Month: January 2019

EPO normally functions by activating the EPOR, a single-pass transmembrane cytokine receptor required for erythroid differentiation and red blood cell production. TC2-3, the traptamer that activates the hEPOR, supports limited erythroid differentiation in primary human hematopoietic progenitor cells in vitro in the absence of EPO. TC2-3 consists of a 19-amino acid random transmembrane segment flanked by 25 amino acids from E5, forms a homodimer, and displays no sequence or biochemical Talatisamine similarity to EPO. TC2-3 does not activate the PDGFbR or the murine EPOR, and the transmembrane domain of the hEPOR is required for TC2-3 action. We reasoned that isolation of a more active mutant of TC2-3 would facilitate the analysis of small transmembrane activators of the hEPOR and allow the identification of specific features of these proteins that are important for their activity. Here, we used a directed evolution approach to isolate a mutant of TC2-3 with increased activity. A library encoding thousands of TC2-3 mutants was subjected to selection under stringent conditions to isolate a traptamer with enhanced activity, EBC516, which contains a single amino acid substitution that increases dimerization. When expressed in hHPCs, EBC5-16 induces cellsurface expression of the erythroid-specific, differentiation marker, glycophorin A,Crassicauline-A to the same extent as in cells stimulated with EPO. These results suggest that dimerization of EBC5-16 plays a key role in its ability to induce erythroid differentiation. As a first step in understanding the molecular basis for the activity of EBC516, we conducted genetic analysis to identify and characterize its homodimer interface. These experiments provide evidence that increased dimerization of EBC5-16 is responsible for its enhanced activity. This work represents a novel approach to isolate and characterize potent, specific, biologically active proteins not found in nature, which have the potential to modulate the activity of a diverse array of cellular transmembrane proteins of research and clinical importance. In addition, study of these proteins will provide insight into protein-protein interactions occurring in membranes.

Moreover, the mini-V3 in a non-scaffolded context failed to be recognized by broadly neutralizing antibodies, indicating that the structural integrity provided by the scaffold was critical to the glycan V3-supersite transplants. Only a portion of the glycan V3 supersite was transplanted, and the degree of antigenic mimicry against antibodies from a specific donor correlated with Cycloastragenol the portion of the transplanted supersite recognized by antibodies from that specific donor. A design that incorporates the transplantation of a larger portion of the supersite may have greater antigenic breadth. In this regard, it should be noted that the glycan V3-supersite is one of the most promiscuously recognized of the HIV-1 Env supersites of vulnerability. Antibodies that recognize this supersite range from PGT122, which binds a glycopeptide V1-V3 spanning epitope,Calycosin-7-glucoside to PGT135, which binds a glycopeptide V3-V4 spanning epitope, and to 2G12, which binds a glycan-only epitope. It remains to be seen whether transplants comprising greater portions of the glycan V3 supersite would make more effective probes or immunogens; we note in this context that it remains to be shown whether the glycan V3-supersite transplants described here can elicit effective HIV-1-neutralizing antibodies – either alone, as cocktails, or as components in prime/boost regimens. Nevertheless, the structural and antigenic analyses described here, along with the successful oligomerization of the glycan V3supersite transplants in the ferritin nanoparticle context, do demonstrate that supersite transplants with antigenic mimicry of the template supersite can be achieved through transplantation of only a portion of the recognized supersite. Overall, the design framework and described glycan V3-supersite transplants provide both conceptual context and initial immunogens for a supersitefocused vaccine effort. The disease is known to cause progressive fatal disease in poor people particularly belonging to North-eastern part of Indian subcontinent. An average of more than 90% of VL cases in India is reported from Bihar alone. Recent epidemics of VL in Sudan and India have resulted in over 100,000 deaths.