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.Procollagen folding in the endoplasmic reticulum (ER) 1 is a unique pathway involving unique post-translational modifications and the assistance of molecular chaperones. Nascent procollagen ␣-chains entering the lumen of the ER are modified by specific enzymes including prolyl 4-hydroxylase. After completion of translocation, three C-terminal propeptides selectively associate, and the trimer is stabilized by disulfide bridges. The long triple helix (300 nm for type I collagen) comprised of tandem Xaa-Yaa-Gly repeats is believed to propagate from the C-terminal nucleus, like fastening a zipper (1). HSP47 is only one heat-shock protein found in the ER, and it specifically interacts with various types of collagen (2-4). This protein has been recognized as a collagen-specific molecular chaperone that facilitates normal procollagen biosynthesis (5). HSP47 is essential to normal embryogenesis and normal collagen biosynthesis. Disruption of the hsp47 gene in both alleles causes an embryonic lethal phenotype in mice with severe impairment of collagen-based tissue structures (6). To unveil the mechanism of HSP47 function, the elucidation of the molecular basis of the chaperone-substrate interaction is required.The recent investigations on substrate recognition by HSP47 provided information about primary and tertiary structures of procollagen required for interaction with HSP47. We (7) previously found that HSP47 binds to classical collagen model peptides with the sequence of (Pro-Pro-Gly) n and that the binding was negatively affected by replacements of Pro residues at Yaa positions with 4-hydroxyproline (Hyp) residues. Triple-helical conformation of the substrate was also shown to be important in recognition by HSP47 (8, 9). However, we have not yet determined th...

Overproduction of the Her2 oncoprotein has been found in Ϸ30% of breast tumors, and patients who have Her2 excesses typically have more aggressive disease. Here we show that the expression of the Her2 gene can be decreased by inhibiting the interaction of the two cancer-linked proteins, DRIP130͞CRSP130͞Sur-2 (a Raslinked subunit of human mediator complexes) and ESX (an epithelial-restricted transcription factor). Disruption of the interaction by a short cell-permeable peptide reduced the expression of the Her2 gene and specifically impaired the growth and viability of Her2-overexpressing breast cancer cells. The association of ESX with DRIP130 is mediated by a small hydrophobic face of an 8-aa helix in ESX, suggesting a therapeutic approach to incapacitating the Her2 gene by small organic molecules. O verexpression of the oncogene Her2 (neu͞ErbB2) has been found in Ϸ30% of breast tumors (1, 2). It is unclear why overexpression of this transmembrane receptor occurs in breast cancers, but patients who have Her2 excesses typically have more aggressive disease with enhanced metastasis and increased resistance to chemotherapy (1-6). Monoclonal antibodies against the Her2 protein have been successful in treating these Her2-positive patients (7-10). A humanized antibody called Herceptin has demonstrated tumor-inhibitory and chemosensitizing effects in clinical studies and is the only drug that the Food and Drug Administration has approved for treatment of Her2-overexpressing breast tumors (9, 10). The clinical success of the Her2-antibody therapy was an excellent example of the translation of basic cancer biology into clinical cancer treatment. However, the antibody therapy alone may not be ideal for therapeutic intervention of Her2-overexpressing breast cancers. In theory, down-regulation of Her2 may be accomplished efficiently by inhibiting the expression of the Her2 gene rather than targeting elevated levels of the Her2 proteins that are already overexpressed. By analogy with treatments for AIDS, cocktails of drugs, each with different mechanisms of action, might also be more effective at achieving complete remission of breast tumors. Thus, discovering a means to provide external control over Her2 expression, particularly through small organic molecules, remains appealing.One of the critical transcription factors that activate the Her2 gene in breast cancers is ESX (ESE-1͞ELF3͞ERT͞Jen), an Ets factor that is expressed specifically in epithelial cells including mammary glands (11-13). ESX binds and strongly activates the Her2 promoter (11), and the ESX-binding site in the Her2 promoter is absolutely required for the high-level expression of Her2 in breast cancer cells (14). The expression of ESX is boosted in Her2-overexpressing breast cancers (11); a simple correlation seems to exist between the expression levels of ESX and Her2 in breast cancer cells. Here we isolate DRIP130͞ CRSP130͞Sur-2, a Ras-linked metazoan-specific subunit of human mediator complexes, as a nuclear cofactor that binds specifically to the trans...

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.

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-ProGly 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.Collagen is the most abundant protein in vertebrate bodies and is folded as procollagen in the endoplasmic reticulum (ER). 1 The folding of procollagen in cells is unique, as is the final structure itself. In the folding of type I collagen, for instance, three ␣ chains (two ␣1(I) and one ␣2(I)) first associate at C-terminal propeptides, and then approximately 340 repeats of the Xaa-Yaa-Gly triplet form the 300-nm length triple helix (1). In the triple helix, three left-handed polyproline type II (PP-II) helices are supercoiled (2). The helix-forming process is believed to be coupled with prolyl 4-hydroxylation at Yaa positions (3). HSP47 transiently interacts with procollagen molecules in the ER in the process of folding and/or modification (4, 5). HSP47 is not secreted with procollagen from the cells, because it possesses the ER retention signal, Arg-Asp-Glu-Leu, at the carboxyl end (5, 6). The expression of HSP47 is well correlated with that of various types of collagen in cultured cells, in various tissues during development, and in pathophysiological conditions (7-12). Thus HSP47 is assumed to be a collagen-specific molecular chaperone (13). However, the binding sites or motif in the procollagen molecule have not been well elucidated. The molecular basis of HSP47 function, including the regulation of the substrate association and dissociation, also remains an enigma.Here, we studied substrate recognition of HSP47 using the recombinant chaperone protein and synthetic collagen model peptides and found that a very simple collagen model peptide mimics the native substrate of HSP47. The difference in substrate recognition between HSP47 and prolyl 4-hydroxylase (P4-H) was discussed in both primary and secondary s...

Activation domains are functional modules that enable DNA-binding proteins to stimulate transcription. Characterization of these essential modules in transcription factors has been hampered by their low sequence homology. Here we delineate the peptide sequences that are required for transactivation and interaction with hTAF II 31, a classical target of the acidic class of activation domains. Our analyses indicate that hTAF II 31 recognizes a diverse set of sequences for transactivation. This information enabled the identification of hTAF II 31-binding sequences that are critical for the activity of the activation domains of five human transcription factors: NFAT1, ALL1, NF-IL6, ESX, and HSF-1. The interaction surfaces are localized in short peptide segments of activation domains. The brevity and heterogeneity of the motifs may explain the low sequence homology among acidic activation domains.Transcription factors typically have distinct domains for binding specific DNA sequences and for activating transcription through protein-protein interactions (1-3). Although a large number of activation domains are known, these functional modules share little sequence homology and have only loosely been classified by the preponderance of amino acid residues such as acidic residues, glutamine, and proline (1, 4). This low homology of activation domains has made it difficult to characterize these essential modules in transcription factors. Identification of functional sequence motifs that are hidden in activation domains would dissect the functions of activation domains and help to understand their composite regulations.Multiple target proteins for each class of activation domains have been proposed, including the basal transcriptional factors, mediators, and chromatin-remodeling factors. One such direct target for acidic activators is hTAF II 31 (a human TFIID TATA box-binding protein-associated factor) (5-9). Functional inactivation of its yeast homolog, yTAF II 17, results in the loss of transcription for approximately 67% of the actively expressed yeast genes (10 -13). Moreover, hTAF II 31 has been found in a human histone-acetylase complex in addition to TFIID (14, 15). These previous results collectively suggest a general role of hTAF II 31 and its homologs in the regulation of eukaryotic gene transcription both at the level of chromatin modification and RNA polymerase recruitment (16).It has been reported that hTAF II 31 makes direct contacts with the activation domains of VP16, p53, and NF-B p65 and that the strength of the interactions correlates with the ability to activate transcription (5-9, 17, 18). NMR and biochemical studies have shown that the activation domains of VP16 and p53 undergo an induced transition from random coil to ␣-helix upon interaction with hTAF II 31, with key hydrophobic residues along one face of the nascent helix (17, 18). The pattern of such hydrophobic residues, FXX⌽⌽ (where X represents any residue and ⌽ represents any hydrophobic residue) is conserved among the activation domains of VP1...

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Development of synthetic molecules that provide external control over the transcription of a given gene represents a challenge in medicinal and bioorganic chemistry. Here we report design and analysis of wrenchnolol, a wrench-shaped synthetic molecule that impairs the transcription of the Her2 oncogene by disrupting association of transcription factor ESX with its coactivator Sur-2. The "jaw" part of the compound mimics the alpha-helical interface of the activation domain of ESX, and the "handle" region accepts chemical modifications for a range of analysis. A water-soluble handle permitted NMR study in aqueous solution; a biotinylated handle verified the selectivity of the interaction, and a fluorescent handle confirmed the cell permeability of the compound. The case study of wrenchnolol foreshadows the promise and the challenge of targeting protein-protein interactions in the nucleus and may lead to the development of unique synthetic modulators of gene transcription.

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. Heat-shock protein 47 (HSP47)3 is an essential molecular chaperone for normal procollagen biosynthesis in mammals. Disruption of both alleles of the hsp47 gene in mice causes abnormal procollagen folding in the endoplasmic reticulum (ER), resulting in an embryonic lethal phenotype (1, 2). Although the molecular function of HSP47 remains unclear, it has been suggested that HSP47 could stabilize correctly folded triple-helical intermediates of procollagen that are otherwise unstable at body temperature (3, 4). HSP47 has also been suggested to inhibit lateral associations that form insoluble aggregates in the ER (5). Efforts toward elucidating the collagen-recognition mechanism of HSP47 have been made by our group and others, and to date, Arg residues at Yaa positions in the collagenous Gly-Xaa-Yaa repeats have been shown to be necessary for interaction with HSP47 (6, 7). The importance of the triple-helical structure for HSP47 binding has also been strongly suggested, and it was finally proven in the preceding paper by using conformationally constrained collagenous peptides (8). The minimal number of Arg residues per triple helix required for the specific interaction was further determined to be one. In addition, the Yaa residue occupying position Ϫ3 (denoted as Yaa Ϫ3 , and similar nomenclatures are used later), based on the essential Arg residue at a Yaa position (whose position is defined as position 0), has also been suggested to be important for the interaction, since replacement of the 4-hydoxy-Lproline (Hyp) residue at position Ϫ3 in (Pro-Hyp-Gly) 2 -Pro-Hyp Ϫ3 -Gly-Pro-Arg 0 -Gly-(Pro-Hyp-Gly) 4 -Pro-Hyp-amide with p-benzoyl-Lphenylalanine (Bpa) abolished the binding, even though the peptide had a triple-helical structure and contained an Arg residue at a Yaa position.In the present study, we focused on the structure of Yaa Ϫ3 , which was expected to be another structural determinant for HSP47 binding, together with the side chain structure of Arg 0 . A structure-activity relationship study was performed using coll...