We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. The virus-cell membrane fusion event is an essential step in the entry process of all enveloped animal viruses, including important human pathogens such as influenza virus, human immunodeficiency virus (HIV) (8, 23), and the newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV) (9). Following the binding to their receptors on the cell surface, virus-encoded membrane fusion proteins mediate the fusion process. In many but not all cases, the viral fusion proteins are proteolytically processed by host proteases into 2 subunits that remain closely associated with each other: a surface subunit with a receptor-binding site and a transmembrane subunit with a fusion peptide consisting of two or more heptad repeat domains. Upon interaction of the fusion protein with a cellular receptor, the buried fusion peptide is exposed and inserted into the membrane of the target cell. A series of conformational changes trigger virus-cell fusion activity (9) and lead to the unloading of the viral genome into cells. Additionally, many viral fusion proteins also induce cell-cell fusion, i.e., the formation of multinucleated syncytia, facilitating the rapid spread of virus infection.TThe spike (S) protein of coronaviruses is responsible for receptor binding and membrane fusion. It shares similarity with class I virus fusion proteins (2, 3). Typically, it is a type I integral membrane protein, which is N-glycosylated and trimerized in the endoplasmic reticulum. The N-terminal S1 protein contains the receptor-binding site (10,18,22,34). The C-terminal S2 protein is a fusion subunit and anchors on the viral envelope through a transmembrane domain. The S2 protein ectodomain contains two 4,3 hydrophobic heptad repeats (HR1 and HR2) and a putative, internal fusion peptide (3, 23). For the SARS-CoV S protein, the HR2 is located adjacent to the transmembrane domain, whereas the HR1 is 140 to 170 residues upstream of the HR2.Crystallographic, biophysical, and biochemical analysis of the fusion core of SARS-CoV S protein (2,12,19,27,30,35) and other class I fusion proteins (8, 25) supports a model of membrane fusion probably adopted by these enveloped viruses. After the attachment of the receptor-binding subunit to the receptor, the HR1 and HR2 domains in the membrane fusion subunit interact with each other and form a six-helix bundle, a complex consisting of a homotrimeric HR1 coiled coil surrounded by three HR2 helices. The spacer domain (or link, or interhelical domain) between HR1 and HR2 forms a loop and reverses the direction of the polypeptide chain so that the HR2 helices pack against the HR1 coiled coil in an antiparallel manner. This conformational change results in a close apposition of the fusion peptide, already exposed and inserted into th...

CTL clear virus-infected cells and tumorigenic cells by releasing potent cytotoxic enzymes stored in preformed lytic granules. The exocytosis process includes polarization of lytic granules toward the immunological synapse, tethering of lytic granules to the plasma membrane and finally fusion of lytic granules with the plasma membrane to release cytotoxic enzymes. Although much is known about the molecular machineries necessary for the earlier steps in lytic granule exocytosis, the molecular machinery governing the final step in the fusion process has not been identified. Here, we show using control and VAMP8 KO mice that VAMP8 is localized to the CTL lytic granules. While the immunological synapse and granule polarization appears normal in both VAMP8 KO and control CTL, CTL-mediated killing was reduced for the Vamp8 -/-CTL. Analysis of lytic enzyme secretion demonstrated that granzyme A and granzyme B secretion is significantly compromised in VAMP8 -/-CTL, while the levels of the lytic enzymes in the cells are unaffected. Our results clearly show that VAMP8 is one of the v-SNARE that regulate the lytic ability of CTL by influencing the ability of the lytic granules to fuse with the plasma membrane and release its contents.

The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1 isolate (A/ chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus-insect cell system. As expected, full-length HA5 elicits strong neutralizing antibodies, as evaluated in microneutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C-terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum against the N-terminal fragment is only four times lower than the antiserum against the full-length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities.

The large number of mutations identified across all cancers represents an untapped reservoir of targets that can be useful for therapeutic targeting if highly selective, mutation-specific reagents are available. We report here our attempt to generate such reagents: monoclonal antibodies against the most common R175H, R248Q, and R273H hotspot mutants of the tumor suppressor p53. These antibodies recognize their intended specific alterations without any cross-reactivity against wild-type (WT) p53 or other p53 mutants, including at the same position (as exemplified by anti-R248Q antibody, which does not recognize the R248W mutation), evaluated by direct immunoblotting, immunoprecipitation, and immunofluorescence methods on transfected and endogenous proteins. Moreover, their clinical utility to diagnose the presence of specific p53 mutants in human tumor microarrays by immunohistochemistry is also shown. Together, the data demonstrate that antibodies against specific single-amino-acid alterations can be generated reproducibly and highlight their utility, which could potentially be extended to therapeutic settings.

The integral membrane glycoprotein BARP associates with voltage-gated calcium channel Cavβ subunits, disrupting their association with Cavα1 subunits and suppressing overall channel activity.

Recepteur d'origine nantais (RON) receptor tyrosine kinase (RTK) and its ligand, serum macrophage-stimulating protein (MSP), are well-established oncogenic drivers for tumorigenesis and metastasis. RON is often found to be alternatively spliced resulting in various isoforms that are constitutively active. RON is therefore an attractive target for cancer therapeutics, including small molecular inhibitors and monoclonal antibodies. While small molecule inhibitors of RON may inhibit other protein kinases including the highly similar MET kinase, monoclonal antibodies targeting RON are more specific, potentially inducing fewer side effects. Although anti-RON monoclonal antibody therapies have been developed and tested in clinical trials, they were met with limited success. Cancer cells have been associated with aberrant glycosylation mechanisms. Notably for RON, the loss of N-bisected glycosylation is a direct cause for tumorigenesis and poorer prognosis in cancer patients. Particularly in gastric cancer, aberrant RON glycosylation augments RON activation. Here, we present a novel panel of monoclonal antibodies which potentially widens the specific targeting of not only the glycosylated RON, but also unglycosylated and aberrantly glycosylated RON. Our antibodies can bind strongly to deglycosylated RON from tunicamycin treated cells, recognise RON in IHC/IF and possess superior therapeutic efficacy in RON expressing xenograft tumours. Our most potent antibody in xenograft assays, is directed to the RON alpha chain and targets a sulfhydryl bond constrained epitope that appears to be cryptic in the crystal structure. This establishes the paradigm that such constrained and cryptic epitopes represent good targets for therapeutic antibodies.

Helicobacter pylori causes cellular vacuolation in host cells, a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT), a constitutively expressed secretory enzyme of H. pylori, in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium, thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay, we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) or in the presence of a competitive GGT inhibitor, serine-borate complex. Furthermore, the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT), although rGGT itself did not induce vacuolation independently. Similarly, the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally, we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively, our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases.

Chemotoxicity due to unwanted p53 activation in the bone marrow remains an unmet clinical challenge. Doxorubicin, a firstline chemotherapy drug, often causes myelosuppression in patients, thus limiting its effectiveness. In this study, we discovered that C646, a reversible p300 inhibitor, downregulates p53 transcription and selectively protects noncancerous cells from p53-dependent apoptosis. C646 treatment blocked acetylation of specific lysine residues that regulate p53 activity. Exploitation of differential p53 genetic backgrounds between human hematopoietic and colorectal cancer cells improved the therapeutic index of doxorubicin with C646 cotreatment. C646 administration in mice afflicted with p53-mutant tumors protected them from doxorubicin-induced neutropenia and anemia while retaining antitumor efficacy. We deduce that temporary and reversible inhibition of p53 acetylation in cancer subjects, especially those with p53-mutant tumors, may protect them from severe chemotoxicity while allowing treatment regimens to effectively proceed.Cancer Res; 77(16); 4342-54. Ó2017 AACR.