, our data supports the wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting several DNA repair proteins to transcribed gene bodies.

The Prolyl Isomerase Pin1 Is a Novel Target of 6,7,4'-Trihydroxyisoflavone for Suppressing Esophageal Cancer Growth.

Intake of soy isoflavones is inversely associated with the risk of esophageal cancer. Numerous experimental results have supported the anticancer activity of soy isoflavones. This study aimed to determine the anti-esophageal cancer activity of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF), a major metabolite of daidzein, which is readily metabolized in the human body. Notably, 6,7,4'-THIF inhibited proliferation and increased apoptosis of esophageal cancer cells. On the basis of a virtual screening analysis, Pin1 was identified as a target protein of 6,7,4'-THIF. Pull-down assay results using 6,7,4'-THIF Sepharose 4B beads showed a direct interaction between 6,7,4'-THIF and the Pin1 protein. Pin1 is a critical therapeutic and preventive target in esophageal cancer because of its positive regulation of β-catenin and cyclin D1. The 6,7,4'-THIF compound simultaneously reduced Pin1 isomerase activity and the downstream activation targets of Pin1. The specific inhibitory activity of 6,7,4'-THIF was analyzed using Neu/Pin1 wild-type (WT) and Neu/Pin1 knockout (KO) MEFs. 6,7,4'-THIF effected Neu/Pin1 WT MEFs, but not Neu/Pin1 KO MEFs. Furthermore, the results of a xenograft assay using Neu/Pin1 WT and KO MEFs were similar to those obtained from the in vitro assay. Overall, we found that 6,7,4'-THIF specifically reduced Pin1 activity in esophageal cancer models. Importantly, 6,7,4'-THIF directly bound to Pin1 but not FKBP or cyclophilin A, the same family of proteins. Because Pin1 acts like an oncogene by modulating various carcinogenesis-related proteins, this study might at least partially explain the underlying mechanism(s) of the anti-esophageal cancer effects of soy isoflavones. Cancer Prev Res; 10(5); 308-18. ©2017 AACR.

Gαi3 nuclear translocation causes irradiation resistance in human glioma cells.

We have previously shown that Gαi3 is elevated in human glioma, mediating Akt activation and cancer cell proliferation. Here, we imply that Gαi3 could also be important for irradiation resistance. In A172 human glioma cells, Gαi3 knockdown (by targeted shRNAs) or dominant-negative mutation significantly potentiated irradiation-induced cell apoptosis. Reversely, forced over-expression of wild-type or constitutively-active Gαi3 inhibited irradiation-induced A172 cell apoptosis. Irradiation in A172 cells induced Gαi3 translocation to cell nuclei and association with local protein DNA-dependent protein kinase (DNA-PK) catalytic subunit. This association was important for DNA damage repair. Gαi3 knockdown, depletion (using Gαi3 knockout MEFs) or dominant-negative mutation potentiated irradiation-induced DNA damages. On the other hand, expression of the constitutively-active Gαi3 in A172 cells inhibited DNA damage by irradiation. Together, these results indicate a novel function of Gαi3 in irradiation-resistance in human glioma cells.