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.

Targeting defective proteostasis in the collagenopathies.

The collagenopathies are a diverse group of diseases caused primarily by mutations in collagen genes. The resulting disruptions in collagen biogenesis can impair development, cause cellular dysfunction, and severely impact connective tissues. Most existing treatment options only address patient symptoms. Yet, while the disease-causing genes and proteins themselves are difficult to target, increasing evidence suggests that resculpting the intracellular proteostasis network, meaning the machineries responsible for producing and ensuring the integrity of collagen, could provide substantial benefit. We present a proteostasis-focused perspective on the collagenopathies, emphasizing progress toward understanding how mechanisms of collagen proteostasis are disrupted in disease. In parallel, we highlight recent advances in small molecule approaches to tune endoplasmic reticulum proteostasis that may prove useful in these disorders.

XBP1s activation can globally remodel N-glycan structure distribution patterns.

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.

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.

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.

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.