A human embryonic kidney 293T cell line mutated at the Golgi -mannosidase II locus

Disruption of Golgi -mannosidase II activity can result in type II congenital dyserythropoietic anemia and can induce lupus-like autoimmunity in mice. Here, we isolate a mutant human embryonic kidney (HEK) 293T cell line, called Lec36, that displays sensitivity to ricin that lies between the parental HEK 293T cells, whose secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which only produce oligomannose-type N-linked glycans. The stem cell marker, 19A, was transiently expressed in the HEK 293T Lec36 cells, and in parental HEK 293T cells with and without the potent Golgi -mannosidase II inhibitor, swainsonine. Negative-ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi -mannosidase II: a point mutation in one allele mapping to the active site and an in-frame deletion of twelve-nucleotides in the other. Expression of wild-type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and provides a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia

Functional characterization of a short peptidoglycan recognition protein from Chinese giant salamander (Andrias davidianus)

This work was supported by the National Natural Science Foundation of China (Grant no. 31302221, 31172408 and 31272666) and Jiangsu Province (Grant no. BK20171274 and BK2011418), and partially by the Opening Project of Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland (Grant no. K2016-08). QZ was supported by the “Qinglan” project of Jiangsu province of China.Peer reviewedPublisher PD

The role of influenza neuraminidase transmembrane domain on budding and virus morphology

Influenza A virus neuraminidase (NA), a type II transmembrane glycoprotein plays a role in the cleavage of sialic acids and facilitating the release of mature virions from the surface of infected cells. NA has also previously been shown to play a role in virion formation during influenza A virus budding, although the exact mechanisms by which NA contributes to influenza virion formation and morphology is currently unknown. Previous research has shown that mutations within the transmembrane domain (TMD) of NA can result in alteration in virion morphology, particularly in the production of filament like influenza virions. In this research project we examined if the TMD does indeed play a role in influenza virus budding and morphology. We utilised both full and partial mutations of the TMD of NA from A/WSN/33, a primarily spherical lab adapted influenza strain, with the TMD of a primarily filamentous strain A/California/09. To evaluate the effects of TMD on the morphology of a primarily spherical strain with that of filamentous strain. This study used a transfection based virus like particle (VLP) system to examine the effects of TMD alterations on morphology, utilising various biochemical and microscopy methods. Our findings show that as previously indicated mutations within the TMD do result in alterations to virion morphology, as well as showing that despite previous theories both NA and NA’s TMD may play a more active role in in budding and morphology than previously though

NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways

Cysteine oxidation targets peroxiredoxins 1 and 2 for exosomal release through a novel mechanism of redox-dependent secretion

Non-classical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of non-classical secretion. We have recently shown that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as LPS or TNF-α. The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms as has been postulated for the inflammatory mediators IL-1β and HMGB1. We show here that circulating Prdx1 and 2 are present exclusively as disulphide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α and this release can be induced with an oxidant. In contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway; instead, both Prdx1 and 2 are released in exosomes from both HEK cells and monocytic cells. Serum Prdx1 and 2 are also associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signalling mechanisms in inflammation

Evaluation of cell culture in microfluidic chips for application in monoclonal antibody production

Microfluidic chips are useful devices for cell culture that allow cell growth under highly controlled conditions, as is required for production of therapeutic recombinant proteins. To understand the optimal conditions for growth of cells amenable of recombinant protein expression in these devices,we culturedHEK-293T cells under different microfluidic experimental conditions. The cells were cultured in polymethyl methacrylate (PMMA) and polydi-methylsiloxane (PDMS)microdevices, in the absence or presence of the cell adhesion agent poly-D-lysine. Different microchannel geometries and thicknesses, as well as the influence of the flow rate have also been tested, showing their great influence in cell adhesion and growth. Results show that the presence of poly-D-lysine improves the adhesion and viability of the cells in continuous or discontinuous flow. Moreover, the optimal adhesion of cells was observed in the corners of themicrochannels, as well as in wide channels possibly due to the decrease in the flow rate in these areas. These studies provide insight into the optimal architecture of microchannels for long-term culture of adherent cells in order to use microfluidics devices as bioreactors for monoclonal antibodies production.Fil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Payés, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sierra Rodero, Marina. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vega, M.. Universidad Tecnológica Nacional; ArgentinaFil: Rosero, G.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pérez, M. S.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; Argentin

Orexin-Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking

Mammalian EAK-7 activates alternative mTOR signaling to regulate cell proliferation and migration.

Nematode EAK-7 (enhancer-of-akt-1-7) regulates dauer formation and controls life span; however, the function of the human ortholog mammalian EAK-7 (mEAK-7) is unknown. We report that mEAK-7 activates an alternative mechanistic/mammalian target of rapamycin (mTOR) signaling pathway in human cells, in which mEAK-7 interacts with mTOR at the lysosome to facilitate S6K2 activation and 4E-BP1 repression. Despite interacting with mTOR and mammalian lethal with SEC13 protein 8 (mLST8), mEAK-7 does not interact with other mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2) components; however, it is essential for mTOR signaling at the lysosome. This phenomenon is distinguished by S6 and 4E-BP1 activity in response to nutrient stimulation. Conventional S6K1 phosphorylation is uncoupled from S6 phosphorylation in response to mEAK-7 knockdown. mEAK-7 recruits mTOR to the lysosome, a crucial compartment for mTOR activation. Loss of mEAK-7 results in a marked decrease in lysosomal localization of mTOR, whereas overexpression of mEAK-7 results in enhanced lysosomal localization of mTOR. Deletion of the carboxyl terminus of mEAK-7 significantly decreases mTOR interaction. mEAK-7 knockdown decreases cell proliferation and migration, whereas overexpression of mEAK-7 enhances these cellular effects. Constitutively activated S6K rescues mTOR signaling in mEAK-7-knocked down cells. Thus, mEAK-7 activates an alternative mTOR signaling pathway through S6K2 and 4E-BP1 to regulate cell proliferation and migration

Improved split fluorescent proteins for endogenous protein labeling.

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split GFP1-10/11; as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use

Cognitive impairment induced by delta9-tetrahydrocannabinol occurs through heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors

Delta-9-tetrahydrocannabinol (THC), the main psychoactive compound of marijuana, induces numerous undesirable effects, including memory impairments, anxiety, and dependence. Conversely, THC also has potentially therapeutic effects, including analgesia, muscle relaxation, and neuroprotection. However, the mechanisms that dissociate these responses are still not known. Using mice lacking the serotonin receptor 5-HT2A, we revealed that the analgesic and amnesic effects of THC are independent of each other: while amnesia induced by THC disappears in the mutant mice, THC can still promote analgesia in these animals. In subsequent molecular studies, we showed that in specific brain regions involved in memory formation, the receptors for THC and the 5-HT2A receptors work together by physically interacting with each other. Experimentally interfering with this interaction prevented the memory deficits induced by THC, but not its analgesic properties. Our results highlight a novel mechanism by which the beneficial analgesic properties of THC can be dissociated from its cognitive side effects