{"id":203,"date":"2018-12-27T09:52:15","date_gmt":"2018-12-27T08:52:15","guid":{"rendered":"http:\/\/mapkinhibitorlibrary.com\/?p=203"},"modified":"2022-01-17T11:38:04","modified_gmt":"2022-01-17T02:38:04","slug":"lines-evidence-suggest-enhanced-activity-ebc5-16","status":"publish","type":"post","link":"http:\/\/mapkinhibitorlibrary.com\/index.php\/2018\/12\/27\/lines-evidence-suggest-enhanced-activity-ebc5-16\/","title":{"rendered":"Several lines of evidence suggest that the enhanced activity of EBC5-16"},"content":{"rendered":"<p>Like TC2-3, EBC5-16 is dimeric, can serve as a transmembrane domain, and functionally interacts with the transmembrane domain of the hEPOR. The high activity of EBC5-16 in inducing erythroid differentiation is particularly striking because it is so dissimilar to EPO,<a href=\"www.abmole.com\/products\/puerarin.html\">Puerarin<\/a> which is monomeric, soluble, and binds the extracellular domain of the EPOR. We used a similar directed evolution strategy to optimize traptamers that down-regulate CCR5. These results demonstrate the utility of random mutagenesis and selection to optimize artificial transmembrane domains that target single-pass and multi-pass transmembrane proteins. Several lines of evidence suggest that the enhanced activity of EBC5-16 is due to increased homodimerization caused by the substitution of a serine for an isoleucine. First, EBC5-16 exists in cells as a disulfide bond-linked homodimer. Second, mutation of the cysteines that mediate covalent dimerization abolishes activity. Third, the serine substitution increases the fraction of EBC5-16 in the dimeric form, as assessed by non-reducing gel electrophoresis and TOXCAT experiments. Although the TOXCAT result indicated the transmembrane domain of EBC5-16 is sufficient for dimerization in bacterial membranes,<a href=\"www.abmole.com\/products\/dipsacoside-b.html\">Dipsacoside B<\/a> the defect caused by the cysteine mutations implies that in mammalian cells the dimer is stabilized by disulfide bonds. Similarly, the transmembrane domain of BPV E5 lacking the C-terminal cysteines has intrinsic dimerization potential, but the presence of the cysteines or fusion to a heterologous dimerization domain is required for high-level activity in mammalian cells. Finally, we identified Gly11, Gly15, Ile18, Pro22, Ser25, and Phe29 as the residues constituting the homodimer interface of EBC5-16. Importantly, insertion of these interfacial residues into an inactive variant of EBC5-16 containing a monomeric poly-leucine transmembrane domain was sufficient to reconstitute a dimeric protein that activates the hEPOR. Although our results show unequivocally that dimerization of EBC5-16 is required for activity, it remains possible that alterations in amino acid side-chain orientation caused by the I25S substitution has a direct effect on the increased activity of EBC5-16 compared to TC2-3. <\/p>\n","protected":false},"excerpt":{"rendered":"<p>Like TC2-3, EBC5-16 is dimeric, can serve as a transmembrane domain, and functionally interacts with the transmembrane domain of the hEPOR. The high activity of EBC5-16 in inducing erythroid differentiation is particularly striking because it is so dissimilar to EPO,Puerarin which is monomeric, soluble, and binds the extracellular domain of the EPOR. We used a &hellip; <a href=\"http:\/\/mapkinhibitorlibrary.com\/index.php\/2018\/12\/27\/lines-evidence-suggest-enhanced-activity-ebc5-16\/\" class=\"more-link\">Continue reading <span class=\"screen-reader-text\">Several lines of evidence suggest that the enhanced activity of EBC5-16<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[1],"tags":[],"_links":{"self":[{"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/203"}],"collection":[{"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/comments?post=203"}],"version-history":[{"count":1,"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/203\/revisions"}],"predecessor-version":[{"id":204,"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/203\/revisions\/204"}],"wp:attachment":[{"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/media?parent=203"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/categories?post=203"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/mapkinhibitorlibrary.com\/index.php\/wp-json\/wp\/v2\/tags?post=203"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}