RESUMEN
Collagen is the most abundant protein in mammals. A unique feature of collagen is its triple-helical structure formed by the Gly-Xaa-Yaa repeats. Three single chains of procollagen make a trimer, and the triple-helical structure is then folded in the endoplasmic reticulum (ER). This unique structure is essential for collagen's functions in vivo, including imparting bone strength, allowing signal transduction, and forming basement membranes. The triple-helical structure of procollagen is stabilized by posttranslational modifications and intermolecular interactions, but collagen is labile even at normal body temperature. Heat shock protein 47 (Hsp47) is a collagen-specific molecular chaperone residing in the ER that plays a pivotal role in collagen biosynthesis and quality control of procollagen in the ER. Mutations that affect the triple-helical structure or result in loss of Hsp47 activity cause the destabilization of procollagen, which is then degraded by autophagy. In this review, we present the current state of the field regarding quality control of procollagen.
Asunto(s)
Colágeno/química , Fibrosis/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Procolágeno/química , Procolágeno/metabolismo , Animales , Colágeno/metabolismo , Retículo Endoplásmico/metabolismo , Fibrosis/genética , Proteínas del Choque Térmico HSP47/química , Proteínas del Choque Térmico HSP47/genética , Humanos , Hidroxilación , Chaperonas Moleculares/metabolismo , Prolina/química , Prolina/metabolismo , Conformación Proteica , Pliegue de Proteína , Procesamiento Proteico-PostraduccionalRESUMEN
Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Proteínas del Choque Térmico HSP47/fisiología , Humanos , Ratones , Chaperonas Moleculares/metabolismo , Transducción de Señal , Estrés Fisiológico , Factores de Transcripción/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are one of the two types of tetrameric ion channels that release calcium ion (Ca2+) from the endoplasmic reticulum (ER) into the cytosol. Ca2+ released via IP3Rs is a fundamental second messenger for numerous cell functions. Disturbances in the intracellular redox environment resulting from various diseases and aging interfere with proper calcium signaling, however, the details are unclear. Here, we elucidated the regulatory mechanisms of IP3Rs by protein disulfide isomerase family proteins localized in the ER by focusing on four cysteine residues residing in the ER lumen of IP3Rs. First, we revealed that two of the cysteine residues are essential for functional tetramer formation of IP3Rs. Two other cysteine residues, on the contrary, were revealed to be involved in the regulation of IP3Rs activity; its oxidation by ERp46 and the reduction by ERdj5 caused the activation and the inactivation of IP3Rs activity, respectively. We previously reported that ERdj5 can activate the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b) using its reducing activity [Ushioda et al., Proc. Natl. Acad. Sci. U.S.A. 113, E6055-E6063 (2016)]. Thus, we here established that ERdj5 exerts the reciprocal regulatory function for IP3Rs and SERCA2b by sensing the ER luminal Ca2+ concentration, which contributes to the calcium homeostasis in the ER.
Asunto(s)
Calcio , Inositol , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Calcio/metabolismo , Inositol/metabolismo , Cisteína/metabolismo , Retículo Endoplásmico/metabolismo , Señalización del Calcio/fisiología , Oxidación-Reducción , Inositol 1,4,5-Trifosfato/metabolismoRESUMEN
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase family member that accelerates ERAD by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates. Biochemical analyses using purified proteins revealed that J-chain oligomers were reduced to monomers by ERdj5 in a stepwise manner via trimeric and dimeric intermediates, and BiP synergistically enhanced this action in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using high-speed atomic force microscopy, demonstrated the stochastic release of small J-chain oligomers through repeated actions of ERdj5 on peripheral and flexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected at the molecular level.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Degradación Asociada con el Retículo Endoplásmico , Chaperonas Moleculares , Chaperón BiP del Retículo Endoplásmico/química , Chaperón BiP del Retículo Endoplásmico/genética , Chaperón BiP del Retículo Endoplásmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Células HEK293 , Cadenas J de Inmunoglobulina/metabolismo , Dominios ProteicosRESUMEN
ER-associated degradation (ERAD) is a protein clearance mechanism by which misfolded, misassembled, or metabolically regulated proteins are specifically dislocated from the ER into the cytosol and degraded by the ubiquitin proteasome system. ERAD very likely evolved to maintain proteostasis and sterol homeostasis in the ER. However, the ironic truth is that membrane-penetrating transportation and protein degradation machineries in ERAD are preferably hijacked by exogenous pathogens such as viruses and toxins for their invasion and evasion from immunological surveillance. In this Review, we provide an overview of our current understanding of the pathogenic hijacking of the host cell ERAD, in which pathogens exploit the complex ERAD machinery in a variety of manners for their own use, suggesting flexibility and plasticity of the molecular machinery of ERAD.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Proteínas Virales/metabolismo , Virus/metabolismo , Animales , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Proteínas/química , Proteínas/metabolismo , Proteolisis , Virosis/patología , Virosis/virologíaRESUMEN
Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell-platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell-platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell-platelet interaction and cancer colonization in secondary organs.
Asunto(s)
Plaquetas/metabolismo , Neoplasias de la Mama/metabolismo , Colágeno/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Células Neoplásicas Circulantes/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Transición Epitelial-Mesenquimal , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Amplificación de Genes , Proteínas del Choque Térmico HSP47/genética , Humanos , Ratones SCID , Metástasis de la NeoplasiaRESUMEN
Although collagens are the most abundant proteins implicated in various disease pathways, essential mechanisms required for their proper folding and assembly are poorly understood. Heat-shock protein 47 (HSP47), an ER-resident chaperone, was mainly reported to fulfill key functions in folding and secretion of fibrillar collagens by stabilizing pro-collagen triple-helices. In this study, we demonstrate unique functions of HSP47 for different collagen subfamilies. Our results show that HSP47 binds to the N-terminal region of procollagen I and is essential for its secretion. However, HSP47 ablation does not majorly impact collagen VI secretion, but its lateral assembly. Moreover, specific ablation of Hsp47 in murine keratinocytes revealed a new role for the transmembrane collagen XVII triple-helix formation. Incompletely folded collagen XVII C-termini protruding from isolated HSP47 null keratinocyte membrane vesicles could be fully restored upon the application of recombinant HSP47. Thus, our study expands the current view regarding the client repertoire and function of HSP47, as well as emphasizes its importance for transmembrane collagen folding.
Asunto(s)
Proteínas del Choque Térmico HSP47/metabolismo , Queratinocitos/metabolismo , Procolágeno/metabolismo , Pliegue de Proteína , Animales , Proteínas del Choque Térmico HSP47/genética , Ratones , Procolágeno/genéticaRESUMEN
Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ubiquitinadas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteínas Portadoras/genética , Núcleo Celular/genética , Células HEK293 , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Proteínas de la Membrana/genética , Ratones , Células 3T3 NIH , Proteínas Nucleares/genética , Proteínas Transportadoras de Solutos , Proteínas Ubiquitinadas/genéticaRESUMEN
Heat shock protein 47 (Hsp47) is an endoplasmic reticulum (ER)-resident molecular chaperone essential for correct folding of procollagen in mammalian cells. In this Review, we discuss the role and function of Hsp47 in vertebrate cells and its role in connective tissue disorders. Hsp47 binds to collagenous (Gly-Xaa-Arg) repeats within triple-helical procollagen in the ER and can prevent its local unfolding or aggregate formation, resulting in accelerating triple-helix formation of procollagen. Hsp47 pH-dependently dissociates from procollagen in the cis-Golgi or ER-Golgi intermediate compartment and is then transported back to the ER. Although Hsp47 belongs to the serine protease inhibitor (serpin) superfamily, it does not possess serine protease inhibitory activity. Whereas general molecular chaperones such as Hsp70 and Hsp90 exhibit broad substrate specificity, Hsp47 has narrower specificity mainly for procollagens. However, other Hsp47-interacting proteins have been recently reported, suggesting a much broader role for Hsp47 in the cell that warrants further investigation. Other ER-resident stress proteins, such as binding immunoglobulin protein (BiP), are induced by ER stress, whereas Hsp47 is induced only by heat shock. Constitutive expression of Hsp47 is always correlated with expression of various collagen types, and disruption of the Hsp47 gene in mice causes embryonic lethality due to impaired basement membrane and collagen fibril formation. Increased Hsp47 expression is associated with collagen-related disorders such as fibrosis, characterized by abnormal collagen accumulation, highlighting Hsp47's potential as a clinically relevant therapeutic target.
Asunto(s)
Colágeno/biosíntesis , Pérdida del Embrión/metabolismo , Estrés del Retículo Endoplásmico , Proteínas del Choque Térmico HSP47/metabolismo , Animales , Colágeno/genética , Pérdida del Embrión/genética , Pérdida del Embrión/patología , Fibrosis , Proteínas del Choque Térmico HSP47/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Estructura Secundaria de Proteína , Secuencias Repetitivas de AminoácidoRESUMEN
Molecular chaperones perform pivotal roles in proteostasis by engaging in protein-protein interactions (PPIs). The collagen-specific molecular chaperone Hsp47 (heat shock protein 47) interacts with procollagen in the endoplasmic reticulum (ER) and plays crucial roles in collagen synthesis. PPIs between Hsp47 and collagen could offer a therapeutic target for fibrosis, which is characterized by abnormal collagen accumulation in the extracellular matrix of fibrotic organs. Herein, we established a bioluminescence resonance energy transfer (BRET) system for assessing Hsp47-collagen interaction dynamics within the ER. After optimization and validation of the method, we could demonstrate inhibition of the interaction between Hsp47 and collagen by a small molecule (Col003) in the ER. Using the BRET system, we also found that Hsp47 interacts not only with the Gly-Pro-Arg motif but also weakly with Gly-Pro-Hyp motifs of triple-helical collagen in cells. Moreover, we found that the serpin loop of Hsp47 (SerpinH1) contributes to its binding to collagen. We propose that the method developed here can provide valuable information on PPIs between Hsp47 and collagen and on the effects of PPI inhibitors important for the management of fibrotic disorders.
Asunto(s)
Colágeno/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Sitios de Unión , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Colágeno/química , Retículo Endoplásmico/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP47/antagonistas & inhibidores , Proteínas del Choque Térmico HSP47/química , Humanos , Unión ProteicaRESUMEN
The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9), decreased epithelial CD1d expression--as observed in inflammatory bowel disease--may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP), as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.
Asunto(s)
Antígenos CD1d/inmunología , Células Epiteliales/inmunología , Inmunidad Mucosa/inmunología , Interleucina-10/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Animales , Proteínas Portadoras/metabolismo , Colitis/inmunología , Colitis/patología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Femenino , Proteínas del Choque Térmico HSP110/genética , Proteínas del Choque Térmico HSP110/metabolismo , Humanos , Inflamación/inmunología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Interleucina-10/genética , Masculino , Ratones , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Oxazolona , Factor de Transcripción STAT3/metabolismoRESUMEN
This study demonstrates the structure-activity relationship of Col-003, a potent collagen-heat-shock protein 47 (Hsp47) interaction inhibitor. Col-003 analogues were successfully synthesized by Pd(0)-catalyzed cross-coupling reactions of 5-bromosalicylaldehyde derivatives with alkyl-metal species, and the inhibitory activities of the synthetic analogues were evaluated using surface plasmon resonance analysis (BIAcore). We succeeded in discovering two potent inhibitors that showed 85 and 81% inhibition at a concentration of 1.9 µM against the collagen-Hsp47 interaction. This indicates that elongation of an alkyl linker between two aromatic rings could considerably improve inhibitory activity due to the adjustment of a pendant phenyl moiety to an appropriate position, in addition to the hydrophobic interaction with an alkyl linker moiety.
Asunto(s)
Aldehídos/química , Colágeno/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Aldehídos/síntesis química , Aldehídos/farmacología , Animales , Catálisis , Colágeno/antagonistas & inhibidores , Proteínas del Choque Térmico HSP47/antagonistas & inhibidores , Paladio/química , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
Hsp47, a collagen-specific molecular chaperone that localizes in the endoplasmic reticulum (ER), is indispensable for molecular maturation of collagen. Hsp47, which is encoded by the SERPINH1 gene, belongs to the serpin family and has the serpin fold; however, it has no serine protease inhibitory activity. Hsp47 transiently binds to procollagen in the ER, dissociates in the cis-Golgi or ER-Golgi intermediate compartment (ERGIC) in a pH-dependent manner, and is then transported back to the ER via its RDEL retention sequence. Hsp47 recognizes collagenous (Gly-Xaa-Arg) repeats on triple-helical procollagen and can prevent local unfolding and/or aggregate formation of procollagen. Gene disruption of Hsp47 in mice causes embryonic lethality due to impairments in basement membrane and collagen fibril formation. In Hsp47-knockout cells, the type I collagen triple helix forms abnormally, resulting in thin and frequently branched fibrils. Secretion of type I collagens is slow and plausible in making aggregates of procollagens in the ER of hsp47-knocked out fibroblasts, which are ultimately degraded by autophagy. Mutations in Hsp47 are causally associated with osteogenesis imperfecta. Expression of Hsp47 is strongly correlated with expression of collagens in multiple types of cells and tissues. Therefore, Hsp47 represents a promising target for treatment of collagen-related disorders, including fibrosis of the liver, lung, and other organs.
Asunto(s)
Colágeno/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Animales , Enfermedad , Proteínas del Choque Térmico HSP47/química , Humanos , Modelos Biológicos , Fenotipo , Unión ProteicaRESUMEN
The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub-cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter- and intra-molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle-specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.
Asunto(s)
Envejecimiento/metabolismo , Caenorhabditis elegans/metabolismo , Retículo Endoplásmico/metabolismo , Deficiencias en la Proteostasis/metabolismo , Envejecimiento/genética , Animales , Caenorhabditis elegans/genética , Retículo Endoplásmico/genética , Humanos , Oxidación-Reducción , Deficiencias en la Proteostasis/genéticaRESUMEN
ER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters. Our systematic biochemical analyses indicated that two thioredoxin domains that constitute the C-terminal cluster form the highly reducing platform that interacts with EDEM1 and reduces EDEM1-recruited substrates, leading to their facilitated degradation. The pulse-chase experiment further provided direct evidence for the sequential movement of an ERAD substrate from calnexin to the downstream EDEM1-ERdj5 complex, and then to the retrotranslocation channel, probably through BiP. We present a detailed molecular view of how ERdj5 mediates ERAD in concert with EDEM1.
Asunto(s)
Retículo Endoplásmico/enzimología , Proteínas del Choque Térmico HSP40/química , Chaperonas Moleculares/química , Proteína Disulfuro Reductasa (Glutatión)/química , Animales , Células Cultivadas , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformación Proteica , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Pliegue de Proteína , Transducción de Señal , TransfecciónRESUMEN
Extracellular matrix (ECM) proteins are biosynthesized in the rough endoplasmic reticulum (rER) and transported via the Golgi apparatus to the extracellular space. The coat protein complex II (COPII) transport vesicles are approximately 60-90 nm in diameter. However, several ECM molecules are much larger, up to several hundreds of nanometers. Therefore, special COPII vesicles are required to coat and transport these molecules. Transmembrane Protein Transport and Golgi Organization 1 (TANGO1) facilitates loading of collagens into special vesicles. The Src homology 3 (SH3) domain of TANGO1 was proposed to recognize collagen molecules, but how the SH3 domain recognizes various types of collagen is not understood. Moreover, how are large noncollagenous ECM molecules transported from the rER to the Golgi? Here we identify heat shock protein (Hsp) 47 as a guide molecule directing collagens to special vesicles by interacting with the SH3 domain of TANGO1. We also consider whether the collagen secretory model applies to other large ECM molecules.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Translocador Nuclear del Receptor de Aril Hidrocarburo/química , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento , Colágeno/metabolismo , Retículo Endoplásmico/metabolismo , Matriz Extracelular , Fibrilina-1/metabolismo , Expresión Génica , Aparato de Golgi/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Humanos , Espacio Intracelular/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Recombinantes , Dominios Homologos src/genéticaRESUMEN
Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+]i) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+]ER), whereas a higher [Ca2+]ER induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+]ER-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Homeostasis , Chaperonas Moleculares/metabolismo , Oxidación-Reducción , Animales , Señalización del Calcio , Línea Celular , Activación Enzimática , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Humanos , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Unión Proteica , Multimerización de Proteína , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
Fibrosis can disrupt tissue structure and integrity and impair organ function. Fibrosis is characterized by abnormal collagen accumulation in the extracellular matrix. Pharmacological inhibition of collagen secretion therefore represents a promising strategy for the management of fibrotic disorders, such as liver and lung fibrosis. Hsp47 is an endoplasmic reticulum (ER)-resident collagen-specific molecular chaperone essential for correct folding of procollagen in the ER. Genetic deletion of Hsp47 or inhibition of its interaction with procollagen interferes with procollagen triple helix production, which vastly reduces procollagen secretion from fibroblasts. Thus, Hsp47 could be a potential and promising target for the management of fibrosis. In this study, we screened small-molecule compounds that inhibit the interaction of Hsp47 with collagen from chemical libraries using surface plasmon resonance (BIAcore), and we found a molecule AK778 and its cleavage product Col003 competitively inhibited the interaction and caused the inhibition of collagen secretion by destabilizing the collagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the collagen-binding site on Hsp47. We propose that these structural insights could provide a basis for designing more effective therapeutic drugs for managing fibrosis.
Asunto(s)
Colágeno/química , Fibrosis/tratamiento farmacológico , Proteínas del Choque Térmico HSP47/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Sitios de Unión , Unión Competitiva , Diseño de Fármacos , Fibrosis/prevención & control , Humanos , Procolágeno/antagonistas & inhibidores , Procolágeno/química , Procolágeno/metabolismo , Bibliotecas de Moléculas PequeñasRESUMEN
Heat shock protein 47â¯kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis.
Asunto(s)
Colágeno/química , Proteínas del Choque Térmico HSP47/química , Imagen Molecular/métodos , Mapeo de Interacción de Proteínas/métodos , Espectrometría de Fluorescencia/métodos , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad por SustratoRESUMEN
Thiol-based redox control is among the most important mechanisms for maintaining cellular redox homeostasis, with essential participation of cysteine thiols of oxidoreductases. To explore cellular redox regulatory networks, direct interactions among active cysteine thiols of oxidoreductases and their targets must be clarified. We applied a recently described thiol-ene crosslinking-based strategy, named divinyl sulfone (DVSF) method, enabling identification of new potential redox relay partners of the cytosolic oxidoreductases thioredoxin (TXN) and thioredoxin domain containing 17 (TXNDC17). Applying multiple methods, including classical substrate-trapping techniques, will increase understanding of redox regulatory mechanisms in cells.