Wild-type Overexpression Research Articles

METTL3 Dysregulates RNA Splicing by Translational Control of Splicing Factors via m 6A Modification in CLL

METTL3 Dysregulates RNA Splicing by Translational Control of Splicing Factors via m 6A Modification in CLL

Two Novel Hydroxymethylbilane Synthase Splicing Mutations Predispose to Acute Intermittent Porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant genetic disease caused by a lack or decrease in hydroxymethylbilane synthase (HMBS) activity. It is characterized by acute nerve and visceral attacks caused by factors in the process of heme synthesis. The penetrance rate of this disease is low, and the heterogeneity is strong. Here, we reported two novel HMBS mutations from two unrelated Chinese AIP patients and confirmed the pathogenicity of these two mutations. We found the HMBS c.760–771+2delCTGAGGCACCTGGTinsGCTGCATCGCTGAA and HMBS c.88-1G>C mutations by second-generation sequencing and Sanger sequencing. The in vitro expression analysis showed that these mutations caused abnormal HMBS mRNA splicing and premature termination or partial missing of HMBS protein. Homologous modeling analysis showed that the HMBS mutants lacked the amino acids which are crucial for the enzyme activity or the protein stability. Consistently, enzyme activity analysis confirmed that the HMBS mutants’ overexpression cells exhibited the reduced enzyme activity compared with the HMBS wildtype overexpression cells. Our study identified and confirmed two novel pathogenic HMBS mutations which will expand the molecular heterogeneity of AIP and provide further scientific basis for the clinical diagnosis of AIP.

Perturbation of TM6SF2 Expression Alters Lipid Metabolism in a Human Liver Cell Line.

Non-alcoholic fatty liver disease (NAFLD) is caused by excess lipid accumulation in hepatocytes. Genome-wide association studies have identified a strong association of NAFLD with non-synonymous E167K amino acid mutation in the transmembrane 6 superfamily member 2 (TM6SF2) protein. The E167K mutation reduces TM6SF2 stability, and its carriers display increased hepatic lipids and lower serum triglycerides. However, the effects of TM6SF2 on hepatic lipid metabolism are not completely understood. We overexpressed wild-type or E167K variant of TM6SF2 or knocked down TM6SF2 expression in lipid-treated Huh-7 cells and used untargeted lipidomic analysis, RNAseq transcriptome analysis, and fluorescent imaging to determine changes in hepatic lipid metabolism. Both TM6SF2 knockdown and E167K overexpression increased hepatic lipid accumulation, while wild-type overexpression decreased acylglyceride levels. We also observed lipid chain remodeling for acylglycerides by TM6SF2 knockdown, leading to a relative increase in species with shorter, more saturated side chains. RNA-sequencing revealed differential expression of several lipid metabolizing genes, including genes belonging to AKR1 family and lipases, primarily in cells with TM6SF2 knockdown. Taken together, our data show that overexpression of TM6SF2 gene or its loss-of-function changes hepatic lipid species composition and expression of lipid metabolizing genes. Additionally, our data further confirms a loss-of-function effect for the E167K variant.

Serine mutations in overexpressed Hsp27 abrogate the protection against doxorubicin-induced p53-dependent cardiac apoptosis in mice.

Small heat shock proteins (sHsps) protect the heart from chemotherapeutics-induced heart failure by inhibiting p53-dependent apoptosis. However, mechanism of such protection has not been elucidated yet. Here we test a hypothesis that serine phosphorylation of sHsps is essential to inhibit the doxorubicin-induced and p53-dependent apoptotic pathway. Three transgenic mice (TG) lines with cardiomyocyte-specific overexpression of human heat shock protein 27 (hHsp27), namely, wild-type [myosin heavy chain (MHC)-hHsp27], S82A single mutant [MHC-mut-hHsp27(S82A)], and trimutant [MHC-mut-hHsp27(S15A/S78A/S82A)] were generated. TG mice were treated with Dox (6 mg/kg body wt; once in a week; 4 wk) along with age-matched nontransgenic (non-TG) controls. The Dox-treated MHC-hHsp27 mice showed improved survival and cardiac function (both MRI and echocardiography) in terms of contractility [ejection fraction (%EF)] and left ventricular inner diameter (LVID) compared with the Dox-treated non-TG mice. However, both MHC-mut-hHsp27(S82A) and MHC-mut-hHsp27(S15A/S78A/S82A) mutants overexpressing TG mice did not show such a cardioprotection. Furthermore, transactivation of p53 was found to be attenuated only in Dox-treated MHC-hHsp27 mice-derived cardiomyocytes in vitro, as low p53 was detected in the nuclei, not in mutant hHsp27 overexpressing cardiomyocytes. Similarly, only in MHC-hHsp27 overexpressing cardiomyocytes, low Bax, higher mechanistic target of rapamycin (mTOR) phosphorylation, and low apoptotic poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (89 kDa fragment) were detected. Pharmacological inhibition of p53 was more effective in mutant TG mice compared with MHC-hHsp27 mice. We conclude that phosphorylation of overexpressed Hsp27 at S82 and its association with p53 are essential for the cardioprotective effect of overexpressed Hsp27 against Dox-induced dilated cardiomyopathy. Only phosphorylated Hsp27 protects the heart by inhibiting p53 transactivation.NEW & NOTEWORTHY Requirement of serine phosphorylation in Hsp27 for cardioprotective effect against Dox is tested in various mutants overexpressing mice. Cardioprotective effect was found to be compromised in Hsp27 serine mutants overexpressed mice compared with wild-type overexpressing mice. These results indicate that cancer patients, who carry these mutations, may have higher risk of aggravated cardiomyopathy on treated with cardiotoxic chemotherapeutics such as doxorubicin.

Integrative Analysis of 1001 Diffuse Large B Cell Lymphoma Identifies Novel Oncogenic Roles for Rhoa

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common hematologic malignancy and is characterized by a striking degree of clinical and molecular heterogeneity. Gene expression profiling has long been used to define subtypes and understand the heterogeneity within DLBCL. Microarray-based gene expression studies have identified cell of origin subgroups (Alizadeh et al., 2000), as well as gene expression signatures derived from host inflammatory response (Monti et al., 2005), stromal tissues (Lenz et al., 2008) as well as other biological processes. However, the connections between these gene expression signatures and genetic alterations is largely unknown. In this study, we sought to define connections between gene expression signatures and genetic alterations to better understand the biology of DLBCL. We enrolled a total of 1001 DLBCL patients and comprehensively defined the landscape of genetic mutations, copy number alterations and expression through whole-exome and transcriptome sequencing. We identified 150 genetic drivers of DLBCL including many novel, clinically relevant genes (e.g. SPEN, SETD1B and KLHL14). We began the gene expression analysis with a comprehensive universe of nearly 9500 annotated gene sets from widely used gene set databases (Kegg, Reactome, MSigDB), several lymphoma-specific gene signature sources (Lenz et al. 2008, Monti et al. 2005, and Shaffer et al.) and other published sources. From this large universe, we identified 1228 gene sets that comprised genes with high intra gene set correlation. The vast majority of these 1228 gene sets also exhibited high inter-gene set correlation indicating a high degree of redundancy between these gene sets. We developed an approach to singular value decomposition to define 31 exemplar gene sets that essentially replicated the patterns of the 1228 described gene sets. Our results recapitulated a number of described patterns underlying DLBCL gene expression including distinct gene sets reflecting cell of origin, host response, BCR and Oxidative Phosphorylation (Monti et al., 2005), as well as the “Stromal 1” and “Stromal 2” signatures (Lenz et al., 2008) signatures. There was no association between the expression of stromal signatures and overall mutational burden (p=0.4). Our data identified the genetic underpinnings of each of these distinct gene sets that provide a better understanding of the underlying biology affected by different genetic mutations. Interestingly, genetic alterations in RHOA were strongly associated with the proliferation gene signature and poor survival in DLBCL. We sought to better define the role of RHOA mutations in DLBCL which are mostly unknown. We knocked out the expression of RHOA in vitro through the CRISPR-Cas9 approach and found that RHOA deletion was lethal in both ABC DLBCL (LY3, TMD8, HBL1) and GCB DLBCLs (Pfeiffer and SUDHL4), results were validated with shRNAs. Conversely, cells overexpressing R5Q mutant proliferated 30% faster than vector control cells and more than 10% faster than wildtype overexpressing cells, directly implicating these mutations and RHOA overexpression in the growth of DLBCL cells. We further investigated the effects of RHOA knockout in vivo using two complementary mouse models. We bred Rhoa conditional knockout mice with Mb1-Cre transgenic mice, in which Cre is restricted to the B cell lineage as well as AID-Cre transgenic mice in which Cre is restricted to germinal center (GC) B cells. While B cells represented the vast majority of cells in the lymphoid organs of Rhoa wildtype mice, we found a dramatic reduction in B cell population and a resulting increase in T cell percentages in Rhoa deleted mice (p Collectively, our data define the genetic basis for observed changes in gene expression in DLBCL. Our experimental data define RHOA as an oncogene with roles in B cell development, germinal center maintenance, and DLCBL growth. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Merck: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen Cilag: Consultancy, Research Funding. Flowers: Abbvie: Consultancy, Research Funding; Infinity: Research Funding; Clinical Care Options: Research Funding; National Cancer Institute: Research Funding; Spectrum: Consultancy; Onyx: Research Funding; OptumRx: Consultancy; Prime Oncology: Research Funding; Janssen Pharmaceutical: Research Funding; Acerta: Research Funding; Gilead: Consultancy; Seattle Genetics: Consultancy; Eastern Cooperative Oncology Group: Research Funding; Research to Practice: Research Funding; Burroughs Welcome Fund: Research Funding; Celgene: Consultancy, Research Funding; V Foundation: Research Funding; Bayer: Consultancy; Educational Concepts: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Genentech/Roche: Consultancy, Research Funding; Millennium/Takeda: Research Funding; National Institutes Of Health: Research Funding; TG Therapeutics: Research Funding. Gribben: TG Therapeutics: Honoraria; Kite: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria; Abbvie: Honoraria; Acerta: Honoraria; Karyopharm: Honoraria; Genentech/Roche: Honoraria. Hsi: Cellerant Therapeutics: Research Funding; Abbvie: Research Funding; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Eli Lilly and Co.: Research Funding. Evens: Merck: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Millennium: Consultancy; AbbVie: Consultancy; Affimed: Consultancy; Pharmacyclics: Consultancy; Celgene: Consultancy; Kite Pharma: Consultancy. Reddy: Abbvie: Consultancy; BMS: Consultancy; Celgene: Consultancy; Gilead: Speakers Bureau. Gordon: Janssen: Other: Data Monitoring Committee.

MAP2 is differentially phosphorylated in schizophrenia, altering its function

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz—that a large proportion of individuals have a “MAP2opathy”—in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

Recurrent XPO1 mutations alter pathogenesis of chronic lymphocytic leukemia

BackgroundExportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood.MethodsWe performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor).ResultsWe report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL.ConclusionsThese findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.

Asymmetrical arginine dimethylation of histone H4 by 8-oxog/OGG1/PRMT1 is essential for oxidative stress-induced transcription activation

It has been established that 8-oxoguanine DNA glycosylase 1 (OGG1) is the main enzyme removing oxidized guanine under oxidative stress. However, increasing evidence has shown that OGG1 is not only a base excision repair protein but also a new transcriptional coactivator involved in oxidative stress-induced gene expression. Its downstream target genes and the underlying regulatory mechanisms still need to be discerned. Here, it was discovered that c-Myc is a downstream target of OGG1 under oxidative stress and that H4R3me2a is involved in this transcriptional regulation. The increased level of H4R3me2a induced by H2O2 is regulated by OGG1, which may directly interact with the specific arginine methyltransferase PRMT1 and promote the asymmetrical dimethylation of H4R3me1. H4R3me2a enrichment on the promoter of c-Myc can recruit YY1 and activate c-Myc transcription. Moreover, knocking down OGG1 or PRMT1 suppresses c-Myc transcription under oxidative stress by downregulating H4R3me2a formation. Furthermore, the overexpression of wild type (WT) H4R3 promotes c-Myc transcription, but the expression of mutant H4R3Q does not have this effect. Taken together, our data show that the 8-oxoG/OGG1/PRMT1/H4R3me2a/YY1 axis senses oxidative stress and promotes gene transcription.

Abstract 117: Elucidating the role of Flotillin-2 in EGFR signaling and cancer growth

Abstract Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl ubiquitinates the activated EGFR, targeting it for degradation, and acts as an adaptor, recruiting proteins required for EGFR trafficking. To study Cbl adaptor function, we used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in 3 groups: without or with EGF stimulation, and with EGF stimulation in the presence of an endosomal trafficking inhibitor. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed with the treatments. Secondary screening with an in cell Elisa assay demonstrated that siRNA mediated knockdown of Flotillin-2 (FLOT2) resulted in increased phosphorylation of FLOT2. Further evaluation of knockdown of FLOT2 in HeLa cells by immunoblotting showed that loss of FLOT2 led to increased phosphorylation, increased Cbl recruitment to the EGFR, increased ubiquitination of the EGFR, and degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. CRISPR knockout of FLOT2 in HeLa cells reduced steady-state levels of EGFR, but increased EGF-stimulated phosphorylation and signaling downstream of EGFR. In lung adenocarcinoma H441 cells, knockdown of FLOT2 increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling. This effect was reversed by the EGFR inhibitor erlotinib. Conversely, stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2 inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. Conversely, overexpression of a soluble G2A FLOT2 mutant (which does not get recruited to the plasma membrane) did not inhibit EGF induced EGFR phosphorylation. In order to determine the effect of FLOT2 on EGFR-dependent cancer cell growth, both HeLa and H441 cells were treated in the presence or absence of both EGF and erlotinib in FLOT2 knockdown or knockout cells, and growth was monitored by cell counts over 7 days. HeLa cells exhibited increased growth and soft-agar colony formation in response to FLOT2 knockout, however this growth was not reduced by erlotinib treatment suggesting this was EGFR independent. However, EGF treatment greatly increased HeLa growth in the FLOT2 knockout group as compared to the control CRISPR and this increase was abrogated by erlotinib, indicating that FLOT2 knockout sensitizes HeLa cells to increased EGF-induced HeLa cell growth. H441 cells had an increase in growth over time in response to FLOT2 knockdown, and this growth was erlotinib sensitive, showing that the increase in growth was EGFR-dependent. In summary, these data demonstrate that FLOT2 negatively regulates EGFR activation and its subsequent ubiquitination and degradation. Further, we present evidence that FLOT2 negatively regulates cancer cell growth in an EGFR-dependent manner in H441 cells. Citation Format: David John Wisniewski, Mariya S. Liyasova, Xu Zhang, Samuel F. Gilbert, Alexis Catalano, Donna Voeller, Udayan Guha, Stanley Lipkowitz. Elucidating the role of Flotillin-2 in EGFR signaling and cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 117.

Enhanced psychostimulant response, but not social avoidance, depends on GluA1 AMPA receptors in VTA dopamine neurons following intermittent social defeat stress in rats.

Evidence from both human and animal studies demonstrates the importance of social stress in the development of addiction‐related behaviour. In rats, intermittent social defeat stress causes long‐lasting psychostimulant cross‐sensitization. Our recent data reveal heightened expression of AMPA receptor (AMPAR) GluA1 subunit in rat ventral tegmental area (VTA), which occurs concurrently with social stress‐induced amphetamine (AMPH) cross‐sensitization. In addition, social stress in rats induced social avoidance behaviour. The present study evaluated the effects of intermittent social defeat stress on GluA1 expression in VTA dopamine (DA) neurons, then utilized Cre‐dependent virus‐mediated gene transfer to determine the functional role of homomeric GluA1‐AMPARs in these neurons. Social defeat stress exposure induced GluA1 expression in VTA DA neurons, as demonstrated by a greater density of GluA1/tyrosine hydroxylase (TH) double‐labelling in VTA neurons in stressed rats. Additionally, functional inactivation of VTA GluA1 AMPARs in DA neurons prevented stress‐induced cross‐sensitization, or augmented locomotor response to low dose AMPH challenge (1.0 mg/kg, i.p.), but had no effect on social stress‐induced social avoidance behaviour. Furthermore, wild‐type overexpression of GluA1 in VTA DA neurons had the opposite effect; locomotor‐activating effects of AMPH were significantly augmented, even in the absence of stress. Taken together, these results suggest that stress‐induced GluA1 expression in VTA DA neurons is necessary for psychostimulant cross‐sensitization, but not for social avoidance. This differential effect suggests that different neural pathways are implicated in these behaviours. These findings could lead to novel pharmacotherapies to help prevent stress‐induced susceptibility to substance abuse.

Expression and Functional Study of Single Mutations of Carbonic Anhydrase 8 in Neuronal Cells.

Carbonic anhydrase 8 (CA8), an isozyme of α-carbonic anhydrases, lacks the ability to catalyze the reversible hydration of CO2 to bicarbonate and proton. Previous studies have shown that single point mutations of CA8, CA8-S100P, and CA8-G162R, are associated with novel syndromes including congenital ataxia and mild cognitive impairment. Our previous results demonstrated that overexpression of wild type (WT) CA8 promoted cell proliferation, neurite outgrowth, anti-apoptosis, invasion and migration abilities in neuronal cells. In this study, we examined the expressions and functions of CA8-S100P and CA8-G162R in neuroblastoma cells lines, compared with those of WT CA8. Our results show that the protein expressions of mutant CA8-S100P and CA8-G162R were significantly decreased in Neuro-2a and SK-N-SH cells. Interestingly, CA8-S100P demonstrated a significant increase in cell proliferation in both Neuro-2a and SK-N-SH cells. However, both CA8 mutations showed significantly decreased effects on cell protection and migration in SK-N-SH cells. Surprisingly, a significant increase of invasive ability was observed in SK-N-SH cells with overexpression of CA8-S100P as compared with those with overexpression of WT CA8 under retinoic acid (RA) treatment. In addition, we found that Neuro-2a cells with overexpression of CA8-S100P and CA8-G162R showed significantly increased neurite outgrowth. Taken together, our data suggest that the expressions of CA8-S100P and CA8-G162R in neuronal cells alter cell morphology, proliferation, mobility and viability; indicating that the homozygous point mutations of CA8 lead to not only the loss of WT CA8 function, but also the gain of novel functions leading to neuromuscular dysfunction.

Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain

Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.

Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress.

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium

AimsMammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation. Methods and resultsWestern blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1−/− mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC. ConclusionsThe study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.

Alterations in α-synuclein and PINK1 expression reduce neurite length and induce mitochondrial fission and Golgi fragmentation in midbrain neurons

Accumulation of α-synuclein is a pathological hallmark of Parkinson’s disease (PD) and has been linked to reductions in neurite length and axonal degeneration of midbrain dopaminergic neurons. Mutations in SNCA, which encodes α-synuclein, and loss of function mutations in PTEN-induced putative kinase-1 (PINK1) cause familial PD. There is a need to identify the mechanisms by which α-synuclein overexpression and the loss of PINK1 induce neurodegeneration in PD. To do this, we employed rat ventral midbrain cultures to investigate the effects of overexpression of wildtype or mutant (A53T) α-synuclein, and of siRNA knockdown of PINK1, on neurite length and on mitochondrial and Golgi integrity. We found reduced neurite length and increased levels of both Golgi fragmentation and mitochondrial fission in response to overexpression of wildtype or mutant α-synuclein, and to PINK1 knockdown. Reductions in neurite length induced by these two PD risk genes were significantly correlated with increases in Golgi fragmentation and mitochondrial fission. Combined α-synuclein overexpression and PINK1 knockdown induced a greater reduction in neurite length and increase in Golgi fragmentation, than either alone. This study provides novel evidence that α-synuclein overexpression and PINK1 deletion converge to induce significant increases in Golgi fragmentation and mitochondrial fission in midbrain neurons, that are correlated with decreases in neurite length. This highlights the need for further studies on these converging mechanisms in dopaminergic neurodegeneration in PD.

ISG15 suppresses translation of ABCC2 via ISGylation of hnRNPA2B1 and enhances drug sensitivity in cisplatin resistant ovarian cancer cells

Cisplatin-based chemotherapies have long been considered as a standard chemotherapy in ovarian cancer. However, cisplatin resistance restricts beneficial therapy for patients with ovarian cancer. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, that is implicated in the post-translational modification of diverse proteins. In this work, we found that ISG15 was downregulated in cisplatin resistant tissues and cell lines of ovarian cancer. Functional studies demonstrated that overexpression of wild type (WT) ISG15, but not nonISGylatable (Mut) ISG15 increased cell responses to cisplatin in resistant ovarian cancer cells. Furthermore, we found that WT ISG15 decreased ABCC2 expression at the protein level. Importantly, overexpression of ABCC2 blocked sensitizing effect of ISG15 on cisplatin. In addition, we identified that hnRNPA2B1 was recruited to 5′UTR of ABCC2 mRNA and promoted its translation, which was blocked by ISG15. We further demonstrated that hnRNPA2B1 could be ISGylated, and ISGylation blocked its recruitment to ABCC2 mRNA, thereby suppressed translation of ABCC2. Altogether, our data support targeting ISG15 might be a potential therapeutic strategy for patients with cisplatin-resistant ovarian cancer.

Dimethylation of eEF1A at Lysine 55 Plays a Key Role in the Regulation of eEF1A2 on Malignant Cell Functions of Acute Myeloid Leukemia.

Objective:This study aimed to explore whether eukaryotic translation elongation factor 1 alpha 2 affected cell proliferation, migration, and apoptosis via regulating the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia.Methods:The expressions of eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia cell lines and human normal bone marrow mononuclear cells (as control) were assessed. Control CRISPR-Cas9 lentivirus, eukaryotic translation elongation factor 1 alpha 2 knockout CRISPR-Cas9 lentivirus, vector plasmid, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression plasmid, and eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression plasmid were transfected into AML-193 and Kasumi-1 cells combined or alone, and were accordingly divided into 4 groups (Sgcontrol + vector group, SgeEF1A2 + vector group, SgeEF1A2 + eEF1A2WT group, and SgeEFIA2 + eEF1A2K55R group).Results:Eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expressions were higher in AML-193, Kasumi-1, and KG-1 cell lines compared to the control. In AML-193 and Kasumi-1 cells, the knockout and compensated experiments revealed that eukaryotic translation elongation factor 1 alpha 2 promoted cell proliferation and migration but repressed apoptosis. Additionally, the knockout of eukaryotic translation elongation factor 1 alpha 2 decreased dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression, meanwhile, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression enhanced while eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression. Furthermore, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression promoted cell proliferation, enhanced migration, and decreased apoptosis, but eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence these cellular functions in AML-193 and Kasumi-1 cells, suggesting the implication of dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in eukaryotic translation elongation factor 1 alpha 2 mediated oncogenesis of acute myeloid leukemia.Conclusion:Eukaryotic translation elongation factor 1 alpha 2 and its dimethylated product may serve as therapeutic targets, and these findings may provide support for exploring novel strategies in acute myeloid leukemia treatment.

Prdx6 is required to protect human corneal epithelial cells against ultraviolet B injury.

The protective role of Prdx6 on rat corneal tissue against ultraviolet B injury in vivo has been confirmed previously. We further investigated the function and molecular mechanism of Prdx6 in human corneal epithelial cells under ultraviolet B radiation. The experimental groups were designed as follows: (1) Prdx6 RNAi, (2) Prdx6 RNAi + ultraviolet B radiation, (3) normal human corneal epithelial cells, (4) normal human corneal epithelial cells + ultraviolet B radiation, (5) wild-type Prdx6 overexpression, (6) wild-type Prdx6 overexpression + ultraviolet B radiation, (7) mutant-type Prdx6 overexpression, and (8) mutant-type Prdx6 overexpression + ultraviolet B radiation. The cell survival rate was detected by a Thiazolyl Blue Tetrazolium Bromide assay. Apoptosis, reactive oxygen species, and malondialdehyde were detected with a commercial kit. Gene expression was detected by real-time polymerase chain reaction. We found the following results. (1) Compared to normal cells, the survival rates were 32%, 87%, and 58% under ultraviolet B radiation in the Prdx6 interference, wild-type overexpression, and mutant-type overexpression groups, respectively. The survival rates were decreased to 50% at 24 h and 31% at 48 h when the phospholipase A2 activity of Prdx6 was inhibited after ultraviolet B radiation. (2) Apoptosis, reactive oxygen species content, and malondialdehyde levels were increased when Prdx6 was downregulated. This phenomenon became more severe under ultraviolet B radiation. (3) The expression levels of apoptosis-related and antioxidant genes all changed along with the changes in expression of Prdx6. (1) Both peroxidase and phospholipase A2 activities of Prdx6 are crucial for its protective role in corneal tissue. (2) Downregulated expression of Prdx6 resulted in high endoplasmic reticulum stress. (3) Apoptosis in human corneal epithelial cells with downregulated Prdx6 coupled with ultraviolet B radiation was related to the pathways of DNA damage and the death receptor. (4) Low levels of antioxidants are sufficient for maintaining homeostasis in human corneal epithelial cells without external stimuli. Under the condition that Prdx6 was downregulated, human corneal epithelial cells were more sensitive to ultraviolet B radiation.

CEBPA Gene Mutations Interfere with Expressions of NKG2D Ligands Ulbps in AML Cells and Modulate Their Susceptibility to NK-Mediated Lysis

Introduction: CEBPA gene encodes CCAAT/enhancer-binding protein-alpha (C/EBPα), a crucial granulocytic differentiation factor and tumor suppressor in hematologic and many non-hematologic malignancies. We previously reported a donor-derived relapse of AML patient after allogeneic hematopoietic stem cell transplantation with multiple mutations in CEBPA gene: a N-terminal frameshift mutation (247dupC causing overproduction of truncated 30-KDa isoform, lacking the TAD1 domain, p30), a N-terminal germ-line mutation (584_589dup disrupting the TAD2 domain of protein, NM2), and a C-terminal mutation (914_916dup disrupting the bZIP domain, CM) (Blood 2011; 117: 5257-5260). Although studies from multiple laboratories have contributed immensely to our understanding that how different CEBPA mutations disturb C/EBPα functions including granulopoiesis and leukemic transformation in AML, whether C/EBPα might regulate immunosurveillance remains unknown. Methods: AML cell line cells infected with lentivirus to over-express of wild type C/EBPα as well as 3 types of C/EBPα mutants were co-cultured with NK92MI cells and detected cytotoxic lysis through FCM. NK92MI cells were stained with CD107a to detect degranulation.We performed gene expression microarray profiling analysis in AML cell line cells with over-expression of wild type C/EBPα and mutants . Flag tagged wide type C/EBPα was over-expressed in 293T cells and ChIP with anti-Flag antibody followed by sequencing assay was performed to explore candidate gene binding sites of C/EBPα. Finally, independent ChIP-qPCR of candidate sequences were performed to further verify the transcription factor binding sites of C/EBPα. Results: ULBPs expressed on the surface of tumor or infected cells are important ligands of NK cell receptor NKG2D. Our gene expression microarray profiling analysis showed that wild type C/EBPα could up-regulate the expression of ULBP2/5/6 in AML cell line cells. Consistent with the results of gene expression microarray profiling analysis, over-expression of wild type C/EBPα and a N-terminal germ-line mutant (NM2) can up-regulate ULBP2/5/6 expression in NB4 cells, whose endogenous expression of ULBPs was low. Meanwhile, the sensitivities of NB4 cells to the cytotoxicity of NK92MI cells were also increased by over-expression of wide type C/EBPα and NM2 mutant. In contrast, leukemia-associated somatic mutations, C/EBPα p30 and C-terminal mutant (CM), were disabled to up-regulate ULBPs expression. In dual-luciferase reporter assay, the ratio of level of firefly luciferase and renilla luciferase significantly increased when co-transduced report plasmid with wide type C/EBPα expressing plasmid compared with vector control, indicating that C/EBPα could up-regulate the transcription of ULBP2 as a transcription factor. Through ChIP-seq assay we identified 12 peaks nearby ULBP genes in chromosome 6. We further performed ChIP-qPCR to target the sequences acting as enhancers of ULBP genes, which located +7kb upstream of transcription start site of ULBP2 gene, +11kb upstream of ULBP5 gene, -9kb downstream of ULBP6 gene and -40kb downstream of ULBP1 gene. Wide type C/EBPα showed higher binding affinity to the ULBP2/5 enhancers with more than 50 folds' enrichment and to the ULBP6/1 enhancers 9 folds' enrichment compared with IgG control. The N-terminal germ-line mutant (NM2) conserved part of the binding affinity, but the enrich fold was lower than wide type. As expected, leukemia-associated C-terminal mutant (CM) totally lost its binding ability to both sequences due to the damage of DNA binding domain. Althoughleukemia-associated truncated 30-KDa isoform partly conserved its binding ability to these DNA sequences, the mutant lost the function of regulating ULBPs expression. Conclusions: C/EBPα played an important role in innate immunosurveillance of AML. C/EBPα could bind to the promoter and potential enhancers of ULBP genes as a transcription factor, up-regulate expression of ULBPs and eventually induce AML cells to be recognized and killed by NK cells. Mutations in TAD2 domain did not affect this regulation function, while mutations in TAD1 and bZIP domain lost the specific ability. Leukemia cells with N-terminal frameshift mutations (p30), or C-terminal mutations could escape from surveillance of NK cells and may play pivotal roles in leukemia relapse. Disclosures No relevant conflicts of interest to declare.

The Clinical and Functional Relevance of the Transforming AKT E17K Mutation in T-ALL

INTRODUCTION. The importance of IL-7R signaling has been thoroughly studied in the context of both normal T-cell development and leukemogenesis. Upon IL-7 induced heterodimerization of the IL-7Rα and the γc chain, downstream signaling routes regulate survival and proliferation of differentiating T-cells. JAK-STAT, MAPK-ERK and PI3K-AKT pathways are aberrantly activated in T-cell acute lymphoblastic leukemia (T-ALL) patients by recurrent gain-of-function mutations of the IL-7R signaling pathway. IL-7R signaling mutations are a result of mutations in IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS, PI3K or AKT that are found in nearly 35% of pediatric T-ALL patients and are associated with steroid resistance and inferior event-free survival. Here we focus on downstream AKT activation in relation to cellular transformation and steroid resistance. We previously demonstrated that AKTE17K transforms IL3-dependent BaF3 cells in contrast to wild type AKT, while wild type AKT drives steroid resistance in SUP-T1 T-ALL cells in contrast to mutant AKTE17K. AIM. Our aim was to identify functional differences between AKT and AKTE17K molecules in respect to cellular transformation and steroid resistance. We assessed activation level, cellular localization and downstream signaling events that may explain these functional differences. RESULTS. For this study, we used doxycycline-inducible SUP-T1 cell lines that overexpress wild type AKT or mutant AKTE17K molecules. Using immunohistochemistry, we revealed abundant cytoplasmic localization of wild type AKT but predominant plasma membrane localization of AKTE17K and extends previous findings by others. Furthermore, we found evidence for phosphorylation of wild type AKT on serine-473 (S473) and threonine-308 (T308) residues in both cytoplasmic and nuclear fractions, indicating that part of induced AKT becomes activated. In contrast, expression of AKTE17K was accompanied with abundant phosphorylation of T308 while having relatively low levels of S473 phosphorylation. The T308/S473 phosphorylation ratio was 5 times higher for AKTE17K compared to wild type AKT. These results are in line with current views on AKT activation that S473 phosphorylation facilitates the opening of the AKT pleckstrin homology domain and membrane docking, after which AKT is phosphorylated (activated) on the T308 residue by PDK1. The constitutive plasma membrane localization and open conformation of AKTE17K as a result of its E17K pleckstrin homology domain mutation leaves it independent of S473 phosphorylation and results in enhanced T308 phosphorylation by PDK1. Consequently, AKTE17K mutant cells may exhibit reduced cellular sensitivity to allosteric AKT inhibitors but not to type I ATP-competitive inhibitors. Indeed, SUP-T1 cells expressing wild type AKT or mutant AKTE17K are equally sensitivity to the ATP-competitive AZD5363 inhibitor, but AKTE17K cells display reduced sensitivity to the MK2206 allosteric inhibitor compared to AKT wild type cells. In an attempt to explain enhanced steroid resistance by expression of wild type AKT but not mutant AKTE17K, we found no differences in AKT-mediated S134 phosphorylation of the NR3C1 steroid receptor or impaired nuclear import of NR3C1 following steroid treatment (Piovan et al., Cancer Cell 2013). This indicates that AKT induces steroid resistance by other mechanisms in our SUP-T1 cell system. CONCLUSIONS. In contrast to wild type AKT overexpression, we demonstrated that AKTE17K has (i) transforming activity, (ii) does not yield cellular resistance to steroid treatment, (iii) is predominantly localized at the plasma membrane, (iv) has high T308 over S473 phosphorylation and (v) is less sensitive to the MK2206 allosteric AKT-inhibitor. These observed differences are likely caused by the constitutively open AKTE17K conformation. Since no differences in NR3C1 phosphorylation or nuclear shuttling were observed in our model, we hypothesize other mechanisms underlie the provoked steroid resistance by AKT wild type overexpression. To identify these novel mechanisms, we are currently performing an unbiased phospho-protein array to identify differentially phosphorylated AKT substrates. Disclosures No relevant conflicts of interest to declare.