2011 Vol. 2, No. 12

News and views
Lupus at the molecular level
Mayami Sengupta, Laurence Morel
2011, 2(12): 941-943. doi: 10.1007/s13238-011-1123-1
In vitro reconstitution of germ cell development
Yun Li
2011, 2(12): 944-945. doi: 10.1007/s13238-011-1122-2
The role of Pbx1 in T cells
Mayami Sengupta, Laurence Morel
2011, 2(12): 946-949. doi: 10.1007/s13238-011-1136-9
Find and replace: editing human genome in pluripotent stem cells
Huize Pan, Weiqi Zhang, Weizhou Zhang, Guang-Hui Liu
2011, 2(12): 950-956. doi: 10.1007/s13238-011-1132-0
Genetic manipulation of human pluripotent stem cells (hPSCs) provides a powerful tool for modeling diseases and developing future medicine. Recently a number of independent genome-editing techniques were developed, including plasmid, bacterial artificial chromosome, adeno-associated virus vector, zinc finger nuclease, transcription activator-like effecter nuclease, and helper-dependent adenoviral vector. Gene editing has been successfully employed in different aspects of stem cell research such as gene correction, mutation knock-in, and establishment of reporter cell lines (Raya et al., 2009; Howden et al., 2011; Li et al., 2011; Liu et al., 2011b; Papapetrou et al., 2011; Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a). These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.
Protein-protein complexation in bioluminescence
Maxim S. Titushin, Yingang Feng, John Lee, Eugene S. Vysotski, Zhi-Jie Liu
2011, 2(12): 957-972. doi: 10.1007/s13238-011-1118-y
In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca2+-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.
Gene therapy: light is finally in the tunnel
Huibi Cao, Robert S. Molday, Jim Hu
2011, 2(12): 973-989. doi: 10.1007/s13238-011-1126-y
After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber's congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adenoassociated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.
ERp44 C160S/C212S mutants regulate IP3R1 channel activity
Congyan Pan, Ji Zheng, Yanyun Wu, Yingxiao Chen, Likun Wang, Zhansong Zhou, Wenxuan Yin, Guangju Ji
2011, 2(12): 990-996. doi: 10.1007/s13238-011-1116-0
Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (ⅡCR) via IP3R1, but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an ⅡCR model, Ca2+ image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP3Rs (ⅡCR) and the ⅡCR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on ⅡCR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP3R1 (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP3R1 but exhibit a weak inhibition of IP3R1 channel activity in Hela cells.
Research articles
Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines
Chunling Xuan, Yi Shi, Jianxun Qi, Wei Zhang, Haixia Xiao, George F. Gao
2011, 2(12): 997-1005. doi: 10.1007/s13238-011-1134-y
There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs. The main component of the current split influenza A virus vaccine is viral hemagglutinin (HA) which induces a strong antibody-mediated immune response. To develop a modern vaccine against influenza A viruses, the current research has been focused on the universal vaccines targeting viral M2, NP and HA proteins. Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface, and each monomer consists of a globular head (HA1) and a "rod-like" stalk region (HA2), the latter being more conserved among different HA subtypes and being the primary target for universal vaccines. In this study, we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A (H1N1) virus HA as a model, tested its immunogenicity in mice, solved its crystal structure and further examined its immunological characteristics. The results show that the HA globular head can be easily prepared by in vitro refolding in an E. coli expression system, which maintains its intact structure and allows for the stimulation of a strong immune response. Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.
Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure
Cheng Zhu, Changsheng Zhang, Huanhuan Liang, Luhua Lai
2011, 2(12): 1006-1013. doi: 10.1007/s13238-011-1121-3
Functional proteins designed de novo have potential application in chemical engineering, agriculture and healthcare. Metal binding sites are commonly used to incorporate functions. Based on a de novo designed protein DS119 with a βαβ structure, we have computationally engineered zinc binding sites into it using a home-made searching program. Seven out of the eight designed sequences tested were shown to bind Zn2+ with micromolar affinity, and one of them bound Zn2+ with 1:1 stoichiometry. This is the first time that metalloproteins with an α, β mixed structure have been designed from scratch.