Publications by authors named "Hans Peter Bächinger"

92 Publications

Type I and type V procollagen triple helix use different subsets of the molecular ensemble for lysine post-translational modifications in the rER.

J Biol Chem 2021 Feb 22:100453. Epub 2021 Feb 22.

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon, USA.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily post-translationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity, however the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null and wild type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.
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http://dx.doi.org/10.1016/j.jbc.2021.100453DOI Listing
February 2021

Aberrant binding of mutant HSP47 affects posttranslational modification of type I collagen and leads to osteogenesis imperfecta.

PLoS Genet 2021 Feb 1;17(2):e1009339. Epub 2021 Feb 1.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Heat shock protein 47 (HSP47), encoded by the SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta leading to early demise. p.R222 is a highly conserved residue located within the collagen interacting surface of HSP47. Binding assays show a significantly reduced affinity of HSP47-R222S for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen produced by dermal fibroblasts, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since we also observed a normal intracellular folding and secretion rate of type I procollagen, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to the modifying hydroxyl- and glycosyltransferases, as is commonly observed in other types of OI. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR. In addition, we showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains in a gelatin sepharose pulldown assay results in increased binding of other chaperones and modifying enzymes. The elevated expression and binding of this molecular ensemble to type I procollagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I procollagen chains. Together, these results illustrate the importance of HSP47 for proper posttranslational modification and provide insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype.
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http://dx.doi.org/10.1371/journal.pgen.1009339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7877763PMC
February 2021

The novel missense mutation Met48Lys in FKBP22 changes its structure and functions.

Sci Rep 2020 01 16;10(1):497. Epub 2020 Jan 16.

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA.

Mutations in the FKBP14 gene encoding FKBP22 (FK506 Binding Protein 22 kDa) cause kyphoscoliotic Ehlers-Danlos Syndrome (kEDS). The first clinical report showed that a lack of FKBP22 protein due to mutations causing nonsense-mediated decay of the mRNA leads to a wide spectrum of clinical phenotypes including progressive kyphoscoliosis, joint hypermobility, hypotonia, hyperelastic skin, hearing loss and aortic rupture. Our previous work showed that these phenotypic features could be correlated with the functions of FKBP22, which preferentially binds to type III, VI and X collagens, but not to type I, II or V collagens. We also showed that FKBP22 catalyzed the folding of type III collagen through its prolyl isomerase activity and acted as a molecular chaperone for type III collagen. Recently, a novel missense mutation Met48Lys in FKBP22 was identified in a patient with kEDS. In this report, we expand the list of substrates of FKBP22 and also demonstrate that the Met48Lys mutation diminishes the activities of FKBP22, indicating that pathology can arise from absence of FKBP22, or partial loss of its function.
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http://dx.doi.org/10.1038/s41598-019-57374-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965642PMC
January 2020

Sex effects of dietary protein source and acid load on renal and bone status in the Pah mouse model of phenylketonuria.

Physiol Rep 2019 10;7(20):e14251

Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin.

The low-phenylalanine (Phe) diet with amino acid (AA) medical foods is associated with low bone mineral density (BMD) and renal dysfunction in human phenylketonuria (PKU). Our objective was to determine if diets differing in dietary protein source and acid load alter bone and renal outcomes in Pah and wild-type (WT) mice. Female and male Pah (Pah ) and WT littermates (C57BL/6 background) were fed high-acid AA, buffered AA (BAA), glycomacropeptide (GMP), or high-Phe casein diets from 3 to 24 weeks of age. The BAA diet significantly reduced the excretion of renal net acid and ammonium compared with the AA diet. Interestingly, the BAA diet did not improve renal dilation in hematoxylin and eosin (H&E) stained renal sections, femoral biomechanical parameters, or femoral bone mineral content (BMC). Significantly lower femoral BMC and strength occurred in Pah versus WT mice, with greater decline in female Pah mice. Polyuria and mild vacuolation in the proximal convoluted tubules were observed in male Pah and WT mice fed the high-acid AA diet versus absent/minimal cortical vacuolation in males fed the GMP, BAA, or casein diets. Vacuole contents in male mice were proteinaceous. Cortical vacuolation was absent in female mice. Dilated medullary tubules were observed in all Pah mice, except for male Pah mice fed the GMP diet. In summary, the PKU genotype and diet showed differential effects on renal and bone status in male and female mice. Renal status improved in male Pah mice fed the GMP diet.
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http://dx.doi.org/10.14814/phy2.14251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813258PMC
October 2019

A ribose modification of Spinach aptamer accelerates lead(ii) cation association in vitro.

Chem Commun (Camb) 2019 May 30;55(42):5882-5885. Epub 2019 Apr 30.

Department of Biochemistry & Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA.

Spinach aptamer fluorescence requires formation of a tripartite complex composed of folded RNA, a GFP-like fluorophore, and selective cation coordination. 2'F pyrimidine modified Spinach has retained fluorescence, increased chemical stability, and accelerated cation association via increased G-quadruplex dynamics, thereby reducing readout time and enhancing Spinach utility for aqueous Pb detection.
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http://dx.doi.org/10.1039/c9cc01697jDOI Listing
May 2019

The HDAC3-SMARCA4-miR-27a axis promotes expression of the fusion oncogene in rhabdomyosarcoma.

Sci Signal 2018 11 20;11(557). Epub 2018 Nov 20.

Children's Cancer Therapy Development Institute, Beaverton, OR 97005, USA.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I-specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3-SMARCA4-miR-27a-PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.
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http://dx.doi.org/10.1126/scisignal.aau7632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432638PMC
November 2018

The endoplasmic reticulum-resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican.

J Biol Chem 2018 08 12;293(35):13707-13716. Epub 2018 Jul 12.

the Department for Medical Biochemistry and Microbiology, Uppsala University, Uppsala 75237, Sweden

The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM.
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http://dx.doi.org/10.1074/jbc.RA117.000758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120207PMC
August 2018

Type III collagen affects dermal and vascular collagen fibrillogenesis and tissue integrity in a mutant Col3a1 transgenic mouse model.

Matrix Biol 2018 09 15;70:72-83. Epub 2018 Mar 15.

Center for Medical Genetics, Ghent University and University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address:

Type III collagen is a major fibrillar collagen consisting of three identical α(III)-chains that is particularly present in tissues exhibiting elastic properties, such as the skin and the arterial wall. Heterozygous mutations in the COL3A1 gene result in vascular Ehlers-Danlos syndrome (vEDS), a severe, life-threatening disorder, characterized by thin, translucent skin and propensity to arterial, intestinal and uterine rupture. Most human vEDS cases result from a missense mutation substituting a crucial glycine residue in the triple helical domain of the α(III)-chains. The mechanisms by which these mutant type III collagen molecules cause dermal and vascular fragility are not well understood. We generated a transgenic mouse line expressing mutant type III collagen, containing a typical helical glycine substitution (p.(Gly182Ser)). This Col3a1 mouse line displays a phenotype recapitulating characteristics of human vEDS patients with signs of dermal and vascular fragility. The Col3a1 mice develop severe transdermal skin wounds, resulting in early demise at 13-14weeks of age. We found that this phenotype was associated with a reduced total collagen content and an abnormal collagen III:I ratio, leading to the production of severely malformed collagen fibrils in the extracellular matrix of dermal and arterial tissues. These results indicate that expression of the glycine substitution in the α(III)-chain disturbs formation of heterotypic type III:I collagen fibrils, and thereby demonstrate a key role for type III collagen in collagen fibrillogenesis in dermal and arterial tissues.
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http://dx.doi.org/10.1016/j.matbio.2018.03.008DOI Listing
September 2018

Post-translational modification of type IV collagen with 3-hydroxyproline affects its interactions with glycoprotein VI and nidogens 1 and 2.

J Biol Chem 2018 04 28;293(16):5987-5999. Epub 2018 Feb 28.

From the Research Department, Shriners Hospital for Children, Portland, Oregon 97239,

Type IV collagen is a major component of the basement membrane and interacts with numerous other basement membrane proteins. Many of these interactions are poorly characterized. Type IV collagen is abundantly post-translationally modified with 3-hydroxyproline (3-Hyp), but 3-Hyp's biochemical role in type IV collagen's interactions with other proteins is not well established. In this work, we present binding data consistent with a major role of 3-Hyp in interactions of collagen IV with glycoprotein VI and nidogens 1 and 2. The increased binding interaction between type IV collagen without 3-Hyp and glycoprotein VI has been the subject of some controversy, which we sought to explore, whereas the lack of binding of nidogens to type IV collagen without 3-Hyp is novel. Using tandem MS, we show that the putative glycoprotein VI-binding site is 3-Hyp-modified in WT PFHR-9 type IV collagen, but not in PFHR-9 cells in which prolyl-3-hydroxylase 2 (P3H2) has been knocked out (KO). Moreover, we observed altered 3-Hyp occupancy across many other sites. Using amino acid analysis of type IV collagen from the WT and P3H2 KO cell lines, we confirm that P3H2 is the major, but not the only 3-Hyp-modifying enzyme of type IV collagen. These findings underscore the importance of post-translational modifications of type IV collagen for interactions with other proteins.
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http://dx.doi.org/10.1074/jbc.RA117.000406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912474PMC
April 2018

Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum.

J Biol Chem 2017 10 31;292(42):17216-17224. Epub 2017 Aug 31.

From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University and Shriners Hospital for Children, Research Department, Portland, Oregon 97239

Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/) and 65-kDa FK506-binding protein (FKBP65/), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes.
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http://dx.doi.org/10.1074/jbc.M117.802298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655501PMC
October 2017

Osteogenesis imperfecta.

Nat Rev Dis Primers 2017 Aug 18;3:17052. Epub 2017 Aug 18.

Children's Hospital, University of Cologne, Cologne, Germany.

Skeletal deformity and bone fragility are the hallmarks of the brittle bone dysplasia osteogenesis imperfecta. The diagnosis of osteogenesis imperfecta usually depends on family history and clinical presentation characterized by a fracture (or fractures) during the prenatal period, at birth or in early childhood; genetic tests can confirm diagnosis. Osteogenesis imperfecta is caused by dominant autosomal mutations in the type I collagen coding genes (COL1A1 and COL1A2) in about 85% of individuals, affecting collagen quantity or structure. In the past decade, (mostly) recessive, dominant and X-linked defects in a wide variety of genes encoding proteins involved in type I collagen synthesis, processing, secretion and post-translational modification, as well as in proteins that regulate the differentiation and activity of bone-forming cells have been shown to cause osteogenesis imperfecta. The large number of causative genes has complicated the classic classification of the disease, and although a new genetic classification system is widely used, it is still debated. Phenotypic manifestations in many organs, in addition to bone, are reported, such as abnormalities in the cardiovascular and pulmonary systems, skin fragility, muscle weakness, hearing loss and dentinogenesis imperfecta. Management involves surgical and medical treatment of skeletal abnormalities, and treatment of other complications. More innovative approaches based on gene and cell therapy, and signalling pathway alterations, are under investigation.
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http://dx.doi.org/10.1038/nrdp.2017.52DOI Listing
August 2017

Ziploc-ing the structure 2.0: Endoplasmic reticulum-resident peptidyl prolyl isomerases show different activities toward hydroxyproline.

J Biol Chem 2017 06 6;292(22):9273-9282. Epub 2017 Apr 6.

From the Department of Biochemistry and Molecular Biology, Oregon Health & Science University and

Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the / isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl / isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen. We previously described this process as "Ziploc-ing the structure" and now provide additional information on the activity of individual rER PPIases. We investigated the substrate preferences of these PPIases using type III collagen, the unhydroxylated quarter fragment of type III collagen, and synthetic peptides as substrates. We observed changes in activity of six rER-resident PPIases, cyclophilin B (encoded by the gene), FKBP13 (), FKBP19 (), FKBP22 (), FKBP23 (), and FKBP65 (), due to posttranslational modifications of proline residues in the substrate. Cyclophilin B and FKBP13 exhibited much lower activity toward post-translationally modified substrates. In contrast, FKBP19, FKBP22, and FKBP65 showed increased activity toward hydroxyproline-containing peptide substrates. Moreover, FKBP22 showed a hydroxyproline-dependent effect by increasing the amount of refolded type III collagen and FKBP19 seems to interact with triple helical type I collagen. Therefore, we propose that hydroxyproline modulates the rate of Ziploc-ing of the triple helix of collagen in the rER.
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http://dx.doi.org/10.1074/jbc.M116.772657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454108PMC
June 2017

A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone. Inhibition of Collagen Fibril Assembly.

Am J Respir Cell Mol Biol 2017 07;57(1):77-90

1 Comprehensive Pneumology Center, and.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular, collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study, we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. Primary human fibroblasts from patients with IPF and healthy donors were treated with nintedanib (0.01-1.0 μM) or pirfenidone (100-1,000 μM) in the absence or presence of transforming growth factor-β1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron microscopy, and the kinetics of collagen fibril assembly was assessed using a light-scattering approach. In IPF fibroblasts, nintedanib reduced the expression of collagen I and V, fibronectin, and FKBP10 and attenuated the secretion of collagen I and III. Pirfenidone also down-regulated collagen V but otherwise showed fewer and less pronounced effects. By and large, the effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. In summary, both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused a reduction in and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.
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http://dx.doi.org/10.1165/rcmb.2016-0217OCDOI Listing
July 2017

Structural insight for chain selection and stagger control in collagen.

Sci Rep 2016 11 29;6:37831. Epub 2016 Nov 29.

Research Department, Shriners Hospital for Children, Portland, Oregon 97239, USA.

Collagen plays a fundamental role in all known metazoans. In collagens three polypeptides form a unique triple-helical structure with a one-residue stagger to fit every third glycine residue in the inner core without disturbing the poly-proline type II helical conformation of each chain. There are homo- and hetero-trimeric types of collagen consisting of one, two or three distinct chains. Thus there must be mechanisms that control composition and stagger during collagen folding. Here, we uncover the structural basis for both chain selection and stagger formation of a collagen molecule. Three distinct chains (α1, α2 and α3) of the non-collagenous domain 2 (NC2) of type IX collagen are assembled to guide triple-helical sequences in the leading, middle and trailing positions. This unique domain opens the door for generating any fragment of collagen in its native composition and stagger.
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http://dx.doi.org/10.1038/srep37831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126661PMC
November 2016

Intracellular mechanisms of molecular recognition and sorting for transport of large extracellular matrix molecules.

Proc Natl Acad Sci U S A 2016 10 27;113(41):E6036-E6044. Epub 2016 Sep 27.

Research Department, Shriners Hospital for Children, Portland, OR 97239; Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239;

Extracellular matrix (ECM) proteins are biosynthesized in the rough endoplasmic reticulum (rER) and transported via the Golgi apparatus to the extracellular space. The coat protein complex II (COPII) transport vesicles are approximately 60-90 nm in diameter. However, several ECM molecules are much larger, up to several hundreds of nanometers. Therefore, special COPII vesicles are required to coat and transport these molecules. Transmembrane Protein Transport and Golgi Organization 1 (TANGO1) facilitates loading of collagens into special vesicles. The Src homology 3 (SH3) domain of TANGO1 was proposed to recognize collagen molecules, but how the SH3 domain recognizes various types of collagen is not understood. Moreover, how are large noncollagenous ECM molecules transported from the rER to the Golgi? Here we identify heat shock protein (Hsp) 47 as a guide molecule directing collagens to special vesicles by interacting with the SH3 domain of TANGO1. We also consider whether the collagen secretory model applies to other large ECM molecules.
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http://dx.doi.org/10.1073/pnas.1609571113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068301PMC
October 2016

Bone matrix hypermineralization in prolyl-3 hydroxylase 1 deficient mice.

Bone 2016 Apr 22;85:15-22. Epub 2016 Jan 22.

Shriners Hospital for Children and McGill University, Montreal, Quebec, Canada. Electronic address:

Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces. Quantitative backscattered electron imaging revealed that the bone mineralization density distribution was shifted towards higher values, indicating hypermineralization of bone matrix. It thus appears that P3H1 deficiency leads to decreased deposition of extracellular matrix by osteoblasts and increased incorporation of mineral into the matrix.
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http://dx.doi.org/10.1016/j.bone.2016.01.018DOI Listing
April 2016

TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response.

PLoS One 2015 15;10(6):e0129566. Epub 2015 Jun 15.

Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States of America.

A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129566PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468210PMC
March 2016

Recessive osteogenesis imperfecta caused by missense mutations in SPARC.

Am J Hum Genet 2015 Jun 28;96(6):979-85. Epub 2015 May 28.

Shriners Hospital for Children, Montréal, QC H3G 1A6, Canada. Electronic address:

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.
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http://dx.doi.org/10.1016/j.ajhg.2015.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457955PMC
June 2015

Conformational change from rigid rod to star: a triple-helical peptide with a linker domain at the C-terminal end.

J Phys Chem B 2015 Mar 23;119(9):3714-9. Epub 2015 Feb 23.

Department of Macromolecular Science, Osaka University , 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.

Small-angle X-ray scattering and circular dichroism measurements were made for a triple-helical peptide of which one end was linked by the thermally stable trimerization domain of type XIX collagen. The radius of gyration decreased steeply around the transition temperature while the scattering intensity at zero angle did not significantly change, indicating no molar mass change through the conformational transition. Thus, the data were analyzed in terms of the rigid cylinder model for the data at low temperatures and the wormlike star model at high temperatures. It was confirmed that the peptide molecules behave as a rod-like cylinder at low temperature and a semi flexible three-arm star-like chain at high temperature of which the single-coil peptide chain is appreciably extended by the high segment density nearby the linking domain.
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http://dx.doi.org/10.1021/jp5129172DOI Listing
March 2015

Cole-Carpenter syndrome is caused by a heterozygous missense mutation in P4HB.

Am J Hum Genet 2015 Mar 12;96(3):425-31. Epub 2015 Feb 12.

Shriners Hospital for Children, Montréal, QC H3G 1A6, Canada.

Cole-Carpenter syndrome is a severe bone fragility disorder that is characterized by frequent fractures, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features. To identify the cause of Cole-Carpenter syndrome in the two individuals whose clinical results were presented in the original description of this disorder, we performed whole-exome sequencing of genomic DNA samples from both individuals. The two unrelated individuals had the same heterozygous missense mutation in exon 9 of P4HB (NM_000918.3: c.1178A>G [p.Tyr393Cys]), the gene that encodes protein disulfide isomerase (PDI). In one individual, the P4HB mutation had arisen de novo, whereas in the other the mutation was transmitted from the clinically unaffected father who was a mosaic carrier of the variant. The mutation was located in the C-terminal disulfide isomerase domain of PDI, sterically close to the enzymatic center, and affected disulfide isomerase activity in vitro. Skin fibroblasts showed signs of increased endoplasmic reticulum stress, but despite the reported importance of PDI for collagen type I production, the rate of collagen type I secretion appeared normal. In conclusion, Cole-Carpenter syndrome is caused by a specific de novo mutation in P4HB that impairs the disulfide isomerase activity of PDI.
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http://dx.doi.org/10.1016/j.ajhg.2014.12.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375435PMC
March 2015

Ziploc-ing the structure: Triple helix formation is coordinated by rough endoplasmic reticulum resident PPIases.

Biochim Biophys Acta 2015 Oct 10;1850(10):1983-93. Epub 2015 Jan 10.

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA; Shriners Hospital for Children, Research Department, Portland, OR 97239, USA. Electronic address:

Background: Protein folding is crucial for proteins' specific functions and is facilitated by various types of enzymes and molecular chaperones. The peptidyl prolyl cis/trans isomerases (PPIase) are one of these families of enzymes. They ubiquitously exist inside the cell and there are eight PPIases in the rough endoplasmic reticulum (rER), a compartment where the folding of most secreted proteins occurs.

Scope Of Review: We review the functional and structural aspects of individual rER resident PPIases. Furthermore, we specifically discuss the role of these PPIases during collagen biosynthesis, since collagen is the most abundant protein in humans, is synthesized in the rER, and contains a proportionally high number of proline residues.

Major Conclusions: The rER resident PPIases recognize different sets of substrates and facilitate their folding. Although they are clearly catalysts for protein folding, they also have more broad and multifaceted functions. We propose that PPIases coordinate collagen biosynthesis in the rER.

General Significance: This review expands our understanding of collagen biosynthesis by explaining the influence of novel indirect mechanisms of regulating folding and this is also explored for PPIases. We also suggest future directions of research to obtain a better understanding of collagen biosynthesis and functions of PPIases in the rER. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.
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http://dx.doi.org/10.1016/j.bbagen.2014.12.024DOI Listing
October 2015

A substrate preference for the rough endoplasmic reticulum resident protein FKBP22 during collagen biosynthesis.

J Biol Chem 2014 Jun 12;289(26):18189-201. Epub 2014 May 12.

From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and the Research Department, Shriners Hospital for Children, Portland, Oregon 97239

The biosynthesis of collagens occurs in the rough endoplasmic reticulum and requires a large numbers of molecular chaperones, foldases, and post-translational modification enzymes. Collagens contain a large number of proline residues that are post-translationally modified to 3-hydroxyproline or 4-hydroxyproline, and the rate-limiting step in formation of the triple helix is the cis-trans isomerization of peptidyl-proline bonds. This step is catalyzed by peptidyl-prolyl cis-trans isomerases. There are seven peptidyl-prolyl cis-trans isomerases in the rER, and so far, two of these enzymes, cyclophilin B and FKBP65, have been shown to be involved in collagen biosynthesis. The absence of either cyclophilin B or FKBP65 leads to a recessive form of osteogenesis imperfecta. The absence of FKBP22 leads to a kyphoscoliotic type of Ehlers-Danlos syndrome (EDS), and this type of EDS is classified as EDS type VI, which can also be caused by a deficiency in lysyl-hydroxylase 1. However, the lack of FKBP22 shows a wider spectrum of clinical phenotypes than the absence of lysyl-hydroxylase 1 and additionally includes myopathy, hearing loss, and aortic rupture. Here we show that FKBP22 catalyzes the folding of type III collagen and interacts with type III collagen, type VI collagen, and type X collagen, but not with type I collagen, type II collagen, or type V collagen. These restrictive interactions might help explain the broader phenotype observed in patients that lack FKBP22.
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http://dx.doi.org/10.1074/jbc.M114.561944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140264PMC
June 2014

Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta.

Nat Med 2014 Jun 4;20(6):670-5. Epub 2014 May 4.

1] Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA. [2] Howard Hughes Medical Institute, Houston, Texas, USA.

Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap(-/-)) and dominant (Col1a2(tm1.1Mcbr)) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap(-/-) mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity. Anti-TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap(-/-) mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.
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http://dx.doi.org/10.1038/nm.3544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048326PMC
June 2014

Biological role of prolyl 3-hydroxylation in type IV collagen.

Proc Natl Acad Sci U S A 2014 Jan 24;111(1):161-6. Epub 2013 Dec 24.

Research Department, Shriners Hospitals for Children, Portland, OR 97239.

Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.
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http://dx.doi.org/10.1073/pnas.1307597111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890855PMC
January 2014

Structure of human peptidyl-prolyl cis-trans isomerase FKBP22 containing two EF-hand motifs.

Protein Sci 2014 Jan 25;23(1):67-75. Epub 2013 Nov 25.

Research Department, Shriners Hospital for Children, Portland, Oregon, 97239; Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon, 97239.

The FK506-binding protein (FKBP) family consists of proteins with a variety of protein-protein interaction domains and versatile cellular functions. It is assumed that all members are peptidyl-prolyl cis-trans isomerases with the enzymatic function attributed to the FKBP domain. Six members of this family localize to the mammalian endoplasmic reticulum (ER). Four of them, FKBP22 (encoded by the FKBP14 gene), FKBP23 (FKBP7), FKBP60 (FKBP9), and FKBP65 (FKBP10), are unique among all FKBPs as they contain the EF-hand motifs. Little is known about the biological roles of these proteins, but emerging genetics studies are attracting great interest to the ER resident FKBPs, as mutations in genes encoding FKBP10 and FKBP14 were shown to cause a variety of matrix disorders. Although the structural organization of the FKBP-type domain as well as of the EF-hand motif has been known for a while, it is difficult to conclude how these structures are combined and how it affects the protein functionality. We have determined a unique 1.9 Å resolution crystal structure for human FKBP22, which can serve as a prototype for other EF hand-containing FKBPs. The EF-hand motifs of two FKBP22 molecules form a dimeric complex with an elongated and predominantly hydrophobic cavity that can potentially be occupied by an aliphatic ligand. The FKBP-type domains are separated by a cleft and their putative active sites can catalyze isomerazation of two bonds within a polypeptide chain in extended conformation. These structural results are of prime interest for understanding biological functions of ER resident FKBPs containing EF-hand motifs.
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http://dx.doi.org/10.1002/pro.2391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3892300PMC
January 2014

The concave face of decorin mediates reversible dimerization and collagen binding.

J Biol Chem 2013 Dec 29;288(49):35526-33. Epub 2013 Oct 29.

From the Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom.

Decorin, the prototypical small leucine-rich proteoglycan, binds to collagen and thereby regulates collagen assembly into fibrils. The crystal structure of the decorin core protein revealed a tight dimer formed by the association of two monomers via their concave faces (Scott, P. G., McEwan, P. A., Dodd, C. M., Bergmann, E. M., Bishop, P. N., and Bella, J. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 15633-15638). Whether decorin binds collagen as a dimer has been controversial. Using analytical ultracentrifugation, we determined a dissociation constant of 1.37 ± 0.30 μm for the mouse decorin dimer. Dimerization could be abolished by engineering glycosylation sites into the dimer interface; other interface mutants remained dimeric. The monomeric mutants were as stable as wild-type decorin in thermal unfolding experiments. Mutations on the concave face of decorin abolished collagen binding regardless of whether the mutant proteins retained the ability to dimerize or not. We conclude that the concave face of decorin mediates collagen binding and that the dimer therefore must dissociate to bind collagen.
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http://dx.doi.org/10.1074/jbc.M113.504530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853298PMC
December 2013

Deficiency for the ER-stress transducer OASIS causes severe recessive osteogenesis imperfecta in humans.

Orphanet J Rare Dis 2013 Sep 30;8:154. Epub 2013 Sep 30.

Center for Medical Genetics, Ghent University Hospital, 9000 Ghent, Belgium.

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous brittle bone disorder. Whereas dominant OI is mostly due to heterozygous mutations in either COL1A1 or COL1A2, encoding type I procollagen, recessive OI is caused by biallelic mutations in genes encoding proteins involved in type I procollagen processing or chaperoning. Hitherto, some OI cases remain molecularly unexplained. We detected a homozygous genomic deletion of CREB3L1 in a family with severe OI. CREB3L1 encodes OASIS, an endoplasmic reticulum-stress transducer that regulates type I procollagen expression during murine bone formation. This is the first report linking CREB3L1 to human recessive OI, thereby expanding the OI gene spectrum.
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http://dx.doi.org/10.1186/1750-1172-8-154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3850743PMC
September 2013

An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

J Biol Chem 2013 Nov 16;288(44):31437-46. Epub 2013 Sep 16.

From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University and Shriners Hospital for Children, Research Department, Portland, Oregon 97239.

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.
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http://dx.doi.org/10.1074/jbc.M113.498063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814740PMC
November 2013

Posttranslational modifications in type I collagen from different tissues extracted from wild type and prolyl 3-hydroxylase 1 null mice.

J Biol Chem 2013 Aug 16;288(34):24742-52. Epub 2013 Jul 16.

Research Department, Shriners Hospitals for Children, Portland, Oregon 97239, USA.

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.
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http://dx.doi.org/10.1074/jbc.M113.464156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750170PMC
August 2013

Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding.

J Biol Chem 2013 Jun 3;288(26):19166-76. Epub 2013 May 3.

Shriners Hospitals for Children Portland Research Center, Portland, Oregon 97239, USA.

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.
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http://dx.doi.org/10.1074/jbc.M113.462002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696688PMC
June 2013