-
Cellular and Molecular Life Sciences :... Dec 2021Secretion and quality control of large extracellular matrix proteins remain poorly understood and debated, particularly transport intermediates delivering folded...
Secretion and quality control of large extracellular matrix proteins remain poorly understood and debated, particularly transport intermediates delivering folded proteins from the ER to Golgi and misfolded ones to lysosomes. Discrepancies between different studies are related to utilization of exogenous cargo, off-target effects of experimental conditions and cell manipulation, and identification of transport intermediates without tracing their origin and destination. To address these issues, here we imaged secretory and degradative trafficking of type I procollagen in live MC3T3 osteoblasts by replacing a region encoding N-propeptide in endogenous Col1a2 gDNA with GFP cDNA. We selected clones that produced the resulting fluorescent procollagen yet had normal expression of key osteoblast and ER/cell stress genes, normal procollagen folding, and normal deposition and mineralization of extracellular matrix. Live-cell imaging of these clones revealed ARF1-dependent transport intermediates, which had no COPII coat and delivered procollagen from ER exit sites (ERESs) to Golgi without stopping at ER-Golgi intermediate compartment (ERGIC). It also confirmed ERES microautophagy, i.e., lysosomes engulfing ERESs containing misfolded procollagen. Beyond validating these trafficking models for endogenous procollagen, we uncovered a probable cause of noncanonical cell stress response to procollagen misfolding. Recognized and retained only at ERESs, misfolded procollagen does not directly activate the canonical UPR, yet it disrupts the ER lumen by blocking normal secretory export from the ER.
Topics: Animals; Autophagy; COP-Coated Vesicles; Cells, Cultured; Collagen Type I; Endoplasmic Reticulum; Golgi Apparatus; Lysosomes; Mice; Osteoblasts; Procollagen; Protein Transport
PubMed: 34779895
DOI: 10.1007/s00018-021-04017-z -
Journal of Cell Science Apr 2005Collagen fibrils in the extracellular matrix allow connective tissues such as tendon, skin and bone to withstand tensile forces. The fibrils are indeterminate in length,... (Review)
Review
Collagen fibrils in the extracellular matrix allow connective tissues such as tendon, skin and bone to withstand tensile forces. The fibrils are indeterminate in length, insoluble and form elaborate three-dimensional arrays that extend over numerous cell lengths. Studies of the molecular basis of collagen fibrillogenesis have provided insight into the trafficking of procollagen (the precursor of collagen) through the cellular secretory pathway, the conversion of procollagen to collagen by the procollagen metalloproteinases, and the directional deposition of fibrils involving the plasma membrane and late secretory pathway. Fibril-associated molecules are targeted to the surface of collagen fibrils, and these molecules play an important role in regulating the diameter and interactions between the fibrils.
Topics: Animals; Extracellular Matrix; Fibrillar Collagens; Humans; Procollagen; Protein Transport
PubMed: 15788652
DOI: 10.1242/jcs.01731 -
Acta Dermato-venereologica Apr 2023Keloids are skin tumours caused by aberrant growth of dermal fibroblasts. Cellular senescence contributes to aging and various pathological conditions, including cancer,...
Keloids are skin tumours caused by aberrant growth of dermal fibroblasts. Cellular senescence contributes to aging and various pathological conditions, including cancer, atherosclerosis, and fibrotic diseases. However, the effects of cellular senescence and senolytic drugs on keloids remain largely unknown. This study investigated senescent fibroblasts in keloids and assessed the effects of dasatinib on these cells. Tissues acquired from keloid removal surgery were analysed for senescence-associated β-galactosidase-positive cells, p16 expression, and the effects of dasatinib treatment on keloids. Keloid tissue was xenotransplanted into mice, and the effect of intralesional dasatinib injection on keloid growth was observed. The results showed that the numbers of β-galactosidase-positive and p16-expressing cells were higher in the keloids compared with in the controls. Dasatinib induced selective clearance of senescent cells and decreased procollagen expression in cultured keloid fibroblasts. In this xenotransplant keloid mouse model, intralesional injection of dasatinib reduced gross keloid tissue weight and the expression of both procollagen and p16. In addition, dasatinib-treated keloid fibroblasts conditioned medium reduced procollagen and p16 expression in cultured keloid fibroblasts. In conclusion, these results suggest that an increased number of senescent fibroblasts may play an important role in the pathogenesis of keloids. Therefore, dasatinib could be an alternative treatment for patients with keloids.
Topics: Animals; Mice; Keloid; Procollagen; Dasatinib; Cellular Senescence; Fibroblasts; Cells, Cultured
PubMed: 37021598
DOI: 10.2340/actadv.v103.4475 -
Biochimica Et Biophysica Acta.... Sep 2019Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen...
Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ± PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.
Topics: Calorimetry, Differential Scanning; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Endoplasmic Reticulum; Fibroblasts; Humans; Microscopy, Fluorescence; Mutation, Missense; Osteogenesis Imperfecta; Procollagen; Protein Structure, Tertiary
PubMed: 31055083
DOI: 10.1016/j.bbadis.2019.04.018 -
Molecular Biology of the Cell Mar 2022Collagen is the major protein component of the extracellular matrix. Synthesis of procollagens starts in the endoplasmic reticulum (ER), and three α chains form a rigid...
Collagen is the major protein component of the extracellular matrix. Synthesis of procollagens starts in the endoplasmic reticulum (ER), and three α chains form a rigid triple helix 300-400 nm in length. It remains unclear how such a large cargo is transported from the ER to the Golgi apparatus. In this study, to elucidate the intracellular transport of fibril-forming collagens, we fused cysteine-free GFP to the N-telopeptide region of procollagen III (GFP-COL3A1) and analyzed transport by live-cell imaging. We found that the maturation dynamics of procollagen III was largely different from that of network-forming procollagen IV. Proline hydroxylation of procollagen III uniquely triggered the formation of intralumenal droplet-like structures, similarly to events caused by liquid-liquid phase separation, and ER exit sites surrounded large droplets containing chaperones. Procollagen III was transported to the Golgi apparatus via vesicular and tubular carriers containing ERGIC53 and RAB1B; this process required TANGO1 and CUL3, which we previously reported to be dispensable for procollagen IV. GFP-COL3A1 and mCherry-α1AT were cotransported in the same vesicle. Based on these findings, we propose that shortly after ER exit, enlarged carriers containing procollagen III fuse to ERGIC for transport to the Golgi apparatus by conventional cargo carriers.
Topics: Biological Transport; Endoplasmic Reticulum; Golgi Apparatus; Procollagen; Protein Transport
PubMed: 35044867
DOI: 10.1091/mbc.E21-07-0372 -
Life Science Alliance May 2022The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by...
The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.
Topics: Collagen; Endoplasmic Reticulum; Procollagen
PubMed: 35181633
DOI: 10.26508/lsa.202101060 -
Journal of Clinical Pathology.... 1978
Topics: Collagen; Endoplasmic Reticulum; Galactose; Golgi Apparatus; Hydroxylation; Hydroxylysine; Procollagen; Protein Conformation; Tropocollagen
PubMed: 282296
DOI: No ID Found -
Proceedings of the National Academy of... Mar 2021Intracellular procollagen folding begins at the protein's C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and...
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 -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 -glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of -glycosylated versus non--glycosylated collagen type-I, leaving the function of the -glycan unclear. We hypothesized that the collagen -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 -glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The -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 -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 -glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.
Topics: Collagen; Extracellular Matrix; Glycosylation; Humans; Procollagen; Protein Domains; Proteoglycans; Proteostasis
PubMed: 33674390
DOI: 10.1073/pnas.2026608118 -
Biochimica Et Biophysica Acta Nov 2013Extracellular matrix (ECM) proteins create structural frameworks in tissues such as bone, skin, tendon and cartilage etc. These connective tissues play important roles... (Review)
Review
Extracellular matrix (ECM) proteins create structural frameworks in tissues such as bone, skin, tendon and cartilage etc. These connective tissues play important roles in the development and homeostasis of organs. Collagen is the most abundant ECM protein and represents one third of all proteins in humans. The biosynthesis of ECM proteins occurs in the rough endoplasmic reticulum (rER). This review describes the current understanding of the biosynthesis and folding of procollagens, which are the precursor molecules of collagens, in the rER. Multiple folding enzymes and molecular chaperones are required for procollagen to establish specific posttranslational modifications, and facilitate folding and transport to the cell surface. Thus, this molecular ensemble in the rER contributes to ECM maturation and to the development and homeostasis of tissues. Mutations in this ensemble are likely candidates for connective tissue disorders. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.
Topics: Animals; Endoplasmic Reticulum, Rough; Extracellular Matrix Proteins; Humans; Molecular Chaperones; Procollagen
PubMed: 23602968
DOI: 10.1016/j.bbamcr.2013.04.008 -
International Journal of... 2022Glioma is a common type of brain tumor with high incidence and mortality rates. Procollagen C-protease enhancer protein (PCOLCE) has been shown to regulate tumor growth...
Procollagen C-protease enhancer protein is a prognostic factor for glioma and promotes glioma development by regulating multiple tumor-related pathways and immune microenvironment.
OBJECTIVES
Glioma is a common type of brain tumor with high incidence and mortality rates. Procollagen C-protease enhancer protein (PCOLCE) has been shown to regulate tumor growth and metastasis in several cancers. However, the role of PCOLCE in glioma is unknown. This study aims to assess the association between PCOLCE and prognosis of glioma, and investigated the potential mechanisms.
METHODS
The prognostic value of PCOLCE was determined using data from nine publicly available glioma cohorts. We also investigated the relationship between PCOLCE and glioma immune microenvironment and predicted response to immunotherapy based on the expression levels of PCOLCE. The potential roles of PCOLCE in glioma were also explored and validated in cell experiment.
RESULTS
Survival analysis suggested that high-PCOLCE expression was associated with poor prognosis in glioma. Upregulation of PCOLCE enhanced an immune suppressive microenvironment in glioma by regulating immunocyte infiltration and Cancer-Immunity Cycle. Cox and ROC analysis revealed that PCOLCE was a prognostic factor for glioma and could be used to predict survival of the patients. Patients with low-PCOLCE expression were more likely to respond to Immunotherapy with ICI (immune checkpoint inhibitor) and survive longer. Enrichment analysis showed that PCOLCE was associated with multiple tumor-related pathways. Finally, we demonstrated that the knockdown of PCOLCE inhibited glioma development by regulating cell cycle and promoting apoptosis in in vitro experiments.
CONCLUSION
PCOLCE promotes glioma progression by regulating multiple tumor-related pathways and immune microenvironment and can be used as a prognostic factor for glioma.
Topics: Biomarkers, Tumor; Brain Neoplasms; Glioma; Humans; Peptide Hydrolases; Procollagen; Prognosis; Tumor Microenvironment
PubMed: 35609253
DOI: 10.1177/03946320221104548