Sequence-specific recognition of collagen triple helices by the collagen-specific molecular chaperone HSP47

HSP47 is a molecular chaperone that plays an unknown role during the assembly and transport of procollagen. Our previous studies showed that, unlike most chaperones, HSP47 interacts with a correctly folded substrate. We suggested that HSP47 either stabilizes the correctly folded collagen helix from heat denaturation or prevents lateral aggregation prior to its transport from the endoplasmic reticulum. In this study we have addressed the role of triple helix stability in the binding of HSP47 to procollagen by expressing procollagen molecules with differing thermal stabilities and analyzing their ability to interact with HSP47 within the endoplasmic reticulum. Our results show that HSP47 interacts with thermostable procollagen molecules, suggesting that helix stabilization is not the primary function of HSP47 and that the interaction of HSP47 with procollagen depends upon the presence of a minimum of one Gly-X-Arg triplet within the triple helical domain. Interestingly, procollagen chains containing high proportions of stabilizing triplets formed triple helices and interacted with HSP47 even in the absence of proline hydroxylation, demonstrating that recognition does not depend upon this modification. Our results support the view that HSP47 functions early in the secretory pathway by preventing the lateral aggregation of procollagen chains.     

Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation

Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly)(n), possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly)(n) has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-Pro-Gly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly)(n), does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.     

A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone

Hsp47 is regarded as a collagen-specific chaperone with several suggested roles in collagen biosynthesis under normal and disease conditions. We describe here a procedure for the expression and purification of Hsp47 in Escherichia coli using the IMPACT expression system (New England Biolabs) where the guest gene is fused to the adduct, intein, with a chitin-binding domain. Use of this system resulted in relatively high levels of soluble Hsp47 compared to other available protocols, especially when the bacterial cells were induced at 14 degrees C instead of 37 degrees C. The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from intein using beta-mercaptoethanol. Minor degradation products were subsequently removed using a hydroxylapatite column to yield milligram amounts of pure and active protein suitable for structural studies. Gel electrophoretic analysis of the purified protein indicated the presence of a small proportion of trimeric species when non-reducing conditions were used. The ability to form a trimer may be important for its role as a chaperone. The IMPACT system allows for radiolabelling of purified Hsp47 with (35)S for use in binding experiments. Illustrative data on collagen binding by (35)S-Hsp47 are shown.     

Separate cis-acting DNA elements control cell type- and tissue-specific expression of collagen binding molecular chaperone HSP47

HSP47 is a collagen-binding heat shock protein and is assumed to act as a molecular chaperone in the biosynthesis and secretion of procollagen. As the synthesis of HSP47 is closely correlated with that of collagen in various cell lines and tissues, we performed a promoter/reporter assay using HSP47-producing and nonproducing cells. 280 base pairs (bp(s)) of upstream promoter were shown to be necessary for the basal expression but not to be enough for the cell type-specific expression. When the first and the second introns were introduced downstream of this 280-bp region, marked up-regulation of the reporter activity was observed in HSP47-producing cells but not in nonproducing cells. This was confirmed in transgenic mice by staining the lacZ gene product under the control of the 280-bp upstream promoter and the introns. Staining was observed in skin, chondrocytes, precursor of bone, and other HSP47/collagen-producing tissues. A putative Sp1-binding site at -210 bp in the promoter, to which Sp3 and an unidentified protein bind, was shown to be responsible for this up-regulation when combined with the introns. However no difference in the binding to this probe was observed between HSP47-producing and nonproducing cells. The responsible region for cell type-specific up-regulation was found to be located in a 500-bp segment in the first intron. On electrophoresis mobility shift assay using this 500-bp probe, specific DNA-protein complexes were only observed in HSP47-producing cell extracts. These results suggest that two separate elements are necessary for the cell type-specific expression of the hsp47 gene; one is a putative Sp1-binding site at -210 bp necessary for basal expression, and the other is a 500-bp region within the first intron, required for cell type-specific expression.     

Antisense oligonucleotide against collagen-specific molecular chaperone 47-kDa heat shock protein suppresses scar formation in rat wounds

The 47-kDa heat shock protein (HSP47) is a molecular chaperone specifically targeting the processing and quality control of collagen molecules. This study was performed to investigate whether antisense therapy preventing HSP47 expression might affect the scar formation occurring during wound healing of skin. In wound healing of neonatal rat skin, the number of HSP47-positive cells and the amount of HSP47 protein consistently increased up to 7 days after surgical wounding. The increase in HSP47-positive cell number and protein content was efficiently suppressed by daily injections of HSP47-antisense deoxynucleotide (30 nmol) for 7 days. This treatment also suppressed the accumulation of collagen type I in the wound. Moreover, the disorder of collagenous fibers was relieved in the healed portion of the wounds subjected to the antisense treatment. Taken together, the authors propose that HSP47 is an important determinant in scar formation and that the antisense treatment against HSP47 gene may have a therapeutic potential to suppress the scar formation of skin.     

Roles of collagen fibers and its specific molecular chaperone: analysis using HSP47-knockout mice.

Collagen is the most abundant protein of mammals and produces highly organized ultrastructures in the extracellular matrix. There are at least 27 types of collagen in mammalian tissues. While fibrillar collagen (eg. types I, II, III, V and XI) assembles into large fibril structures in the extracellular matrix, type IV collagen produces meshwork-like structures in the basement membranes. As collagen has a distinct triple helix structure composed of Gly-X-Y repeats whose Y position is often hydroxyproline, its folding and maturation process differs considerably from globular proteins. Type I collagen is an assembly of two alpha-1 chains and one alpha-2 chain, and each of the alpha chains contain the N-terminal propeptide, C-terminal propeptide and central triple helical region. The 47-kDa heat shock protein (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes the triple helical region of collagen and is required for productive folding and maturation of collagen molecules. Only in the presence of HSP47, collagen type I molecules can be assembled into the correctly folded triple helices in the ER of mouse embryos without producing misfolded or non-functionally aggregated molecules. HSP47-knockout embryos die just after 10.5 day due to the absence of functional collagen. Recent our data demonstrated that the non-fibrillar network-forming collagen type IV also requires HSP47 for productive folding and maturation. Here, we discuss the role of HSP47 in the folding and maturation of collagen type IV as well as type I.     

Advanced glycation end products increase collagen-specific chaperone protein in mouse diabetic nephropathy

Advanced glycation end products (AGEs) appear to contribute to the diabetic complications. This study reports the inhibitory effect of OPB-9195 (OPB), an inhibitor of AGEs formation, and the role of a collagen-specific molecular chaperone, a 47-kDa heat shock protein (HSP47) in diabetic nephropathy. Transgenic mice carrying nitric-oxide synthase cDNA fused with insulin promoter (iNOSTg) leads to diabetes mellitus. The iNOSTg mice at 6 months of age represented diffuse glomerulosclerosis, and the expression of HSP47 was markedly increased in the mesangial area in parallel with increased expression of types I and IV collagens. OPB treatment ameliorated glomerulosclerosis in the iNOSTg mice associated with the decreased expression of HSP47 and types I and IV collagens. The expression of transforming growth factor-beta (TGF-beta) was increased in glomeruli of iNOSTg mice and decreased after treatment with OPB. To confirm these mechanisms, cultured mesangial cells were stimulated with AGEs. AGEs significantly increased the expression of HSP47, type IV collagen, and TGF-beta mRNA. Neutralizing antibody for TGF-beta inhibited the overexpression of both HSP47 and type IV collagen in vitro. In conclusion, AGEs increase the expression of HSP47 in association with collagens, both in vivo and in vitro. The processes may be mediated by TGF-beta.     

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).     

Hsp47: a molecular chaperone that interacts with and stabilizes correctly-folded procollagen

Hsp47 is a heat-shock protein that interacts transiently with procollagen during its folding, assembly and transport from the endoplasmic reticulum (ER) of mammalian cells. It has been suggested to carry out a diverse range of functions, such as acting as a molecular chaperone facilitating the folding and assembly of procollagen molecules, retaining unfolded molecules within the ER, and assisting the transport of correctly folded molecules from the ER to the Golgi apparatus. Here we define the substrate recognition of Hsp47, demonstrating that it interacts preferentially with triple-helical procollagen molecules. The association of Hsp47 with procollagen coincides with the formation of a collagen triple helix. This demonstrates that Hsp47's role in procollagen folding and assembly is distinct from that of prolyl 4-hydroxylase. These results indicate that Hsp47 acts as a novel molecular chaperone, potentially stabilizing the correctly folded collagen helix from heat denaturation before its transport from the ER.     

Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47.

HSP47 is an essential procollagen-specific molecular chaperone that resides in the endoplasmic reticulum of procollagen-producing cells. Recent advances have revealed that HSP47 recognizes the (Pro-Pro-Gly)(n) sequence but not (Pro-Hyp-Gly)(n) and that HSP47 recognizes the triple-helical conformation. In this study, to better understand the substrate recognition by HSP47, we synthesized various collagen model peptides and examined their interaction with HSP47 in vitro. We found that the Pro-Arg-Gly triplet forms an HSP47-binding site. The HSP47 binding was observed only when Arg residues were incorporated in the Yaa positions of the Xaa-Yaa-Gly triplets. Amino acids in the Xaa position did not largely affect the interaction. The recognition of the Arg residue by HSP47 was specific to its side-chain structure because replacement of the Arg residue by other basic amino acids decreased the affinity to HSP47. The significance of Arg residues in HSP47 binding was further confirmed by using residue-specific chemical modification of types I and III collagen. Our results demonstrate that Xaa-Arg-Gly sequences in the triple-helical procollagen molecule are dominant binding sites for HSP47 and enable us to predict HSP47-binding sites in homotrimeric procollagen molecules.     

The content of heat shock protein 47 (HSP47), a collagen-specific stress protein, changes with gravitational conditions in skeletal muscle.

It is well known that unloading of skeletal muscle with spaceflight or tail suspension leads rat soleus muscle atrophy. Previously, we reported that one of small heat shock protein (sHSP), alpha B-crystallin shows an early dramatic decrease in atrophied rat soleus muscle (Atomi et al, 1991). In this report, we focused to study the gravitational responses of another HSP, which may be reactive to the gravity. HSP47, a collagen-specific stress protein, has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in slow skeletal muscle decreases at 5 days after tail suspension (TS) and increased at 5 days recovery after 10 days of TS as compared with the control level. Hypothetically, HSP47 in slow soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. The content of HSP47 in soleus muscle was strongly affected by gravity conditions.     

The change of HSP47, collagen specific molecular chaperone, expression in rat skeletal muscle may regulate collagen production with gravitational conditions.

It is well known that unloading of skeletal muscle with spaceflight leads skeletal muscle atrophy. However, it remains unclear how the extracellular matrix within the muscle and the connective tissues such as tendon and ligament respond to reduced mechanical load including microgravity, although they have been thought to play important roles in both the transmission of force and the signal transduction between cells and tissues. Type-I collagen and type-IV collagen, both of the major components of extracellular matrix and connective tissues. We focused on change of these collagen synthesis with mechanical load. To obtain an insight into the effects of gravitational changing on the protein metabolism of collagen in skeletal muscle during mechanical unloading, reloading after unloading, we investigated changes in the amount of Heat shock protein 47 (HSP47), has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in rat soleus muscle decreases at 5 days after hindlimb suspension (HS). On the other hand, HSP47 in rat soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. RT-PCR analysis showed HSP47 mRNA decreased with HS earlier, as compared with collagen type-I and type-IV mRNA. From these results, the amount of HSP47 changing by gravitational condition may effect on signal transfers in the primary stage of adaptation and the change of HSP47 expression in skeletal muscle may regulate collagen production with gravitational conditions.     

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 Arg(0)) 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 Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) 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.     

73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin

Although gentamicin (GM) has been used widely as an antibiotic, the specific binding protein of the drug has not yet been understood sufficiently. Here we show that GM specifically associates with the 73-kDa molecular chaperone HSP73 and reduces its chaperone activity in vitro. In the present study, we investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the eluant. Upon immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known to be a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury.     

分子伴侣的功能和应用

本文综述了分子伴侣的分类、功能、作用机理、研究现状及应用前景。分子伴侣是在生物大分子的折叠、组装、转运及降解等过程中起协助作用,参与协助抗原的呈递和遗传物质的复制、转录及构象的确立,但自身并不发生任何变化的一大类广泛存在于生物体内的蛋白质分子。随着对分子伴侣的进一步研究和相关知识的不断深入,分子伴侣在生物产品开发、物种改良、抗衰老,疾病预防、诊断和治疗以及环境监测方面具有广阔的前景。     

Interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 molecular chaperone

At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met(1)-Arg(400)), middle (Glu(401)-Lys(615)), and C-terminal (Asp(621)-Asp(732)) regions. In the present study, we investigated potential subregion structures of these three regions and their roles. Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met(1) to Lys(281) (or Lys(283)) and Glu(282) (or Tyr(284)) to Arg(400). The former is known to carry the ATP-binding domain. The fragments carrying the N-terminal two-thirds (Glu(401)-Lys(546)) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast.     

Sumo-1 function is dispensable in normal mouse development

To elucidate SUMO-1 functions in vivo, we targeted by homologous recombination the last three exons of the murine Sumo-1 gene. Sumo-1 mRNA abundance was reduced to one-half in heterozygotes and was undetectable in Sumo-1(-/-) mice, and SUMO-1-conjugated RanGAP1 was detectable in wild-type mouse embryo fibroblasts (MEFs) but not in Sumo-1(-/-) MEFs, indicating that gene targeting yielded Sumo-1-null mice. Sumo-1 mRNA is expressed in all tissues of wild-type mice, and its abundance is highest in the testis, brain, lungs, and spleen. Sumo-2 and Sumo-3 mRNAs are also expressed in all tissues, but their abundance was not upregulated in Sumo-1-null mice. The development and function of testis are normal in the absence of Sumo-1, and Sumo-1(-)(/)(-) mice of both sexes are viable and fertile. In contrast to a previous report (F. S. Alkuraya et al., Science 313:1751, 2006), we did not observe embryonic or early postnatal demise of Sumo-1-targeted mice; genotypes of embryos and 21-day-old mice were of predicted Mendelian ratios, and there was no defect in lip and palate development in Sumo-1(+/-) or Sumo-1(-/-) embryos. The ability of Sumo-1(-/-) MEFs to differentiate into adipocyte was not different from that of wild-type MEFs. Collectively, our results support the notion that most, if not all, SUMO-1 functions are compensated for in vivo by SUMO-2 and SUMO-3.