VPS37 isoforms differentially modulate the ternary complex formation of ALIX, ALG-2, and ESCRT-I.

The endosomal sorting complex required for transport (ESCRT) system comprises a series of protein complexes that play essential roles in multivesicular body (MVB) sorting of ubiquitylated membrane proteins, enveloped RNA virus budding, and cytokinesis in mammalian cells. The complex, named ESCRT-I, consists of four subunits (TSG101, VPS28, VPS37, and MVB12). There are four VPS37 isoforms. We have reported that ALIX (an ALG-2-interacting protein and accessory protein in the ESCRT system) is physically linked with TSG101 by ALG-2 in a Ca²âº-dependent manner, but the role of ALG-2 as an adaptor protein for the ESCRT-I complex remains unknown. To characterize this adaptor function, initially we investigated the binding of ALG-2 to ESCRT-I complexes containing each one of the four different VPS37 isoforms by two approaches: first, Far-Western blot analysis with biotin-labeled ALG-2 probe, and second, a pulldown assay to determine the binding of the four recombinant ESCRT-I complexes to Strep-tagged ALG-2 after co-expression in HEK293T cells. VPS37B and VPS37C appeared to interact with ALG-2 in a stronger manner than TSG101 does. The results of in vitro binding assays using purified recombinant proteins indicated that ALG-2 functions as a Ca²âº-dependent adaptor protein that bridges ALIX and ESCRT-I to form a ternary complex, ESCRT-I/ALIX/ALG-2.

Mammalian ESCRT-III-related protein IST1 has a distinctive met-pro repeat sequence that is essential for interaction with ALG-2 in the presence of Ca2+.

ALG-2 is an EF-hand-type Ca(2+)-binding protein that interacts with a variety of intracellular proteins that possess Pro-rich regions (PRRs) in mammalian cells. IST1 is an endosomal sorting complex required for transport (ESCRT)-III-related charged multivesicular body protein (CHMP)-like protein, but unlike other ESCRT-III proteins, mammalian IST1 has a PRR and a distinctive sequence of Met-Pro repeats. We found that ALG-2 binds to IST1 by Far-Western analysis using biotinylated ALG-2 as probe, and that the Met-Pro repeat sequence is essential for interaction. The results of pulldown assays using Strep-tagged ALG-2 and lysates of cells expressing GFP-fused IST1 proteins indicated that the binding of ALG-2 to IST1 is Ca(2+)-dependent, and that it is enhanced by co-expression with CHMP1 proteins. Moreover, pulldown assays using various mutants of GST-ALG-2 revealed that the ability of IST1 to bind to mutants is different from those of known ALG-2-interacting proteins, suggesting that IST1 binds to ALG-2 by a different mode of recognition.

Subjective symptoms of female workers sorting goods in summer.

Subjective musculoskeletal symptoms are more frequently complained about in cold store work and in related conditions than those experienced in normal temperature work. This cross sectional study was undertaken to evaluate the effects of indoor cooling and cold storage goods on the prevalence of subjective symptoms in summer. Female workers sorting cold storage goods (exposed group) were the main subjects of this study (n=47). We also included a group of female workers engaged sorting dry goods as the unexposed to cold group (n=86). Work load for the two groups were estimated according to the recommended criteria. A self-administered questionnaire covering age, occupational career, smoking, alcohol drinking and physical exercise, present or past history of diseases, individual protective measures against cold or heat, and subjective symptoms (60 items) was used. The air temperature of the site at the start of working time for the workers sorting cold storage goods was 22.2℃ which was significantly lower than those measured for the other two work places (25.4℃ and 25.4℃) of the unexposed to cold group. Environmental temperatures at the foot level at the sorting workshop of cold storage goods and dry goods were ca.16℃ and 26℃ all day, respectively. The surface temperatures of cold storage goods were between -2.8℃ and 9.4℃. The surface temperature of dry goods was 26.5℃. Among the working characteristic items, only daily working hours in the exposed group (5.6 ± 0.6 h) were significantly longer than those in the unexposed to cold group (4.6 ± 0.9 h) (p<0.01). The prevalence rates of finger cold sensation, stiffness in the fingers, pain in the wrist, pain in the elbow, back dullness, back pain, low-back cold sensation, foot cold sensation and pain in the foot in the exposed group were significantly higher than those in the unexposed to cold group (p<0.05 or p<0.01). Pain in the fingers, numbness in the fingers, pain in the foot and foot numbness due to the cold in the exposed group were significantly higher than those in the unexposed to cold group (p<0.05 or p<0.01). These results suggest that indoor cooling and/or job activities related to cold storage goods could, to some extent, affect peripheral circulatory disturbances; and it could be regarded as a factor related to musculoskeletal symptoms among the exposed workers.

Penta-EF-hand protein ALG-2 functions as a Ca2+-dependent adaptor that bridges Alix and TSG101.

Alix and TSG101, known to physically interact with each other, have Pro-rich regions that are bound by ALG-2 Ca2+-dependently. We investigated the role of ALG-2 in the Alix-TSG101 association by pulldown assays using Strep-tagged Alix and its various mutants. The ALG-2-binding site was required for the Ca2+-dependent pulldown of TSG101 using HEK293T cells, whereas the PSAP sequence, a binding motif for the UEV domain of TSG101, was dispensable. Alix-TSG101 association was not observed using ALG-2-knockdown cells but became detectable by addition of the purified recombinant ALG-2 protein in the assay mixtures. Exogenous expression of mGFP-fused ALG-2 also restored the pulldown capability of Strep-Alix, but an alternatively spliced shorter ALG-2 isoform and a dimerization-defective mutant were incompetent. Based on the X-ray crystal structure model showing the presence of one ligand-binding site in each molecule of an ALG-2 dimer, we propose that Ca2+-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein.

The mechanism of Ca2+-dependent recognition of Alix by ALG-2: insights from X-ray crystal structures.

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] was originally identified as a protein that interacts with ALG-2, a member of the penta-EF-hand Ca(2+)-binding protein family. ALG-2 binds to its C-terminal proline-rich region that contains four tandem repeats of PXY (where X represents an uncharged amino acid). Recent X-ray crystal structural analyses of the Ca(2+)-free and Ca(2+)-bound forms of ALG-2, as well as the complex with an Alix oligopeptide, have revealed a mechanism of Ca(2+)-dependent binding of ALG-2 to its target protein. Binding of Ca(2+) to EF3 (third EF-hand) enables the side chain of Arg(125), present in the loop connecting EF3 and EF4 (fourth EF-hand), to move sufficiently to make a primary hydrophobic pocket accessible to the critical PPYP (Pro-Pro-Tyr-Pro) motif in Alix, which partially overlaps with the GPP (Gly-Pro-Pro) motif for binding to Cep55 (centrosome protein of 55 kDa). The fact that ALG-2 forms a homodimer and each monomer has one peptide-binding site indicates the possibility that ALG-2 bridges two interacting proteins, including Alix and Tsg101 (tumour susceptibility gene 101), and functions as a Ca(2+)-dependent adaptor protein.

Structural basis for Ca2+ -dependent formation of ALG-2/Alix peptide complex: Ca2+/EF3-driven arginine switch mechanism.

ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.

[An analysis of the educational effects of group counseling with visual aids: efforts to prevent diabetes in a business office setting].

We made this report with a view to clarifying the effects of group counseling with visual aids for railway workers enjoying improved health conducted as a part of prevention activities for lifestyle-related diseases such as diabetes. We employed the use of visual aids including slides, samples of blood and the measurement of vascular age on diabetes, and carried out group counseling to improve the interest, knowledge and realization of diabetes and lifestyle. This group counseling was carried out both with and without visual aids. A comparative study of the immediate effects mediated by the use or absence of visual aids was conducted. The workers who accepted our study and cooperated with us totaled 1054 (the average age was 43 +/- 11.2). We divided them at random into the object group and comparative control group and two months later we were able to analyze 190 people among them who could be traced and followed up. As a result of using visual aids, the workers showed much more interest and realization of diabetes and their lifestyles (p < 0.05), and a lower learning decline than the group without visual aids in retention of this knowledge. On the other hand, we have not yet seen improvement in the physical data through group counseling. It is possible that the medical examination data taken beforehand for the object group and the comparative control group were both within the normal range, or the follow-up period was short at only two months after the group counseling was conducted. We conclude that the use of visual aids could lead to a change in consciousness among workers having some confidence in their health as the result of group counseling.