Shinichi Asada

Nucleobindin 1 Controls the Unfolded Protein Response by Inhibiting ATF6 Activation

In response to endoplasmic reticulum (ER) stress, activating transcription factor 6 (ATF6), an ER membrane-anchored transcription factor, is transported to the Golgi apparatus and cleaved by site-1 protease (S1P) to activate the unfolded protein response (UPR). Here, we identified nucleobindin 1 (NUCB1) as a novel repressor of the S1P-mediated ATF6 activation. NUCB1 is an ER stress-inducible gene with the promoter region having functional cis-elements for transcriptional activation by ATF6. Overexpression of NUCB1 inhibits S1P-mediated ATF6 cleavage without affecting ER-to-Golgi transport of ATF6, whereas knock-down of NUCB1 by siRNA accelerates ATF6 cleavage during ER stress. NUCB1 protein localizes in the Golgi apparatus, and disruption of the Golgi localization results in loss of the ATF6-inhibitiory activity. Consistent with these observations, NUCB1 can suppress physical interaction of S1P-ATF6 during ER stress. Together, our results demonstrate that NUCB1 is the first-identified, Golgi-localized negative feedback regulator in the ATF6-mediated branch of the UPR.

Artificial collagen gels via self‐assembly of de novo designed peptides

Development of artificial collagens to replace the animal‐derived collagens presents a challenge in the formation of safer and functional biomaterials. We report here the development of collagen‐like gels by means of the self‐assembly of chemically synthesized peptides. The peptides are disulfide‐linked trimers of collagenous Gly‐X‐Y triplet repeats with self‐complementary shapes. Upon cooling the peptide solutions, hydrogels of peptide supramolecules are formed by spontaneous intermolecular triple helix formation. The thermal gel–sol transition appeared to be reversible, and the transition temperatures were found to be tunable by the design of the peptides. Our systems for the formation of artificial collagen‐like gels will offer possibilities for novel types of biomaterials.

Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation.

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed “flying capstan model” (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).

Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins

The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa-3) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg0) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa-3 and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa-3 determined the binding affinity for HSP47. Maximal binding was observed when Yaa-3 was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr-3 and one Arg0. The results revealed that HSP47 recognizes the Yaa-3 and Arg0 residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.

A collagen-mimetic triple helical supramolecule that evokes integrin-dependent cell responses.

Collagen is an abundantly distributed extracellular matrix protein in mammalian bodies that maintains structural integrity of the organs and tissues. Besides its function as a structural protein, collagen has various biological functions which regulate cell adhesion, migration and differentiation. In order to develop totally synthetic collagen-surrogates, we recently reported a basic concept for preparing collagen-like triple helical supramolecules based on the self-assembly of staggered trimeric peptides with self-complementary shapes. In this paper, we add one of the specific cellular functions of the native collagen to the collagen-mimetic supramolecule. We synthesized a self-assembling peptide unit containing the integrin-binding sequence Gly-Phe-Hyp-Gly-Glu-Arg. The supramolecule carrying the sequence exhibited significant binding activity to human dermal fibroblasts. The supramolecular structure was found to be essential for function in in vitro cell culture. Cell adhesion was shown to be comparable to that of native collagen, and was further demonstrated to be mediated solely by integrin alpha 2 beta 1. Well-grown focal contacts and stress fibers were observed in cells spread on the supramolecular collagen-mimetic. The results demonstrate the potential of peptide-based artificial collagen as a biomaterial for regulating specific cellular function and fate.

A structure-activity relationship study elucidating the mechanism of sequence-specific collagen recognition by the chaperone HSP47

Heat-shock protein 47 (HSP47) is a chaperone that facilitates the proper folding of procollagen. Our previous studies showed that the high-affinity HSP47-binding motif in the collagen triple helix is Xaa-(Thr/Pro)-Gly-Xaa-Arg-Gly. In this study, we further investigated structural requirements for the HSP47-binding motif, using synthetic triple-helical collagen-model peptides with systematic amino acid substitutions at either the Thr/Pro (=Yaa(-3)) or the Arg (=Yaa(0)) position. Results obtained from in vitro binding assays indicated that HSP47 detects the side-chain structure of Arg at the Yaa(0)-position, while the Yaa(-3) amino acid serves as the secondary recognition site that affects affinity to HSP47.

Development of a high-throughput screening system for the compounds that inhibit collagen-protein interactions

Collagen-binding proteins (CBPs) play important roles in various physiological events. Some CBPs are regarded as targets for drug development; for example, platelet glycoprotein VI (GPVI) and heat shock protein 47 (HSP47) are promising targets for the development of novel antiplatelet and antifibrotic drugs, respectively. However, no systematic screening method to search compounds that inhibit collagen-CBP interactions have been developed, and only a few CBP inhibitors have been reported to date. In this study, a facile turbidimetric multiwell plate assay was developed to evaluate inhibitors of CBPs. The assay is based on the finding that CBPs retard spontaneous collagen fibril formation in vitro and that fibril formation is restored in the presence of compounds that interfere with the collagen-CBP interactions. Using the same platform, the assay was performed in various combinations of fibril-forming collagen types and CBPs. This homogeneous assay is simple, convenient, and suitable as an automated high-throughput screening system.

Characterization of Novel Insulin Fibrils That Show Strong Cytotoxicity Under Physiological pH

Amyloid fibrils are β-sheet-rich protein aggregates that are associated with more than 20 diseases. Insulin is known to form amyloid fibrils under a variety of conditions in vitro. Insulin fibrillations have been generally performed under acidic conditions, which are conducive to the formation of fibrils. As insulin is found almost exclusively as a monomer in acidic solutions, insulin fibrillation under acidic conditions is proposed to occur via its monomer. However, insulin fibrils, which cause injection-localized amyloidosis, form under neutral pH conditions in vivo, because both subcutaneous tissue and almost all insulin formulations maintain a neutral pH. In this study, we induced fibrillation under conditions more closely resembling physiological conditions than those used in previous studies with the aim of better understanding the nature of injection-localized amyloidosis in vivo. The results of transmission electron microscopy, structural analyses, and MTT assay show that the fibrils formed under conditions more closely resembling physiological conditions have different properties from the fibrils described to date. The results of this study indicate that fibrils formed under conditions more closely resembling physiological conditions have different properties from insulin fibrils induced under the conditions reported in previous studies.

Activation of the Unfolded Protein Response in Sporadic Inclusion Body Myositis But Not in Hereditary GNE Inclusion Body Myopathy

Abstract Muscle fibers in patients with sporadic inclusion-body myositis (s-IBM),the most common age-associated myopathy, are characterized by autophagic vacuoles and accumulation of ubiquitinated and congophilic multiprotein aggregates that contain amyloid-&bgr; and phosphorylated tau. Muscle fibers of autosomal-recessive hereditary inclusion-body myopathy caused by the GNE mutation (GNE-h-IBM) display similar pathologic features, except with less pronounced congophilia. Accumulation of unfolded/misfolded proteins inside the endoplasmic reticulum (ER) lumen leads to ER stress, which elicits the unfolded protein response (UPR) as a protective mechanism. Here we demonstrate for the first time that UPR is activated in s-IBM muscle biopsies, since there was 1) increased activating transcription factor 4 (ATF4) protein and increased mRNA of its target C/EBP homologous protein; 2) cleavage of the ATF6 and increased mRNA of its target glucose-regulated protein 78; and 3) an increase of the spliced form of X-box binding protein 1 and increased mRNA of ER degradation–enhancing &agr;-mannosidase–like protein, target of heterodimer of cleaved ATF6 and spliced X-box binding protein 1. In contrast, we did not find similar evidence of the UPR induction in GNE-h-IBM patient muscle, suggesting that different intracellular mechanisms might lead to similar pathologic phenotypes. Interestingly, cultured GNE-h-IBM muscle fibers had a robust UPR response to experimental ER stress stimuli, suggesting that the GNE mutation per se is not responsible for the lack of UPR in GNE-h-IBM biopsied muscle.