Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain

Abstract

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that “C-Pro-only” constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies. Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that “C-Pro-only” constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies. Proper folding and assembly of collagen is critical for maintaining the structural integrity of extracellular matrices (1Shoulders M.D. Raines R.T. 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D.J. of fibrillar collagen and Biol. PubMed Scopus Google Scholar). The are for and to critical for formation D.R. M.D. molecular collagen C-propeptide PubMed Scopus Google Scholar). These biochemical and structural insights provide a to how C-Pro mutations collagenopathies. missense mutations in or C-Proα2(I) are known to cause S. D.J. A. procollagen C-propeptide of and of Google Scholar). of these mutations disrupt the key cause defects for it that variants delay secretion or cause collagen-I and in and and in Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. D.J. A. procollagen C-propeptide of and of Google Scholar, of one in the propeptide of the of type procollagen assembly and secretion of but stable procollagen trimers and results in osteogenesis Med. PubMed Google Scholar). to procollagen secretion and cause formation of Colα1(I) C1163R in Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of variants in cells that the cause collagen-I A. C. E. D.R. J.P. Leikin S. E. C-propeptide mutations cause of procollagen and C-terminal procollagen PubMed Scopus Google Scholar). is that remains C-Pro associated with biochemical characterization of collagen quality and M.D. and the collagen-I proteostasis Chem. Biol. PubMed Scopus Google for characterization of proteostasis in the of full-length collagen-I constructs C-Pro variants to biochemical Moreover, it is unclear whether or not quality control directly misfolded C-Pro domains or the presence of a triple-helix collagen stalled the assembly or folding these is how the proteostasis network engages full-length collagen-I M.D. and the collagen-I proteostasis Chem. Biol. PubMed Scopus Google Scholar, molecular in the for procollagen PubMed Scopus Google but which of that network are in C-Pro domain proteostasis remains We to a understanding of how collagen-I folding and assembly is by C-Pro mutations and of how the to C-Pro We C-Proα2(I) variants a of C-Proα2(I) is in assembled collagen-I in then one or C-Proα2(I) of that a and a We a of full-length and Colα2(I) constructs a of C-Pro mutations mutations that or S. D.J. A. procollagen C-propeptide of and of Google and C1163R Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and mutations that or for protein S. D.J. A. procollagen C-propeptide of and of Google and Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google We by that these constructs recapitulate the behavior of the full-length collagen-I that the presence versus the absence of unassembled or triple-helical domains the of these which is by the C-Pro domain itself. the most severe assembly associated with C1163R C-Proα2(I), which the retained in cells. We a of biochemical and approaches to the and quality control behavior of versus C1163R we MS-based to illuminate how the proteostasis network differentially engages versus collagen-I C-Pro domains. results provide insights into the molecular of collagen-I proteostasis and inform to collagen proteostasis in We by a of constructs missense variants of Colα2(I) within the C-Pro domain that are known to cause We and C1163R C-Proα2(I) and an or one of the in the protein Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. D.J. 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We constructs in which full-length Colα2(I) variants the Colα1(I) constructs the of collagen-I of the constructs. We that not disrupt collagen-I assembly M.D. and the collagen-I proteostasis Chem. Biol. PubMed Scopus Google Scholar). We or full-length Colα2(I) with the full-length Colα1(I) in cells. We cells as an not which our and it for our the C-Pro domain itself. a cells with Colα1(I) and of Colα1(I) and Colα2(I) in We the and by of We full-length Colα1(I) and Colα2(I), the full-length Colα2(I) variants in of the the other full-length Colα2(I) variants full-length C1163R Colα2(I) retained We that the full-length Colα1(I) that with full-length C1163R Colα2(I) into the C1163R Colα2(I) with the that prevent collagen-I assembly with Colα1(I) Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these are with to collagen-I by the We whether these by which are to biochemical we whether the presence or absence of a triple-helical domain critical for the we constructs collagen-I triple-helical domains that C-Proα2(I) and variants We a A. M.D. J. the in of and in a 2009; PubMed Scopus Google between the and the domain to and of C-Proα2(I) versus which molecular Upon of these an we to the behavior of the full-length constructs. C1163R C-Proα2(I) the other variants key of constructs is that to biochemical full-length constructs. We of feature to the of these C-Proα2(I) mutations for collagen-I folding and We of to C-Pro in from cells with and or C-Proα2(I) We that C-Proα2(I) and two of the variants and to with for and for the other not with of forming with C-Proα2(I) and that and to protein is with the that the amino collagen-I proteostasis by with S. D.J. 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PubMed Scopus Google Scholar). the proteostasis network that the C-Pro the molecular of C-Pro and how the and to misfolded C-Pro domains K. M.D. proteostasis and the protein Google Scholar). Here, we the and of collagen-I C-Pro domains in cells a in which C-Pro domains in the absence of associated triple-helical domains. a to elucidate how C-Pro domain mutations disrupt collagen-I assembly and We first that the behavior of versus constructs the of the full-length collagen-I constructs. of the and of C-Pro constructs with the full-length constructs secretion and retention is C-Pro domain mutations to to it unclear whether the quality control directly and engages the misfolded C-Pro domain itself or triple-helical domains assembly is stalled by the presence of a misfolded C-Pro A. A. K. of misfolded procollagen in the endoplasmic reticulum as a of Biol. 2009; PubMed Scopus Google Scholar, J. of unassembled type collagen Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for the mutations the presence of a triple-helical domain is not an of the behavior of the C1163R C-Proα2(I) is retained in the absence of the triple-helical misfolded Colα2(I) is the triple-helical domain is that cells to triple-helix domain We the of C-Proα2(I) mutations to in type mutations in the and C1163R amino The and C-Proα2(I) variants with and not collagen-I secretion. These results are with that these variants delay collagen-I in cells and in a Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. D.J. A. procollagen C-propeptide of and of Google Scholar). the other the and C1163R C-Proα2(I) variants from the of collagen C-Proα2(I) but not with formation of collagen-I is a process that assembly of in a by of assembled D.R. M.D. molecular collagen C-propeptide PubMed Scopus Google Scholar). of which is a of a the to prevent the assembly of we that in the formation of and of not prevent secretion of C-Proα2(I), with by and that the not the secretion of full-length Colα2(I) S. D.J. A. procollagen C-propeptide of and of Google Scholar). the amino type with a severe of S. D.J. A. procollagen C-propeptide of and of Google the severe into is are as severe or cellular and not with the presence of Colα2(I) in cells the formation of collagen-I not stable or into extracellular it The C1163R C-Proα2(I) amino from a protein-folding and assembly The of a critical in We that the protein as misfolded by the proteostasis and secretion the disease by the C1163R C-Proα2(I) is type Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the severe folding that or of the C1163R C-Proα2(I) quality control to extracellular matrix the for retained triple helical variants L. A. N.A. A. of and in the of osteogenesis Med. PubMed Scopus Google Scholar, A. A. K. of misfolded procollagen in the endoplasmic reticulum as a of Biol. 2009; PubMed Scopus Google the retained C1163R to in the and or or of the C1163R Colα2(I) with quality and in Colα2(I) with Colα1(I) to it is that Colα1(I) by C1163R the in Colα2(I) by the Leikin S. S. of the of type procollagen molecular assembly and in osteogenesis Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to the C1163R Colα2(I) is not as from a as the Colα2(I) from C1163R The proteostasis network prevent secretion of C1163R Colα2(I) and direct it to degradation. Colα2(I) into quality and is into the extracellular it structural by the retention and with the C1163R C-Proα2(I) we to to how the proteostasis network engages C-Pro domains and to how the and quality control C1163R C-Proα2(I) to it and direct it to of C1163R C-Proα2(I), with with the and as as and The are known to formation and with assembled The of C1163R C-Proα2(I) with these proteostasis network the of network The with the the of to these results the potential of the to provide insights into how cells collagen proteostasis The and are to with of the full-length of the associated with with and of and the of the not for understanding of collagen folding but for whether and how we cellular proteostasis to collagen folding defects M.D. proteostasis in the Chem. Biol. PubMed Scopus Google Scholar). of the cellular proteostasis network is an and for other protein M.D. to illuminate Rev. Biochem. PubMed Scopus Google Scholar). the proteostasis to and or prevent the of collagen-I strands in critical first is as that proteostasis defects in and how the proteostasis network to and with the of an or M.D. and the collagen-I proteostasis Chem. Biol. PubMed Scopus Google Scholar). and and C-Proα2(I), by the C-Pro domains from the K. E. A. K. S. Leikin S. C-propeptide mutations cause osteogenesis 2011; PubMed Scopus Google to the of procollagen that the and for of a and the or The then to a by into the The amino introduced by of versus C-Proα2(I) from variants introduced by the from and the in for the constructs are in the cells in of with and in a for the of full-length and C-Pro collagen-I and to with and for with and then in a and in to and in and and for to a and with and then with the in and with appropriate and or by an with the constructs in a cells with to with and with a of for The by with of for Cell by and then by for with Cell by for in the a of of to a of then to in and for and then the and the with the in of in for to the the from the for our A. A. M.D. the protein to the molecular of mature Biol. 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