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1.
Nazzareno Dimasi Ryan Fleming Kris F. Sachsenmeier Binyam Bezabeh Carl Hay Jincheng Wu 《MABS-AUSTIN》2017,9(3):438-454
We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies. 相似文献
2.
Nazzareno D'Avanzo Sun-Joo Lee Wayland W. L. Cheng Colin G. Nichols 《The Journal of biological chemistry》2013,288(23):16726-16737
Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P2) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96–99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P2 binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P2 involves orientational specificity and that other PIPs inhibit this activation through direct competition. 相似文献
3.
These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland. 相似文献
4.
A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX. 相似文献
5.
Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism. 相似文献
6.
Antonio Zanotti-Gerosa Euro Solari Luca Giannini Carlo Floriani Nazzareno Re Angiola Chiesi-Villa Corrado Rizzoli 《Inorganica chimica acta》1998,270(1-2):298-311
Lithiation of [p-But-calix[4]-(OMe)2(OH)2] (1), followed by reaction with TiCl3(thf)3 or TiCl4(thf)2, led to the corresponding titanium-calix[4]arene complexes [p-But-calix[4]-(OMe)2(O)2]TiCl] (2) and [p-But-calix[4]-(OMe)2(O)2]TiCl2] (3), respectively. Reaction of 1 with TiCl4(thf)2 results in demethylation of the calix[4]arene and the obtention of [p-But-calix[4]-(OMe)2(O)3]TiCl] (4), whose hydrolysis led to [p-But-calix[4]-(OMe)(OH)3] (6). The preparation of 6 can be carried out as a one-pot synthesis. Both 2 and 4 undergo alkylation reactions using conventional procedures, thus forming surprisingly stable organometallic species, namely [p-But-calix[4]-(OMe)2(O)2Ti(R)] (R = Me (7); CH2Ph (8), p-MeC6H4 (9) and [p-But-calix[4]-(OMe)(O)3Ti(R)] (R = Me (10); CH2Ph (11); p-MeC6H4 (12)). Complexes 7 and 9 undergo a thermal oxidative conversion into 10 and 12, occurring with the demethylation of one of the methoxy groups. A solid state structural property of 9 and 12 has been revealed by X-ray analysis showing a self-assembly of the monomeric units into a columnar polymer, where the p-tolyl substituent at the metal functions as a guest group for an adjacent titanium-calixarene. Reductive alkylation of 3 with Mg(CH2Ph)2 gave 8 instead of forming the corresponding dialkyl derivative. Two synthetic routes have been devised for the synthesis of the Ti(III)-Ti(III) dimer [p-But-calix[4]-(OMe)(O)3Ti]2] (13): the reduction of 4 and the reaction of TiCl3(thf)3 with the lithiated form of 6. A very strong antiferromagnetic coupling is responsible for the peculiar magnetic behavior of 13. The proposed structures have been supported by the X-ray analyses of 4, 9, 12 and 13. 相似文献
7.
Márquez JA Galfré E Dupeux F Flot D Moran O Dimasi N 《Journal of molecular biology》2007,367(2):310-318
The immune receptors expressed on myeloid cells (IREM) are type I transmembrane proteins encoded on human chromosome 17 (17q25.1), whose function is believed to be important in controlling inflammation. To date, three IREM receptors have been identified. IREM-1 functions as an inhibitory receptor, whereas IREM-2 and IREM-3 serve an activating function. Here, we report the crystal structure of IREM-1 extracellular domain at 2.6 A resolution. The overall fold of IREM-1 resembles that of a V-type immunoglobulin domain, and reveals overall close homology with immunoglobulin domains from other immunoreceptors such as CLM-1, TREM-1, TLT-1 and NKp44. Comparing the surface electrostatic potential and hydrophobicity of IREM-1 with its murine homologous CLM-1, we observed unique structural properties for the complementary determining region of IREM-1, which suggests that they may be involved in recognition of the IREM-1 ligand. Particularly interesting is the structural conformation and physical properties of the antibody's equivalent CDR3 loop, which we show to be a structurally variable region of the molecule and therefore could be the main structural determinant for ligand discrimination and binding. In addition, the analysis of the IREM-1 structure revealed the presence of four structurally different cavities. Three of these cavities form a continuous hydrophobic groove on the IREM-1 surface, which point to a region of the molecule capable of accommodating potential ligands. 相似文献
8.
Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called 'natural' killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb. 相似文献
9.
Ballatori N 《Experimental biology and medicine (Maywood, N.J.)》2005,230(10):689-698
Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis. 相似文献
10.
Piccoli C Ria R Scrima R Cela O D'Aprile A Boffoli D Falzetti F Tabilio A Capitanio N 《The Journal of biological chemistry》2005,280(28):26467-26476