Browsing by Subject "Nuclear Proteins"
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Item Open Access A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.(Sci Signal, 2013-05-07) Kurokawa, Manabu; Kim, Jiyeon; Geradts, Joseph; Matsuura, Kenkyo; Liu, Liu; Ran, Xu; Xia, Wenle; Ribar, Thomas J; Henao, Ricardo; Dewhirst, Mark W; Kim, Wun-Jae; Lucas, Joseph E; Wang, Shaomeng; Spector, Neil L; Kornbluth, SallyIn the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.Item Open Access Association between the ERCC5 Asp1104His polymorphism and cancer risk: a meta-analysis.(PloS one, 2012-01) Zhu, Mei-Ling; Wang, Mengyun; Cao, Zhi-Gang; He, Jing; Shi, Ting-Yan; Xia, Kai-Qin; Qiu, Li-Xin; Wei, Qing-YiBACKGROUND: Excision repair cross complementing group 5 (ERCC5 or XPG) plays an important role in regulating DNA excision repair, removal of bulky lesions caused by environmental chemicals or UV light. Mutations in this gene cause a rare autosomal recessive syndrome, and its functional single nucleotide polymorphisms (SNPs) may alter DNA repair capacity phenotype and cancer risk. However, a series of epidemiological studies on the association between the ERCC5 Asp1104His polymorphism (rs17655, G>C) and cancer susceptibility generated conflicting results. METHODOLOGY/PRINCIPAL FINDINGS: To derive a more precise estimation of the association between the ERCC5 Asp1104His polymorphism and overall cancer risk, we performed a meta-analysis of 44 published case-control studies, in which a total of 23,490 cases and 27,168 controls were included. To provide additional biological plausibility, we also assessed the genotype-gene expression correlation from the HapMap phase II release 23 data with 270 individuals from 4 ethnic populations. When all studies were pooled, we found no statistical evidence for a significantly increased cancer risk in the recessive genetic models (His/His vs. Asp/Asp: OR = 0.99, 95% CI: 0.92-1.06, P = 0.242 for heterogeneity or His/His vs. Asp/His + Asp/Asp: OR = 0.98, 95% CI: 0.93-1.03, P = 0.260 for heterogeneity), nor in further stratified analyses by cancer type, ethnicity, source of controls and sample size. In the genotype-phenotype correlation analysis from 270 individuals, we consistently found no significant correlation of the Asp1104His polymorphism with ERCC5 mRNA expression. CONCLUSIONS/SIGNIFICANCE: This meta-analysis suggests that it is unlikely that the ERCC5 Asp1104His polymorphism may contribute to individual susceptibility to cancer risk.Item Open Access Association of combined p73 and p53 genetic variants with tumor HPV16-positive oropharyngeal cancer.(PloS one, 2012-01) Wang, Zhongqiu; Sturgis, Erich M; Guo, Wei; Song, Xicheng; Zhang, Fenghua; Xu, Li; Wei, Qingyi; Li, Guojunp53 and p73 interact with human papillomavirus (HPV) E6 and E7 oncoproteins. The interplay between p53 and p73 and HPV16 may lead to deregulation of cell cycle and apoptosis, through which inflammation/immune responses control the HPV clearance and escape of immune surveillance, and subsequently contribute to tumor HPV16 status. In this case-case comparison study, HPV16 status in tumor specimens was analyzed and p53 codon 72 and p73 G4C14-to-A4T14 polymorphisms were genotyped using genomic DNA from blood of 309 oropharyngeal cancer patients. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated in univariate and multivariable logistic regression models to examine the association. The results from this study showed both p53 variant genotypes (Arg/Pro+Pro/Pro) and p73 variant genotypes (GC/AT+AT/AT) were significantly associated with HPV16-positive tumor in oropharyngeal cancer patients (OR, 1.9, 95% CI, 1.1-3.3 and OR, 2.1, 95% CI, 1.2-3.8, respectively), while the combined variant genotypes (p53 Pro carriers and p73 AT carriers) exhibited a significantly greater association with HPV16-positive tumor (OR, 3.2, 95% CI, 1.4-7.4), compared with combined wild-type genotypes (p53 Arg/Arg and p73 GC/GC), and the association was in a statistically significant dose-effect relationship (p = 0.001). Moreover, such association was more pronounced among several subgroups. These findings suggest that variant genotypes of p53 and p73 genes may be individually, or more likely jointly, associated with tumor HPV16-positive oropharyngeal cancer patients, particularly in never smokers. Identification of such susceptible biomarkers would greatly influence on individualized treatment for an improved prognosis.Item Restricted beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.(Mol Cell Biol, 2004-10) Usui, Isao; Imamura, Takeshi; Huang, Jie; Satoh, Hiroaki; Shenoy, Sudha K; Lefkowitz, Robert J; Hupfeld, Christopher J; Olefsky, Jerrold Mbeta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.Item Open Access Candidate genes on murine chromosome 8 are associated with susceptibility to Staphylococcus aureus infection in mice and are involved with Staphylococcus aureus septicemia in humans.(PloS one, 2017-01) Yan, Qin; Ahn, Sun Hee; Medie, Felix Mba; Sharma-Kuinkel, Batu K; Park, Lawrence P; Scott, William K; Deshmukh, Hitesh; Tsalik, Ephraim L; Cyr, Derek D; Woods, Christopher W; Yu, Chen-Hsin Albert; Adams, Carlton; Qi, Robert; Hansen, Brenda; Fowler, Vance GWe previously showed that chromosome 8 of A/J mice was associated with susceptibility to S. aureus infection. However, the specific genes responsible for this susceptibility are unknown. Chromosome substitution strain 8 (CSS8) mice, which have chromosome 8 from A/J but an otherwise C57BL/6J genome, were used to identify the genetic determinants of susceptibility to S. aureus on chromosome 8. Quantitative trait loci (QTL) mapping of S. aureus-infected N2 backcross mice (F1 [C8A] × C57BL/6J) identified a locus 83180780-88103009 (GRCm38/mm10) on A/J chromosome 8 that was linked to S. aureus susceptibility. All genes on the QTL (n~ 102) were further analyzed by three different strategies: 1) different expression in susceptible (A/J) and resistant (C57BL/6J) mice only in response to S. aureus, 2) consistently different expression in both uninfected and infected states between the two strains, and 3) damaging non-synonymous SNPs in either strain. Eleven candidate genes from the QTL region were significantly differently expressed in patients with S. aureus infection vs healthy human subjects. Four of these 11 genes also exhibited significantly different expression in S. aureus-challenged human neutrophils: Ier2, Crif1, Cd97 and Lyl1. CD97 ligand binding was evaluated within peritoneal neutrophils from A/J and C57BL/6J. CD97 from A/J had stronger CD55 but weaker integrin α5β1 ligand binding as compared with C57BL/6J. Because CD55/CD97 binding regulates immune cell activation and cytokine production, and integrin α5β1 is a membrane receptor for fibronectin, which is also bound by S. aureus, strain-specific differences could contribute to susceptibility to S. aureus. Down-regulation of Crif1 with siRNA was associated with increased host cell apoptosis among both naïve and S. aureus-infected bone marrow-derived macrophages. Specific genes in A/J chromosome 8, including Cd97 and Crif1, may play important roles in host defense against S. aureus.Item Open Access CoA synthase regulates mitotic fidelity via CBP-mediated acetylation.(Nature communications, 2018-03-12) Lin, Chao-Chieh; Kitagawa, Mayumi; Tang, Xiaohu; Hou, Ming-Hsin; Wu, Jianli; Qu, Dan Chen; Srinivas, Vinayaka; Liu, Xiaojing; Thompson, J Will; Mathey-Prevot, Bernard; Yao, Tso-Pang; Lee, Sang Hyun; Chi, Jen-TsanThe temporal activation of kinases and timely ubiquitin-mediated degradation is central to faithful mitosis. Here we present evidence that acetylation controlled by Coenzyme A synthase (COASY) and acetyltransferase CBP constitutes a novel mechanism that ensures faithful mitosis. We found that COASY knockdown triggers prolonged mitosis and multinucleation. Acetylome analysis reveals that COASY inactivation leads to hyper-acetylation of proteins associated with mitosis, including CBP and an Aurora A kinase activator, TPX2. During early mitosis, a transient CBP-mediated TPX2 acetylation is associated with TPX2 accumulation and Aurora A activation. The recruitment of COASY inhibits CBP-mediated TPX2 acetylation, promoting TPX2 degradation for mitotic exit. Consistently, we detected a stage-specific COASY-CBP-TPX2 association during mitosis. Remarkably, pharmacological and genetic inactivation of CBP effectively rescued the mitotic defects caused by COASY knockdown. Together, our findings uncover a novel mitotic regulation wherein COASY and CBP coordinate an acetylation network to enforce productive mitosis.Item Open Access Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.(Genes Dev, 1998-07-15) Yang, J; Bardes, ES; Moore, JD; Brennan, J; Powers, MA; Kornbluth, SActivation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.Item Open Access Evaluation of an epithelial plasticity biomarker panel in men with localized prostate cancer.(Prostate Cancer Prostatic Dis, 2016-03) Armstrong, AJ; Healy, P; Halabi, S; Vollmer, R; Lark, A; Kemeny, G; Ware, K; Freedland, SJBACKGROUND: Given the potential importance of epithelial plasticity (EP) to cancer metastasis, we sought to investigate biomarkers related to EP in men with localized prostate cancer (PC) for the association with time to PSA recurrence and other clinical outcomes after surgery. METHODS: Men with localized PC treated with radical prostatectomy at the Durham VA Medical Center and whose prostatectomy tissues were included in a tissue microarray (TMA) linked to long-term outcomes. We performed immunohistochemical studies using validated antibodies against E-cadherin and Ki-67 and mesenchymal biomarkers including N-cadherin, vimentin, SNAIL, ZEB1 and TWIST. Association studies were conducted for each biomarker with baseline clinical/pathologic characteristics an risk of PSA recurrence over time. RESULTS: Two hundred and five men contributed TMA tissue and had long-term follow-up (median 11 years). Forty-three percent had PSA recurrence; three died of PC. The majority had high E-cadherin expression (86%); 14% had low/absent E-cadherin expression. N-cadherin was rarely expressed (<4%) and we were unable to identify an E-to-N-cadherin switch as independently prognostic. No associations with clinical risk group, PSA recurrence or Gleason sum were noted for SNAIL, ZEB1, vimentin or TWIST, despite heterogeneous expression between patients. We observed an association of higher Ki-67 expression with Gleason sum (P=0.043), National Comprehensive Cancer Network risk (P=0.013) and PSA recurrence (hazard ratio 1.07, P=0.016). CONCLUSIONS: The expression of EP biomarkers in this cohort of men with a low risk of PC-specific mortality was not associated with aggressive features or PSA relapse after surgery.Item Open Access Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas.(Oncotarget, 2012-07) Jiao, Yuchen; Killela, Patrick J; Reitman, Zachary J; Rasheed, Ahmed B; Heaphy, Christopher M; de Wilde, Roeland F; Rodriguez, Fausto J; Rosemberg, Sergio; Oba-Shinjo, Sueli Mieko; Nagahashi Marie, Suely Kazue; Bettegowda, Chetan; Agrawal, Nishant; Lipp, Eric; Pirozzi, Christopher; Lopez, Giselle; He, Yiping; Friedman, Henry; Friedman, Allan H; Riggins, Gregory J; Holdhoff, Matthias; Burger, Peter; McLendon, Roger; Bigner, Darell D; Vogelstein, Bert; Meeker, Alan K; Kinzler, Kenneth W; Papadopoulos, Nickolas; Diaz, Luis A; Yan, HaiMutations in the critical chromatin modifier ATRX and mutations in CIC and FUBP1, which are potent regulators of cell growth, have been discovered in specific subtypes of gliomas, the most common type of primary malignant brain tumors. However, the frequency of these mutations in many subtypes of gliomas, and their association with clinical features of the patients, is poorly understood. Here we analyzed these loci in 363 brain tumors. ATRX is frequently mutated in grade II-III astrocytomas (71%), oligoastrocytomas (68%), and secondary glioblastomas (57%), and ATRX mutations are associated with IDH1 mutations and with an alternative lengthening of telomeres phenotype. CIC and FUBP1 mutations occurred frequently in oligodendrogliomas (46% and 24%, respectively) but rarely in astrocytomas or oligoastrocytomas ( more than 10%). This analysis allowed us to define two highly recurrent genetic signatures in gliomas: IDH1/ATRX (I-A) and IDH1/CIC/FUBP1 (I-CF). Patients with I-CF gliomas had a significantly longer median overall survival (96 months) than patients with I-A gliomas (51 months) and patients with gliomas that did not harbor either signature (13 months). The genetic signatures distinguished clinically distinct groups of oligoastrocytoma patients, which usually present a diagnostic challenge, and were associated with differences in clinical outcome even among individual tumor types. In addition to providing new clues about the genetic alterations underlying gliomas, the results have immediate clinical implications, providing a tripartite genetic signature that can serve as a useful adjunct to conventional glioma classification that may aid in prognosis, treatment selection, and therapeutic trial design.Item Open Access Functional Variants in Notch Pathway Genes NCOR2, NCSTN, and MAML2 Predict Survival of Patients with Cutaneous Melanoma.(Cancer Epidemiol Biomarkers Prev, 2015-07) Zhang, Weikang; Liu, Hongliang; Liu, Zhensheng; Zhu, Dakai; Amos, Christopher I; Fang, Shenying; Lee, Jeffrey E; Wei, QingyiBACKGROUND: The Notch signaling pathway is constitutively activated in human cutaneous melanoma to promote growth and aggressive metastatic potential of primary melanoma cells. Therefore, genetic variants in Notch pathway genes may affect the prognosis of cutaneous melanoma patients. METHODS: We identified 6,256 SNPs in 48 Notch genes in 858 cutaneous melanoma patients included in a previously published cutaneous melanoma genome-wide association study dataset. Multivariate and stepwise Cox proportional hazards regression and false-positive report probability corrections were performed to evaluate associations between putative functional SNPs and cutaneous melanoma disease-specific survival. Receiver operating characteristic curve was constructed, and area under the curve was used to assess the classification performance of the model. RESULTS: Four putative functional SNPs of Notch pathway genes had independent and joint predictive roles in survival of cutaneous melanoma patients. The most significant variant was NCOR2 rs2342924 T>C (adjusted HR, 2.71; 95% confidence interval, 1.73-4.23; Ptrend = 9.62 × 10(-7)), followed by NCSTN rs1124379 G>A, NCOR2 rs10846684 G>A, and MAML2 rs7953425 G>A (Ptrend = 0.005, 0.005, and 0.013, respectively). The receiver operating characteristic analysis revealed that area under the curve was significantly increased after adding the combined unfavorable genotype score to the model containing the known clinicopathologic factors. CONCLUSIONS: Our results suggest that SNPs in Notch pathway genes may be predictors of cutaneous melanoma disease-specific survival. IMPACT: Our discovery offers a translational potential for using genetic variants in Notch pathway genes as a genotype score of biomarkers for developing an improved prognostic assessment and personalized management of cutaneous melanoma patients.Item Open Access Genes with high penetrance for syndromic and non-syndromic autism typically function within the nucleus and regulate gene expression.(Molecular autism, 2016-01) Casanova, Emily L; Sharp, Julia L; Chakraborty, Hrishikesh; Sumi, Nahid Sultana; Casanova, Manuel FBACKGROUND:Intellectual disability (ID), autism, and epilepsy share frequent yet variable comorbidities with one another. In order to better understand potential genetic divergence underlying this variable risk, we studied genes responsible for monogenic IDs, grouped according to their autism and epilepsy comorbidities. METHODS:Utilizing 465 different forms of ID with known molecular origins, we accessed available genetic databases in conjunction with gene ontology (GO) to determine whether the genetics underlying ID diverge according to its comorbidities with autism and epilepsy and if genes highly penetrant for autism or epilepsy share distinctive features that set them apart from genes that confer comparatively variable or no apparent risk. RESULTS:The genetics of ID with autism are relatively enriched in terms associated with nervous system-specific processes and structural morphogenesis. In contrast, we find that ID with highly comorbid epilepsy (HCE) is modestly associated with lipid metabolic processes while ID without autism or epilepsy comorbidity (ID only) is enriched at the Golgi membrane. Highly comorbid autism (HCA) genes, on the other hand, are strongly enriched within the nucleus, are typically involved in regulation of gene expression, and, along with IDs with more variable autism, share strong ties with a core protein-protein interaction (PPI) network integral to basic patterning of the CNS. CONCLUSIONS:According to GO terminology, autism-related gene products are integral to neural development. While it is difficult to draw firm conclusions regarding IDs unassociated with autism, it is clear that the majority of HCA genes are tightly linked with general dysregulation of gene expression, suggesting that disturbances to the chronology of neural maturation and patterning may be key in conferring susceptibility to autism spectrum conditions.Item Open Access Genetic variants of GADD45A, GADD45B and MAPK14 predict platinum-based chemotherapy-induced toxicities in Chinese patients with non-small cell lung cancer.(Oncotarget, 2016-05) Jia, Ming; Zhu, Meiling; Wang, Mengyun; Sun, Menghong; Qian, Ji; Ding, Fei; Chang, Jianhua; Wei, QingyiThe JNK and P38α pathways play a crucial role in tissue homeostasis, apoptosis and autophagy under genotoxic stresses, but it is unclear whether single nucleotide polymorphisms (SNPs) of genes in these pathways play a role in platinum-based chemotherapy-induced toxicities in patients with advanced non-small cell lung cancer (NSCLC). We genotyped 11 selected, independent, potentially functional SNPs of nine genes in the JNK and P38α pathways in 689 patients with advanced NSCLC treated with platinum-combination chemotherapy regimens. Associations between these SNPs and chemotherapy toxicities were tested in a discovery group of 345 patients and then validated in a replication group of 344 patients. In both discovery and validation groups as well as their pooled analysis, carriers of GADD45B rs2024144T variant allele had a significantly higher risk for severe hematologic toxicity and carriers of MAPK14 rs3804451A variant allele had a significantly higher risk for both overall toxicity and gastrointestinal toxicity. In addition, carriers of GADD45A rs581000C had a lower risk of anemia, while carriers of GADD45B rs2024144T had a significantly higher risk for leukocytopenia or agranulocytosis. The present study provides evidence that genetic variants in genes involved in the JNK and P38α pathways may predict platinum-based chemotherapy toxicity outcomes in patients with advanced NSCLC. Larger studies of other patient populations are needed to validate our findings.Item Open Access How the kinetochore couples microtubule force and centromere stretch to move chromosomes.(Nature cell biology, 2016-04) Suzuki, Aussie; Badger, Benjamin L; Haase, Julian; Ohashi, Tomoo; Erickson, Harold P; Salmon, Edward D; Bloom, KerryThe Ndc80 complex (Ndc80, Nuf2, Spc24 and Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule- and MAP-binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule-binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule-binding interactions with the Ndc80 and Dam1 complexes.Item Open Access Huntingtin is required for normal excitatory synapse development in cortical and striatal circuits.(J Neurosci, 2014-07-09) McKinstry, Spencer U; Karadeniz, Yonca B; Worthington, Atesh K; Hayrapetyan, Volodya Y; Ozlu, M Ilcim; Serafin-Molina, Karol; Risher, W Christopher; Ustunkaya, Tuna; Dragatsis, Ioannis; Zeitlin, Scott; Yin, Henry H; Eroglu, CaglaHuntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.Item Open Access IFI16-STING-NF-κB signaling controls exogenous mitochondrion-induced endothelial activation.(American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2022-06) Li, Shu; Xu, He; Song, Mingqing; Shaw, Brian I; Li, Qi-Jing; Kirk, Allan DMitochondria released from injured cells activate endothelial cells (ECs), fostering inflammatory processes, including allograft rejection. The stimulator of interferon genes (STING) senses endogenous mitochondrial DNA, triggering innate immune activation via NF-κB signaling. Here, we show that exogenous mitochondria exposure induces EC STING-NF-κB activation, promoting EC/effector memory T cell adhesion, which is abrogated by NF-κB and STING inhibitors. STING activation in mitochondrion-activated ECs is independent of canonical cGMP-AMP synthetase sensing/signaling, but rather is mediated by interferon gamma-inducible factor 16 (IFI16) and can be inhibited by IFI16 inhibition. Internalized mitochondria undergo mitofusion and STING-dependent mitophagy, leading to selective sequestration of internalized mitochondria. The exposure of donor hearts to exogenous mitochondria activates murine heart ECs in vivo. Collectively, our results suggest that IFI16-STING-NF-κB signaling regulates exogenous mitochondrion-induced EC activation and mitophagy, and exogenous mitochondria foster T cell-mediated CoBRR. These data suggest a novel, donor-directed, therapeutic approach toward mitigating perioperative allograft immunogenicity.Item Open Access Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.(J Cell Biol, 2005-04-11) Casaletto, Jessica B; Nutt, Leta K; Wu, Qiju; Moore, Jonathan D; Etkin, Laurence D; Jackson, Peter K; Hunt, Tim; Kornbluth, SallyDegradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.Item Open Access Isolation, purification and in vitro differentiation of cytotrophoblast cells from human term placenta.(Reproductive biology and endocrinology : RB&E, 2015-07) Li, Liping; Schust, Danny JBackground
The syncytialization of cytotrophoblast cells to syncytiotrophoblast is central to human placental transport and hormone production. Many techniques for in vitro study of this process have been proposed and new investigators to the field may find the literature in the field daunting. Here, we present a straightforward and reliable method to establish this important model using modern but readily available tools and reagents.Methods
Villous cytotrophoblast cells are obtained from term placenta using mild enzymatic degradation, Percoll gradient centrifugation, negative magnetic cell sorting using an antibody against classical major histocompatibility complex molecules and in vitro culture on a matrix-coated growth surface.Results
The purity of isolated cytotrophoblast cells exceeds 98 % as assessed by cytokeratin-7 expression using flow cytometry. Contamination by mesenchymal cells, extravillous trophoblast cells, leukocytes, Hofbauer and endothelial cells is minimized (less than 2 % when analyzed for vimentin, HLA-G, CD45, CD163 and CD31 using flow cytometry). Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12 h of plating. By 72 h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular E-cadherin expression upon fusion into multinucleated syncytia. After 72 h in culture, nearly every cultured cell expresses syncytiotrophoblast markers, including cytokeratin-7, human chorionic gonadotropin-β (β-hCG) and the fusion-related proteins glial cell missing-1 (GCM-1) and syncytin.Conclusions
We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.Item Open Access Kruppel-like factor 15 is critical for vascular inflammation.(The Journal of clinical investigation, 2013-10) Lu, Yuan; Zhang, Lisheng; Liao, Xudong; Sangwung, Panjamaporn; Prosdocimo, Domenick A; Zhou, Guangjin; Votruba, Alexander R; Brian, Leigh; Han, Yuh Jung; Gao, Huiyun; Wang, Yunmei; Shimizu, Koichi; Weinert-Stein, Kaitlyn; Khrestian, Maria; Simon, Daniel I; Freedman, Neil J; Jain, Mukesh KActivation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle-specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.Item Open Access MDM4 genetic variants and risk of gastric cancer in an Eastern Chinese population.(Oncotarget, 2017-03) Wang, Meng-Yun; Jia, Ming; He, Jing; Zhou, Fei; Qiu, Li-Xin; Sun, Meng-Hong; Yang, Ya-Jun; Wang, Jiu-Cun; Jin, Li; Wang, Ya-Nong; Wei, Qing-YiMDM4 is a p53-interacting protein and plays an important role in carcinogenesis. In this study of 1,077 gastric cancer (GCa) cases and 1,173 matched cancer-free controls, we investigated associations between three tagging single nucleotide polymorphisms (SNPs) (rs11801299 G>A, rs1380576 C>G and rs10900598 G>T) in MDM4 and gastric cancer risk in an Eastern Chinese Population. In logistic regression analysis, a significantly decreased GCa risk was associated with the rs1380576 GG variant genotype (adjusted odds ratio [OR] =0.74, 95% confidence interval [CI] =0.56-0.98) under a recessive model, which remained significant after correction by the false-positive reporting probability. This risk was more evident in subgroups of older subjects, males, never smokers, never drinkers and cancers of non-cardia. We then performed SNP-mRNA expression correlation analysis and found that the GG variant genotype was associated with significantly decreased expression of MDM4 mRNA in normal cell lines for 44 Chinese (P=0.032 for GG vs. CC) as well as for 269 multi-ethnic subjects (P<0.0001 for GG vs. CC). Our results suggest that the MDM4 rs1380576 G variant may be markers for GCa susceptibility. Larger, independent studies are warranted to validate our findings.Item Open Access Mouse double minute 4 variants modify susceptibility to risk of recurrence in patients with squamous cell carcinoma of the oropharynx.(Molecular carcinogenesis, 2018-03) Lu, Zhongming; Lu, Zhongming; Sturgis, Erich M; Zhu, Lijun; Zhang, Hua; Tao, Ye; Wei, Peng; Wei, Qingyi; Li, GuojunGiven the crucial role of Mouse double minute 4 (MDM4) oncoprotein in p53 pathway, single nucleotide polymorphisms (SNPs) could serve as such biomarkers for prediction of SCCOP recurrence. Thus, we investigated associations between three tagging putatively functional variants of MDM4, two in the 3' untranslated region of 3' UTR [rs11801299 (NC_000001.10:g.204529084G>A) and rs10900598(NC_000001.10:g.204525568G>T)] and one in intron 1 [rs1380576(NC_000001.10:g.204488278G>C)], and recurrence risk of SCCOP in 1,008 incident patients. A log-rank test and multivariable Cox models were used to assess associations. Patients with MDM4-rs10900598 GT/TT had a worse disease-free survival (DFS) compared with corresponding GG genotype, while those with rs11801299 AG/AA genotypes had a lower recurrence risk than the cases with rs11801299 GG genotype (both log-rank, P < 0.001). Multivariable analysis showed that significantly different recurrence risk were found among patients with MDM4-rs10900598 GT/TT and rs11801299 AG/AA variant genotypes (HR, 2.0, 95% CI, 1.4-2.9 and HR, 0.4, 95% CI, 0.3-0.6, respectively) compared with their corresponding common homozygous genotypes. Furthermore, after combining the risk genotypes of the three SNPs, patients among low-risk group had a significantly lower risk of SCCOP recurrence than those in high-risk group (HR, 0.2, 95% CI, 0.1-0.3). The risk for both individual SNPs or combined risk genotypes was restricted to HPV-positive SCCOP patients. Our findings suggest that the MDM4 polymorphisms may, individually or in combination, confer an independent risk of SCCOP recurrence, particularly in HPV-positive SCCOP patients. However, larger studies are needed to validate our findings.