An especially mild, safe, efficient, and environmentally responsible reduction of aromatic and heteroaromatic nitro-group-containing educts is reported that utilizes very inexpensive carbonyl iron powder (CIP), a highly active commercial grade of iron powder. These reductions are conducted in the presence of nanomicelles composed of TPGS-750-M in water, a recyclable aqueous micellar reaction medium. This new technology also shows broad scope and scalability and presents opportunities for multistep one-pot sequences involving this reducing agent.
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.
Osteogenesis imperfecta (OI) is an inherited disease most commonly caused by autosomal dominant mutations in collagen type‐I. OI‐causing mutations are found in both the triple helical region and the C‐propeptide region of collagen‐I. Mutations in the triple helical region cause disruption to triple helical folding, stability, and structure. Mutations in the C‐propeptide region, the C‐terminal nucleation domain of collagen‐I, disrupt the initial assembly steps in collagen folding. Currently, the mechanism by which the cellular protein folding machinery recognizes and processes these types of misfolded variants of collagen‐I is not well‐understood. Here, we report how the endoplasmic reticulum (ER) differentially processes normal and misfolding, OI‐causing collagen‐I variants. We employed state‐of‐the‐art, quantitative mass spectrometry‐based proteomic methods to determine the full complement of proteostasis network components that differentially engage wild‐type (wt) and several OI‐causing mutants of collagen‐I. We identified >75 putative proteins that might play a role in collagen‐I proteostasis, in addition to the currently known collagen‐I interactors. Interestingly, we found that while there is no significant difference in the interactomes of wild‐type (wt) and triple‐helical mutant collagen‐I variants, mutant C‐propeptide collagen‐I is well‐recognized by a number of components of the ER proteostasis network. These results indicate that the cell can specifically recognize collagen C‐propeptide misfolding, but has limited capacity to address triple‐helix domain misfolding. Our findings contribute to an understanding of how the proteostasis network reacts to different types of collagen folding defects and maintains proper collagen folding and secretion.
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