Phycobilisome: architecture of a light-harvesting supercomplex

The phycobilisome (PBS) is an extra-membrane supramolecular complex composed of many chromophore (bilin)-binding proteins (phycobiliproteins) and linker proteins, which generally are colorless. PBS collects light energy of a wide range of wavelengths, funnels it to the central core, and then transfers it to photosystems. Although phycobiliproteins are evolutionarily related to each other, the binding of different bilin pigments ensures the ability to collect energy over a wide range of wavelengths. Spatial arrangement and functional tuning of the different phycobiliproteins, which are mediated primarily by linker proteins, yield PBS that is efficient and versatile light-harvesting systems. In this review, we discuss the functional and spatial tuning of phycobiliproteins with a focus on linker proteins.     

Development of an optimized 5-stage protocol for the in vitro preparation of insulin-secreting cells from mouse ES cells

In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage. GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 10⁶ cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.     

Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor

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Sequential Ubiquitination of Ribosomal Protein uS3 Triggers the Degradation of Non-functional 18S rRNA

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RBMX is a novel hepatic transcriptional regulator of SREBP-1c gene response to high-fructose diet

In rodents a high-fructose diet induces metabolic derangements similar to those in metabolic syndrome. Previously we suggested that in mouse liver an unidentified nuclear protein binding to the sterol regulatory element (SRE)-binding protein-1c (SREBP-1c) promoter region plays a key role for the response to high-fructose diet. Here, using MALDI-TOF MASS technique, we identified an X-chromosome-linked RNA binding motif protein (RBMX) as a new candidate molecule. In electrophoretic mobility shift assay, anti-RBMX antibody displaced the bands induced by fructose-feeding. Overexpression or suppression of RBMX on rat hepatoma cells regulated the SREBP-1c promoter activity. RBMX may control SREBP-1c expression in mouse liver in response to high-fructose diet.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

A real-time method of imaging glucose uptake in single, living mammalian cells

This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.     

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.