Tissue-specific extracellular matrices (ECMs) are crucial for normal development and tissue function, and mutations in ECM genes result in a wide range of serious inherited connective tissue disorders. Mutations cause ECM dysfunction by combinations of two mechanisms. First, secretion of the mutated ECM components can be reduced by mutations affecting synthesis or by structural mutations causing cellular retention and/or degradation. Second, secretion of mutant protein can disturb crucial ECM interactions, structure and stability. Moreover, recent experiments suggest that endoplasmic reticulum (ER) stress, caused by mutant misfolded ECM proteins, contributes to the molecular pathology. Targeting ER stress might offer a new therapeutic strategy.
Type I collagen is a major component of the extracellular matrix, and mutations in the collagen gene cause several matrix-associated diseases. These mutant procollagens are misfolded and often aggregated in the endoplasmic reticulum (ER). Although the misfolded procollagens are potentially toxic to the cell, little is known about how they are eliminated from the ER. Here, we show that procollagen that can initially trimerize but then aggregates in the ER are eliminated by an autophagy-lysosome pathway, but not by the ER-associated degradation (ERAD) pathway. Inhibition of autophagy by specific inhibitors or RNAi-mediated knockdown of an autophagy-related gene significantly stimulated accumulation of aggregated procollagen trimers in the ER, and activation of autophagy with rapamycin resulted in reduced amount of aggregates. In contrast, a mutant procollagen which has a compromised ability to form trimers was degraded by ERAD. Moreover, we found that autophagy plays an essential role in protecting cells against the toxicity of the ERAD-inefficient procollagen aggregates. The autophagic elimination of aggregated procollagen occurs independently of the ERAD system. These results indicate that autophagy is a final cell protection strategy deployed against ER-accumulated cytotoxic aggregates that are not able to be removed by ERAD.
The binding of native biglycan and decorin to pepsinextracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore TM 2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K D ) of ϳ30 nM. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the ␣2(VI) chain probably plays a role in the interaction.Collagens are a large family of extracellular structural proteins made up of three ␣ chains that are intracellularly associated and folded into specific structures including characteristic triple helical domains (1). The major class, recognized as the fibril-forming collagens, contains molecules with one large uninterrupted triple helical domain (for review, see Refs. 1 and 2). Other members of the collagen family have one or more non-triple helical domains, which may constitute the major part of the protein. Most of these collagens do not form prominent lateral aggregates in a manner similar to that of the fibril-forming collagens. Instead, they form complex aggregates together with other matrix macromolecules. Collagen VI is one example, forming multimolecular filamentous beaded structures after secretion from the cell (for review, see Ref. 3). This collagen is composed of three different peptide chains (␣1(VI), ␣2(VI), and ␣3(VI)), which form the basic unit consisting of a relatively short triple helical domain flanked by two large multidomain globular regions (4). These are composed primarily of repeating units of von Willebrand type A domains (5). Collagen VI assembles intracellularly into antiparallel, overlapping dimers that then align and form tetramers (6). These structures are stabilized by disulfide bonds. Secreted tetramers assemble extracellularly in a characteristic end-to-end fashion into thin (3-10 nm) beaded filaments with a periodicity of about 100 nm (7-9). Further supramolecular assembly includes lateral associations of the beaded filaments into microfibrils (8, 9).Collagen VI is ubiquitous. It can be found intermingled with fibril-forming collagens and is often enriched in the pericellular matrix (for review, see Refs. 3 and 10). Decreased amounts of secreted collagen VI resulting from mutations in COL6A1 have been shown in Bethlem myopathy (11, 12), a dominantly inherited disorder characterized by progressive muscle weakness and wasting....
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