Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis.

Antibodies are central effectors of the adaptive immune response, widespread used therapeutics, but also potentially disease-causing biomolecules. Antibody folding catalysts in the plasma cell are incompletely defined. Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease with increasingly recognized autoimmune features. We found elevated expression of FK506-binding protein 11 (FKBP11) in IPF lungs where FKBP11 specifically localized to antibody-producing plasma cells. Suggesting a general role in plasma cells, plasma cell-specific FKBP11 expression was equally observed in lymphatic tissues, and in vitro B cell to plasma cell differentiation was accompanied by induction of FKBP11 expression. Recombinant human FKBP11 was able to refold IgG antibody in vitro and inhibited by FK506, strongly supporting a function as antibody peptidyl-prolyl cis-trans isomerase. Induction of ER stress in cell lines demonstrated induction of FKBP11 in the context of the unfolded protein response in an X-box-binding protein 1 (XBP1)-dependent manner. While deficiency of FKBP11 increased susceptibility to ER stress-mediated cell death in an alveolar epithelial cell line, FKBP11 knockdown in an antibody-producing hybridoma cell line neither induced cell death nor decreased expression or secretion of IgG antibody. Similarly, antibody secretion by the same hybridoma cell line was not affected by knockdown of the established antibody peptidyl-prolyl isomerase cyclophilin B. The results are consistent with FKBP11 as a novel XBP1-regulated antibody peptidyl-prolyl cis-trans isomerase and indicate significant redundancy in the ER-resident folding machinery of antibody-producing hybridoma cells.

Defining the Extracellular Matrix of Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy, and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades-underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment. To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and four human sarcomas to analyze expression of seven different collagens, fibrillins, and collagen-modifying proteins, with cross-correlation to RNA deep sequencing. We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1, and PLOD2 in human RMS relative to normal skeletal muscle. These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins, and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally 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.

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

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.

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

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.

Sex effects of dietary protein source and acid load on renal and bone status in the Pahenu2 mouse model of phenylketonuria.

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-/- (Pahenu2/enu2 ) 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.

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

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 Pb2+ detection.

The HDAC3-SMARCA4-miR-27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma.

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 PAX3:FOXO1 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 PAX3:FOXO1 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.

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

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.

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

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.

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

Type III collagen is a major fibrillar collagen consisting of three identical α1(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 α1(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 Col3a1Tg-G182S mouse line displays a phenotype recapitulating characteristics of human vEDS patients with signs of dermal and vascular fragility. The Col3a1Tg-G182S 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 α1(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.

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

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/SERPINH1) and 65-kDa FK506-binding protein (FKBP65/FKBP10), 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 in vitro 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.

Osteogenesis imperfecta.

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.

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

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 cis/trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis/trans 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 in vitro 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 PPIB gene), FKBP13 (FKBP2), FKBP19 (FKBP11), FKBP22 (FKBP14), FKBP23 (FKBP7), and FKBP65 (FKBP10), 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 in vitro 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.

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

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-ß1. 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.

Structural insight for chain selection and stagger control in collagen.

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.

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

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.

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

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.

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

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.