Collagen's enigmatic, highly conserved N-glycan has an essential proteostatic function.

Intracellular procollagen folding begins at the protein's C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single N-glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, the N-glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of N-glycosylated versus non-N-glycosylated collagen type-I, leaving the function of the N-glycan unclear. We hypothesized that the collagen N-glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of the N-glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The N-glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER's lectin-based chaperone machinery. Finally, we show that the C-Pro N-glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen's previously enigmatic N-glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.

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

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.

Adapting Secretory Proteostasis and Function Through the Unfolded Protein Response.

Cells address challenges to protein folding in the secretory pathway by engaging endoplasmic reticulum (ER)-localized protective mechanisms that are collectively termed the unfolded protein response (UPR). By the action of the transmembrane signal transducers IRE1, PERK, and ATF6, the UPR induces networks of genes whose products alleviate the burden of protein misfolding. The UPR also plays instructive roles in cell differentiation and development, aids in the response to pathogens, and coordinates the output of professional secretory cells. These functions add to and move beyond the UPR's classical role in addressing proteotoxic stress. Thus, the UPR is not just a reaction to protein misfolding, but also a fundamental driving force in physiology and pathology. Recent efforts have yielded a suite of chemical genetic methods and small molecule modulators that now provide researchers with both stress-dependent and -independent control of UPR activity. Such tools provide new opportunities to perturb the UPR and thereby study mechanisms for maintaining proteostasis in the secretory pathway. Numerous observations now hint at the therapeutic potential of UPR modulation for diseases related to the misfolding and aggregation of ER client proteins. Growing evidence also indicates the promise of targeting ER proteostasis nodes downstream of the UPR. Here, we review selected advances in these areas, providing a resource to inform ongoing studies of secretory proteostasis and function as they relate to the UPR.

A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production.

Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.

Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes.

We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.

A cysteine-based molecular code informs collagen C-propeptide assembly.

Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that the presence or absence of a single cysteine in type-I collagen's C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca2+ in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.

Mapping and Exploring the Collagen-I Proteostasis Network.

Collagen-I is the most abundant protein in the human body, yet our understanding of how the endoplasmic reticulum regulates collagen-I proteostasis (folding, quality control, and secretion) remains immature. Of particular importance, interactomic studies to map the collagen-I proteostasis network have never been performed. Such studies would provide insight into mechanisms of collagen-I folding and misfolding in cells, an area that is particularly important owing to the prominence of the collagen misfolding-related diseases. Here, we overcome key roadblocks to progress in this area by generating stable fibrosarcoma cells that inducibly express properly folded and modified collagen-I strands tagged with distinctive antibody epitopes. Selective immunoprecipitation of collagen-I from these cells integrated with quantitative mass spectrometry-based proteomics permits the first mapping of the collagen-I proteostasis network. Biochemical validation of the resulting map leads to the assignment of numerous new players in collagen-I proteostasis, and the unanticipated discovery of apparent aspartyl-hydroxylation as a new post-translational modification in the N-propeptide of collagen-I. Furthermore, quantitative analyses reveal that Erp29, an abundant endoplasmic reticulum proteostasis machinery component with few known functions, plays a key role in collagen-I retention under ascorbate-deficient conditions. In summary, the work here provides fresh insights into the molecular mechanisms of collagen-I proteostasis, yielding a detailed roadmap for future investigations. Straightforward adaptations of the cellular platform developed will also enable hypothesis-driven, comparative research on the likely distinctive proteostasis mechanisms engaged by normal and disease-causing, misfolding collagen-I variants, potentially motivating new therapeutic strategies for currently incurable collagenopathies.

XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans.

The molecular architecture of the mature N-glycome is dynamic, with consequences for both normal and pathologic processes. Elucidating cellular mechanisms that modulate the N-linked glycome is, therefore, crucial. The unfolded protein response (UPR) is classically responsible for maintaining proteostasis in the secretory pathway by defining levels of chaperones and quality control proteins. Here, we employ chemical biology methods for UPR regulation to show that stress-independent activation of the UPR's XBP1s transcription factor also induces a panel of N-glycan maturation-related enzymes. The downstream consequence is a distinctive shift toward specific hybrid and complex N-glycans on N-glycoproteins produced from XBP1s-activated cells, which we characterize by mass spectrometry. Pulse-chase studies attribute this shift specifically to altered N-glycan processing, rather than to changes in degradation or secretion rates. Our findings implicate XBP1s in a new role for N-glycoprotein biosynthesis, unveiling an important link between intracellular stress responses and the molecular architecture of extracellular N-glycoproteins.