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Chimeric antigen receptor (CAR)-modified immune cells have emerged as a promising approach for cancer treatment, but single-target CAR therapy in solid tumors is limited by immune escape caused by tumor antigen heterogeneity and shedding. Natural killer group 2D (NKG2D) is an activating receptor expressed in human NK cells, and its ligands, such as MICA and MICB (MICA/B), are widely expressed in malignant cells and typically absent from healthy tissue. NKG2D plays an important role in anti-tumor immunity, recognizing tumor cells and initiating an anti-tumor response. Therefore, NKG2D-based CAR is a promising CAR candidate. Nevertheless, the shedding of MICA/B hinders the therapeutic efficacy of NKG2D-CARs. Here, we designed a novel CAR by engineering an anti-MICA/B shedding antibody 1D5 into the CAR construct. The engineered NK cells exhibited significantly enhanced cytotoxicity against various MICA/B-expressing tumor cells and were not inhibited by NKG2D antibody or NKG2D-Fc fusion protein, indicating no interference with NKG2D-MICA/B binding. Therefore, the developed 1D5-CAR could be combined with NKG2D-CAR to further improve the obstacles caused by MICA/B shedding. • 1D5-CAR targeting MICA/B α3 was designed based on structure. • 1D5-CAR-modified NK cells exhibited potent cytotoxicity. • 1D5-CAR didn't disturb NKG2D-MICA/B interaction. • 1D5-CAR could be combined with NKG2D-CAR. [ABSTRACT FROM AUTHOR]

Abstract Nasopharyngeal carcinoma (NPC) is a prevalent tumor in southern China and southeast Asia. Recent studies have demonstrated that viral infection, somatic genetic changes, and epigenetic changes synergistically contribute to NPC pathogenesis. Genome-wide studies show that epigenetic aberrations likely drive nasopharyngeal carcinoma development and progression. This work is aimed at investigating the effect of histone methyltransferase SUV39H2 in NPC. The elevated expression of SUV39H2 in NPC is observed by analyzing GSE53819 and GSE12452 downloaded from the Gene Expression Omnibus (GEO) database. SUV39H2 knockdown inhibits NPC proliferation and induces the apoptosis of cancer cells. At last, RNaseq analysis identifies a variety of SUV39H2 downstream genes related with cancer, in which, NRIP1 is identified as a critical downstream target of SUV39H2 in NPC. Taken together, these findings provide a theoretical basis for understating the biological roles of SUV39H2 in NPC. Highlights • SUV39H2 is upregualted in GSE53819 and GSE12452 database. • SUV39H2 knockdown inhibits NPC proliferation and induces the apoptosis of cancer cells. • NRIP1 is identified as a critical downstream target of SUV39H2 in NPC. [ABSTRACT FROM AUTHOR]

Highlights: [•] Twelve genes at AC134922 locus show resistance to at least one isolate. [•] Gene stacking is evolutionary strategy for wide spectrum and durable resistance. [•] The mosaic structure and ambiguous relationship of AC134922 genes was observed. [•] Only several amino-acid differences were pivot for blast resistance spectrum. [Copyright &y& Elsevier]

Abstract: The ezrin–radixin–moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton, and also participate in signal-transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies have mapped the PKA-mediated phosphorylation site to Ser66 and established its functional role in parietal cell activation [R. Zhou et al., Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651–35659], but the underlying basis for this regulation is not known. Here, we provide the first evidence that PKA-mediated phosphorylation of Ser66regulates the interaction of ezrin with WWOX, a WW domain-containing protein. Our biochemical study reveals that ezrin directly binds to the first WW domain of WWOX via its C-terminal tyrosine-containing polyproline sequence 470PPPPPPVY477. Mutational analyses further demonstrate that tyrosine477 is essential for the ezrin–WWOX interaction. In addition, our study shows that PKA-mediated phosphorylation of ezrin is essential and sufficient for the apical localization of WWOX protein as disruption of ezrin–WWOX interaction eliminated the apical localization of WWOX. Finally, our study demonstrates the essential role of ezrin–WWOX interaction in the apical membrane remodeling associated with H,K-ATPase recruitment. Taken together, these results define a novel molecular mechanism underlying phospho-regulation of ezrin function by PKA in parietal cell activation. [Copyright &y& Elsevier]

Abstract: Yip1p and Yif1p are essential for transport from ER to Golgi stack during the early secretory pathway in budding yeast. Here, we report the identification and characterization of human Yif1. Sequence analysis revealed that human Yif1 (HsYif1), like most of the other YIP1 protein family members, contains multiple transmembrane segments. Double immunofluorescence study revealed co-distribution of HsYif1 with Golgi marker such as GS27. To delineate the function of HsYif1, we conducted a yeast two-hybrid assay and identified an interaction between human HsYif1 and HsYip1A, a homolog of yeast Yip1. In addition, our immunoprecipitation pull-down assay validates the interaction between HsYif1 and HsYip1A. Moreover, our immunofluorescence study demonstrates the co-distribution of HsYif1 and HsYip1A. Significantly, over-expression of mutant HsYip1A-lacked cytosolic region disrupts the localization of HsYif1 to the Golgi, suggesting that HsYip1A specifies the localization of HsYif1 to the Golgi. Therefore, we conclude that human Yip1A interacts with and determines the localization of HsYif1 to the Golgi apparatus. [Copyright &y& Elsevier]

Hypoxia-inducible factor 1 (HIF-1) is a phosphorylated protein and its phosphorylation is involved in HIF-1α subunit stabilization as well as in the regulation of HIF-1 transcriptional activity. In a variety of cell lines, the phosphorylation of HIF-1α is dependent on ERK or p38, two members of the mitogen-activated protein kinase (MAPK) superfamily. In addition, active MAPK could be inactivated through dephosphorylation by mitogen-activated protein kinase phosphatase-1 (MKP-1). MKP-1 has been identified as a hypoxia responsive gene, but its role in the response of cells to hypoxia is poorly understood. Here we found that hypoxia induces MKP-1 expression in human hepatoma cells HepG2 in a time-dependent manner. Inhibition of MKP-1 expression using siRNA technique could enhance HIF-1α phosphorylation, accompanied by an increase in transcriptionally active HIF-1 as well as a rise in the levels of HIF-1-induced erythropoietin expression. [Copyright &y& Elsevier]

Pleckstrin homology-like domain family A, member 3 (PHLDA3) is a novel tumor-related protein that mediates carcinogenesis of multiple cancers. However, the relevance of PHLDA3 in prostate cancer has not been explored. The purpose of this work was to illustrate the possible roles and mechanisms of PHLDA3 in prostate cancer. Our data showed strikingly lower abundance of PHLDA3 in prostate cancer, and that low levels of PHLDA3 in prostate cancer patients was associated with reduced survival. PHLDA3 was also weakly expressed in prostate cancer cells, and demethylation treatment dramatically up-regulated the expression level of PHLDA3. Up-regulation of PHLDA3 restrained proliferation, induced G1 cell cycle arrest, suppressed epithelial-mesenchymal transition of prostate cancer cells. In addition, up-regulation of PHLDA3 increased the sensitivity of prostate cancer cells to docetaxel In-depth research into the mechanism elucidated that PHLDA3 overexpression decreased the phosphorylation of Akt and suppressed the activation of Wnt/β-catenin signaling. Overexpression of constitutively active Akt strikingly abolished PHLDA3-mediated inactivation of Wnt/β-catenin pathway. A xenograft assay revealed that prostate cancer cells with PHLDA3 overexpression displayed reduced tumorigenicity in vivo. Collectively, these data document that PHLDA3 exerts an outstanding cancer-inhibiting role in prostate cancer by down-regulating Wnt/β-catenin pathway via the inhibition of Akt. This work highlights PHLDA3 as a novel anticancer target for prostate cancer. [Display omitted] • PHLDA3 is decreased in prostate cancer and has a prognostic value. • PHLDA3 exerts a tumor suppressive role in prostate cancer. • PHLDA3 inhibits Wnt/β-catenin pathway via Akt. • PHLDA3 inhibits prostate cancer by inhibiting Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

Engineered natural killer (NK) cell-based therapies have been potentially broadly applicable and exhibited promising results in clinical trials, particularly in the fight against cancers. NK cell immunotherapy however always remains variable. One major obstacle is the inhibitory pathway including PD1/PDL1, providing tumor cells an escape mechanism from immunosurveillance. In this regard, we rationally designed a chimeric switch-receptor (CSR) PD1-DAP10-41BB, which comprising the ectodomain of PD1 fused to the co-stimulatory receptor DAP10 and 41BB. Therefore, by exchanging the transmembrane and cytoplasmic tail of PD1 with positive costimulatory molecules DAP10 and 41BB signaling domains, the negative PD1/PDL1 signal pathway was thus converted into a positive one. This CSR-expressing NK92 cells showed a typical parental NK92 phenotype and improved cytotoxicity against human lung cancer H1299 cells. Besides, the expression of CSR elicited a significant increase of effector molecules such as perforin and granzymes, which can induce apoptosis of H1299 cells. More importantly, in the solid tumor cell H1299-bearing mice model, the CSR-modified NK92 cells significantly inhibited tumor growth. Collectively, we demonstrated that expression of PD1-DAP10-41BB augmented NK92-cell activation and killing in vitro and in vivo , which provides a considerable avenue of using NK-tailored chimeric receptor engineered NK92 cells to treat a wide range of solid tumors. • A novel NK-tailored chimeric switch-receptor PD1-DAP10-41BB is rationally designed. • Modified NK92 cells mediate the clearance of H1299 cells by triggering apoptosis. • PD1-DAP10-41BB-expressing NK92 cells elicit in vivo tumor control of H1299 cells. [ABSTRACT FROM AUTHOR]

Abstract: Protein acetylation is an important and reversible post-translational modification (PTM), and it governs a variety of cellular dynamics and plasticity. Experimental identification of acetylation sites is labor-intensive and often limited by the availability of reagents such as acetyl-specific antibodies and optimization of enzymatic reactions. Computational analyses may facilitate the identification of potential acetylation sites and provide insights into further experimentation. In this manuscript, we present a novel protein acetylation prediction program named PAIL, prediction of acetylation on internal lysines, implemented in a BDM (Bayesian Discriminant Method) algorithm. The accuracies of PAIL are 85.13%, 87.97%, and 89.21% at low, medium, and high thresholds, respectively. Both Jack–Knife validation and n-fold cross-validation have been performed to show that PAIL is accurate and robust. Taken together, we propose that PAIL is a novel predictor for identification of protein acetylation sites and may serve as an important tool to study the function of protein acetylation. PAIL has been implemented in PHP and is freely available on a web server at: http://bioinformatics.lcd-ustc.org/pail. [Copyright &y& Elsevier]

Abstract: Mitotic arrest deficient 2 (MAD2) is an essential component of the mitotic spindle checkpoint pathway. It was previously shown to be associated with drug resistance of tumor cells. To further explore the roles of MAD2 in responses of gastric cancer cells to chemotherapy drugs, we constructed the siRNA vectors of MAD2 and transfected them into gastric cancer SGC7901 cells to inhibit expression of MAD2. MTT assay showed that the downregulation of MAD2 increased the resistance of SGC7901 cells to spindle inhibitors and DNA damaging agents. The apoptosis rates of gastric cancer cells transfected with MAD2-siRNA were 10.7% and 10%, respectively, after treated by 1.0μg/ml VCR and cisplatin. In contrast, the apoptosis rates of SGC7901 and SGC7901/psilencer3.1 induced by VCR were 43.2%, 38.7%; and that induced by cispaltin were 34.1%, 31.4%. The ratio of Bcl-2 to Bax was much higher in the MAD2-siRNA transfectants compared with the SGC7901/psilencer. In SGC7901/psilencer, cytochrome c and cleaved caspase 3 protein levels increased along with the exposure time increased. However, these protein levels of SGC7901/MAD2-siRNA had no changes during the drug treatment. These results indicate that down regulation of MAD2 could promote the drug resistance of gastric cancer cells and inhibit anticancer drugs induced-apoptosis by upregulating Bcl-2 and interfering the mitochondrion apoptosis pathway. [Copyright &y& Elsevier]

Bcl-2 family of proteins plays differential roles in regulation of mitochondria-mediated apoptosis, by either promoting or inhibiting the release of apoptogenic molecules from mitochondria to cytosol. Bcl-2 family proteins modulate the mitochondrial permeability through interaction with adenine nucleotide translocator (ANT), voltage-dependent anion channel (VDAC), ADP/ATP exchange, or oxidative phosphorylation during apoptosis. Although the mitochondrial homeostasis is affected by the relative ratio of pro- and anti-apoptotic Bcl-2 family members, the molecular mechanism underlying the release of mitochondrial intermembrane proteins remains elusive. Here we reported the biochemical evidence that both pro-apoptotic Bax and anti-apoptotic Bcl-XL might simultaneously contact the putative loop regions of human VDAC1, and the existence of VDAC1–Bax–Bcl-XL tertiary complex in vitro suggested that VDAC1 channel conformation and mitochondrial permeability could be determined by the delicate balance between Bax and Bcl-XL. [Copyright &y& Elsevier]

Emerging evidence suggests that microRNAs (miRNAs) participate in hepatocellular carcinoma (HCC) progression. Nevertheless, the mechanism of miR-7-5p in HCC cells has not been researched. In the research, the underlying biological function of miR-7-5p and SPC24 in HCC was explored. qRT-PCR was performed to measure the miR-7-5p and SPC24 level in HCC tissues and cells. The effect of miR-7-5p on HCC progression was detected by performing CCK-8, BrdU, and transwell assay. The relationship between miR-7-5p and SPC24 was determined using luciferase and RNA pull-down assays. Our findings showed that miR-7-5p was downregulated in HCC whereas SPC24 was upregulated in HCC. It was also showed that miR-7-5p upregulation restricted malignant behaviors of HCC cells, but this inhibitory effect of miR-7-5p could be relieved by its target gene SPC24. In conclusion, this research suggested that by inhibiting SPC24, miR-7-5p could act as a tumor inhibitory factor in HCC. • The identification of mRNA and miRNA of interest. • Overexpression of miR-7-5p inhibits the growth, migration and promotes apoptosis of HCC cells. • MiR-7-5p directly targets SPC24. • SPC24 reverses the effect of miR-7-5p on the cellular progression of HCC. [ABSTRACT FROM AUTHOR]

Cell-based immunotherapy continues to be a promising avenue for cancers that standard therapy has failed. Although the specificity, avidity, and efficacy of infused cells have improved, immunocytotherapy still faces substantial hurdles. To this end, we developed a structure-based rational design approach and constructed a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D comprising the truncated ectodomain of PD1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D, which evaluated the capacity of solid tumor cell killing. Retroviral transduction of DTCR dramatically increased NK92 cell surface expression of PD1 and NKG2D, which boosted robust cytotoxicity against human gastric cell SGC-7901. Chimeric receptor DTCR stimulation elicited a significant increase of TNF-α and TRAIL, which can trigger apoptosis of SGC-7901 cells. More importantly, DTCR-NK92 cells had considerable antitumor activity in the solid tumor cell SGC-7901-bearing mice model. Collectively, we demonstrated that expression of DTCR markedly augmented the cytotoxic potential of NK92 cells against solid tumor cells, and this potentially promising treatment modality will facilitate clinical translation of potent NK-tailored chimeric receptor strategy for a generalized cellular therapy that may be conducive to treat a wide range of solid tumors. • A novel NK-tailored bispecific chimeric receptor PD1-DAP10/NKG2D is rationally designed with a structure-based approach. • Engineered NK92 cells can not only trigger apoptosis, but seemingly induce pyroptosis of SGC-7901 solid tumor cells. • Expressing PD1-DAP10/NKG2D mediates robust in vitro cytotoxicity and in vivo tumor control of NK92 cells. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg 2+ , which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics. [ABSTRACT FROM AUTHOR]

The function of subcutaneous adipocytes in promoting wound healing is significantly suppressed in diabetic wounds. Recent studies have demonstrated the ability of mesenchymal stem cell (MSC) to ameliorate impaired diabetic wound healing. We hypothesized that MSC function may involve subcutaneous adipocytes. The abnormal function of subcutaneous adipocytes from STZ induced diabetic mice including glucose uptake and free fatty acid (FFA) secretion level were assessed. Then these cells were co-cultured with MSC via a transwell system to observe the changes of metabolic index and glucose transporter four (GLUT4) as well as phosphoinositide 3-kinase/protein kinase (PI3K/AKT) signaling pathway expression. The results of metabolic index suggest that MSC obviously attenuated the diabetes-induced functional impairment. Both mRNA and protein expression analyses showed that PI3K/AKT insulin signaling pathway and GLUT4 expression were up-regulated. These changes were substantially associated with a increased level of insulin-like growth factor-1 (IGF-1) secretion from MSC. These findings suggest that MSC could attenuate abnormal function of diabetic adipocytes by IGF-1secretion, which was more or less associated with the beneficial effects of MSC on improving diabetic wound healing. [ABSTRACT FROM AUTHOR]

Highlights: [•] β-TrCP1 is a novel interacting protein of TRF1. [•] β-TrCP1 promotes ubiquitination of TRF1. [•] β-TrCP1 regulates the stability of TRF1. [•] β-TrCP1 regulates APB formation in ALT cells. [ABSTRACT FROM AUTHOR]

Abstract: Tara was identified as an interacting partner of guanine nucleotide exchange factor Trio and TRF1. Tara is proposed to be involved in many important fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation. Yet, its exact roles required further elucidation. Here, we identify a novel Tara-binding protein HECTD3, a putative member of HECT E3 ubiquitin ligases. HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo. Overexpression of HECTD3 enhances the ubiquitination of Tara in vivo and promotes the turnover of Tara, whereas depletion of HECTD3 by small interfering RNA decreases Tara degradation. Furthermore, depletion of HECTD3 leads to multipolar spindle formation. All these findings suggest that HECTD3 may facilitate cell cycle progression via regulating ubiquitination and degradation of Tara. [Copyright &y& Elsevier]

Abstract: The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. p150 Glued is the dynactin subunit responsible for binding to dynein and microtubules. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which governs phosphorylation-dependent ubiquitination and subsequent proteolysis. Our recent study showed that the proteolysis of mitotic kinesin CENP-E is mediated by SCF via a direct Skp1 link [D. Liu, N. Zhang, J. Du, X. Cai, M. Zhu, C. Jin, Z. Dou, C. Feng, Y. Yang, L. Liu, K. Takeyasu, W. Xie, X. Yao, Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis, Biochem. Biophys. Res. Commun. 345 (2006) 394–402]. Here we show that F-box protein FBXL5 interacts with p150 Glued and orchestrates its turnover via ubiquitination. FBXL5 binds to p150 Glued in vitro and in vivo. FBXL5 and p150 Glued co-localize primarily in the cytoplasm with peri-nuclear enrichment in HeLa cells. Overexpression of FBXL5 promotes poly-ubiquitination of p150 Glued and protein turnover of p150 Glued . Our findings provide a potential mechanism by which p150 Glued protein function is regulated by SCFs. [Copyright &y& Elsevier]

Abstract: Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Our previous studies show that microtubule motor CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signaling cascade. However, the molecular function of CENP-E at the midbody had remained elusive. Here we show that CENP-E interacts with Skp1 at the midbody and participates in cytokinesis. CENP-E interacts with Skp1 in vitro and in vivo via its coiled-coil domain. Our yeast two-hybrid assays mapped the binding interfaces to the central stalk region of CENP-E (955–1571 aa) and the C-terminal 33 amino acids of Skp1, respectively. Our immunocytochemical studies revealed that CENP-E targets to the midbody prior to Skp1 and the midbody localization of CENP-E becomes diminished as Skp1 arrives at the midbody. Suppression of Skp1 in mitotic HeLa cells by siRNA resulted in accumulation of telophase cells with elongated inter-cell bridges and with midbodies stretched 2–3 times longer than that of normal cells. These Skp1-eliminated or -suppressed cells accumulate higher level of CENP-E, suggesting that spatiotemporal regulation of CENP-E degradation at the midbody is essential for cytokinesis. Over-expression of Skp1 lacking the CENP-E-binding domain confirmed that Skp1–CENP-E interaction is essential for faithful cytokinesis. We hypothesize that CENP-E degradation is essential for faithful mitotic exit and the proteolysis of CENP-E is mediated by SCF via a direct Skp1 link. [Copyright &y& Elsevier]

Zinc ribbon domain-containing 1 (ZNRD1), a transcription-associated gene, was recently found to be downregulated in human gastric cancer tissues as compared to the matched adjacent nonneoplastic tissues. In this study, we constructed the siRNA eukaryotic expression vectors of ZNRD1 and transfected them into normal gastric epithelial cells (GES-1). We also introduced the ZNRD1 gene into gastric cancer cells that do (SGC7901) and do not (AGS) express ZNRD1 endogenously. GES-1 cells stably transfected with the ZNRD1-RNAi were found to exhibit significantly quicker proliferation than empty vector transfectants. AGS cells stably transfected with the ZNRD1 cDNA exhibited significantly decreased growth rate as compared to control vector transfectants, whereas SGC7901 cells did not. Furthermore, ZNRD1 suppresses growth of AGS cells in soft agar and tumor formation in athymic nude mice. This study clearly demonstrates that ZNRD1 may play an important role in the control of human gastric cancer development by regulating cell proliferation. These results provide new insights into the function of ZNRD1 and further validate ZNRD1 as a potential therapeutic target in gastric cancer. [Copyright &y& Elsevier]