Collagen Prolyl Hydroxylase Research Articles

Prolyl 4-Hydroxylase: Substrate Isosteres in Which an (E)- or (Z)-Alkene Replaces the Prolyl Peptide Bond.

Collagen prolyl 4-hydroxylases (CP4Hs) catalyze a prevalent posttranslational modification, the hydroxylation of (2S)-proline residues in protocollagen strands. The ensuing (2S,4R)-4-hydroxyproline residues are necessary for the conformational stability of the collagen triple helix. Prolyl peptide bonds isomerize between cis and trans isomers, and the preference of the enzyme is unknown. We synthesized alkene isosteres of the cis and trans isomers to probe the conformational preferences of human CP4H1. We discovered that the presence of a prolyl peptide bond is necessary for catalysis. The cis isostere is, however, an inhibitor with a potency greater than that of the trans isostere, suggesting that the cis conformation of a prolyl peptide bond is recognized preferentially. Comparative studies with a Chlamydomonas reinhardtii P4H, which has a similar catalytic domain but lacks an N-terminal substrate-binding domain, showed a similar preference for the cis isostere. These findings support the hypothesis that the catalytic domain of CP4Hs recognizes the cis conformation of the prolyl peptide bond and inform the use of alkenes as isosteres for peptide bonds.

Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns

Proline hydroxylation is the most prevalent post-translational modification in collagen. The resulting product trans-4-hydroxyproline (Hyp) is of critical importance for the stability and thus function of collagen, with defects leading to several diseases. Prolyl 4-hydroxylases (P4Hs) are mononuclear non-heme iron α-ketoglutarate (αKG)-dependent dioxygenases that catalyze Hyp formation. Although animal and plant P4Hs target peptidyl proline, prokaryotes have been known to use free l-proline as a precursor to form Hyp. The P4H from Bacillus anthracis (BaP4H) has been postulated to act on peptidyl proline in collagen peptides, making it unusual within the bacterial clade, but its true physiological substrate remains enigmatic. Here we use mass spectrometry, fluorescence binding, x-ray crystallography, and docking experiments to confirm that BaP4H recognizes and acts on peptidyl substrates but not free l-proline, using elements characteristic of an Fe(II)/αKG-dependent dioxygenases. We further show that BaP4H can hydroxylate unique peptidyl proline sites in collagen-derived peptides with asymmetric hydroxylation patterns. The cofactor-bound crystal structures of BaP4H reveal active site conformational changes that define open and closed forms and mimic "ready" and "product-released" states of the enzyme in the catalytic cycle. These results help to clarify the role of BaP4H as well as provide broader insights into human collagen P4H and proteins with poly-l-proline type II helices.

Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits.

Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning.

The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to Wnt5a knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the Wnt5a knock out when compared to control. Expression of a panel of BM components such as laminin and of type IV collagen was also reduced due to the Wnt5a knock out. The P4ha1 gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in Wnt5a function from control. The compound Wnt5a+/-;P4ha1+/- embryos demonstrated Wnt5a-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling in vitro. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease.

Selective Inhibition of Collagen Prolyl 4-Hydroxylase in Human Cells

Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4-hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a "P4H" inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2'-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2-(5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents.

Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase.

The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded β-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells. Structural–mechanistic studies with prolyl hydroxylases have broader implications for understanding mechanisms in the Fe(II)- and 2-OG-dependent dioxygenase superfamily. Here, we describe crystal structures of an N-terminally truncated viral collagen prolyl hydroxylase (vCPH). The crystal structure shows that vCPH contains the conserved DSBH motif and iron binding active site residues of 2-OG oxygenases. Molecular dynamics simulations are used to delineate structural changes in vCPH upon binding its substrate. Kinetic investigations are used to report on reaction cycle intermediates and compare them to the closest homologues of vCPH. The study highlights the utility of vCPH as a model enzyme for broader mechanistic analysis of Fe(II)- and 2-OG-dependent dioxygenases, including those of biomedical interest.

Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells

Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics.

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.

Selective inhibition of prolyl 4-hydroxylases by bipyridinedicarboxylates

Collagen is the most abundant protein in animals. A variety of indications are associated with the overproduction of collagen, including fibrotic diseases and cancer metastasis. The stability of collagen relies on the posttranslational modification of proline residues to form (2S,4R)-4-hydroxyproline. This modification is catalyzed by collagen prolyl 4-hydroxylases (CP4Hs), which are Fe(II)- and α-ketoglutarate (AKG)-dependent dioxygenases located in the lumen of the endoplasmic reticulum. Human CP4Hs are validated targets for treatment of both fibrotic diseases and metastatic breast cancer. Herein, we report on 2,2′-bipyridinedicarboxylates as inhibitors of a human CP4H. Although most 2,2′-bipyridinedicarboxylates are capable of inhibition via iron sequestration, the 4,5′- and 5,5′-dicarboxylates were found to be potent competitive inhibitors of CP4H, and the 5,5′-dicarboxylate was selective in its inhibitory activity. Our findings clarify a strategy for developing CP4H inhibitors of clinical utility.

A novel profibrotic mechanism mediated by TGFβ-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells.

Idiopathic pulmonary fibrosis is a severe chronic lung disease with a high mortality rate. Excessive TGFβ signalling is recognized as a central player in lung fibrosis. However, the related mechanisms remain unclear. Herein we used a novel Tbx4 lung enhancer-driven Tet-On transgenic system to inhibit TGFβ signalling in mouse lung-resident mesenchymal cells at different stages of bleomycin-induced fibrosis, by conditionally knocking out TGFβ receptor II or expressing a dominant-negative TGFβ receptor II. Abrogation of mesenchymal TGFβ signalling markedly attenuated bleomycin-induced fibrotic pathology, which was independent of altered early inflammation. Furthermore, a novel TGFβ downstream target gene P4HA3 (an α-subunit of collagen prolyl hydroxylase) was identified, and its expression was significantly increased in fibroblastic foci of both bleomycin-induced fibrotic mouse lungs and idiopathic pulmonary fibrosis patients' lungs. The relationship between activated TGFβ signalling, up-regulation of P4HA3 and increased hydroxyproline/collagen production was further verified in cultured lung fibroblasts. Moreover, inhibition of collagen prolyl hydroxylase by pyridine-2,5-dicarboxylate attenuated TGFβ-stimulated collagen production in both cultured fibroblasts and bleomycin-induced mouse lung fibrosis. These data indicate that increased expression and activity of collagen prolyl hydroxylase is one of the important mechanisms underlying TGFβ-mediated profibrotic effects. Inhibition of collagen prolyl hydroxylase may be a new, promising approach for preventing and treating pulmonary fibrosis.

Selective and Bioavailable Inhibitors of Collagen Prolyl 4‐Hydroxylase

Collagen is the most abundant protein in animals. A variety of diseases are associated with the overproduction of collagen including cancer metastasis. The stability of collagen relies on post‐translational modifications that occur throughout the secretory pathway. One of the most important modifications is the hydroxylation of collagen strands by collagen prolyl 4‐hydroxylases (CP4Hs). Catalysis by CP4Hs converts proline residues to hydroxyproline residues, which are essential for the conformational stability of mature collagen strands. Importantly, CP4Hs are validated targets for the treatment of metastatic breast cancer, a significant unmet clinical need. Nonetheless, a therapy based upon this strategy has yet to emerge, primarily due to the difficulty in designing selective inhibitors for members of this enzyme family. Herein, we use a chemical approach based upon the classic 2,2′‐bipyridine (bipy) scaffold to probe the active site of human CP4H1 and understand the determinants of selectivity for this enzyme. Then, we redesign the bipy scaffold to enhance its pharmacological properties and generate potent biheteroaryl inhibitors that are selective for human CP4H1. Evaluations of these inhibitors in cell culture suggest that they are bioavailable and inhibit collagen synthesis at concentrations that do not disrupt iron homeostasis or cause general cytotoxicity. These results suggest the potential use of these inhibitors as leads for the development of antimetastatic therapeutics.This work was supported by grant R01 AR044276 (NIH).

CB-01 * TARGETING COLLAGEN REGULATION IN GLIOBLASTOMA MULTIFORME

Extracellular matrices (ECM) are quintessential complex structures that provide cells and tissues of functional support for cell adhesion and migration, cell-to-cell communication and differentiation. Collagens are fibrillar proteins that constitute the major component of ECM and have key structural roles. Intracellular maturation of collagen precursors, prior to secretion, is dependent on the hydroxylation of collagen proline residues. This reaction is mediated by collagen prolyl 4-hydroxylases (P3H and P4H families' enzymes) in the presence of α-ketoglutarate, oxygen, Fe2+ and ascorbate. It is already known that ECM and collagen prolyl-hydroxylases are important players in cancer migration and invasion, but little has been explored in brain tumours and hypoxia. We have focused our investigations on glioblastoma multiforme (GBM) using two independent and specific inhibitors of collagen prolyl-hydroxylases: Ethyl 3,4-dihydroxybenzoate (EDHB), an α-ketoglutarate competitor, and 3,4-Dehydro-DL-proline (L-Proline analogue). The presence of these inhibitors showed a dramatic reduction in cell proliferation in a panel of established GBM cell lines. Similar results were obtained in primary cell lines from GBM tumours and melanoma brain metastasis. The mechanisms behind these effects are independent of apoptosis and we are currently exploring the role of autophagy and cell cycle arrest as possible other mechanisms. Surprisingly, even in low oxygen concentration (1%) there was a significant reduction in proliferation. Collagen regulation and its role in cell proliferation in both normoxic and hypoxic conditions will be discussed as a potential new therapeutic strategy for GBMs.

Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm.

Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-β and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-588) contains supplementary material, which is available to authorized users.

P52 * TARGETING COLLAGEN REGULATION IN GLIOBLASTOMA MULTIFORME

INTRODUCTION: Extracellular matrices (ECM) are quintessential complex structures that provide cells and tissues with functional support for adhesion, migration, cell-to-cell communication and differentiation. Collagens are fibrillar proteins that constitute the major component of ECM and have a key structural role. Intracellular maturation of collagen precursors, prior to secretion, is dependent on the hydroxylation of the collagen proline residues which is mediated by the prolyl 4-hydroxylases (P4H) family of enzymes. Although, ECM and collagen prolyl-hydroxylases have been shown to be important players in cancer migration and invasion, little is known of their role in brain tumours. METHOD: To examine the role of P4H enzymes in the biology of GBM we profiled their expression in a panel of GBM cell lines a variety of techniques including RT-PCR, western blotting and MSP. For functional studies we assesed the effects of two independent inhibitors of P4H on the growth of these lines using proliferation and apoptotic assays (SRB and Annexin V respectively). RESULTS: Pharmacological inhibition of collagen prolyl-hydroxylases dramatically reduced the proliferation of a panel of GBM cell lines. This reduction was independent of apoptosis. CONCLUSION: Our preliminary results show that collagen biosynthesis plays an important role in the growth of GBM and therefore could be a potential therapeutic strategy that needs further investigation.

The miR-124-prolyl hydroxylase P4HA1-MMP1 axis plays a critical role in prostate cancer progression.

Collagen prolyl hydroxylases (C-P4HAs) are a family of enzymes involved in collagen biogenesis. One of the isoforms of P4HA, Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains. Here, we show the overexpression of P4HA1 in aggressive prostate cancer. Immunohistochemical analysis using tissue microarray demonstrated that P4HA1 expression was correlated with prostate cancer progression. Using in vitro studies, we showed that P4HA1 plays a critical role in prostate cancer cell growth and tumor progression. Expression profiling studies using P4HA1 modulated prostate cells suggested regulation of Matrix metalloproteases 1. The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1. Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124. MiR-124 in turn is negatively regulated by transcriptional repressors EZH2 and CtBP1, both of which are overexpressed in aggressive prostate cancer. Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo. Our observations suggest that P4HA1 plays a critical role in prostate cancer progression and could serve as a viable therapeutic target.

Critical Roles Of The IDH2 Mutation In Development and Maintenance Of Acute Myeloid Leukemia

Mutations in genes encoding isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in numerous cancers including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and angioimmunoblastic t-cell lymphoma (AITL). The roles of mutant IDHs in tumorigenesis remain unclear because of a lack of appropriate cancer models. Here we established a mouse AML model harboring an IDH2 mutation. IDH1/2 mutations in AML frequently occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, IDH2/R140Q, NPMc, DNMT3A/R882H and FLT3/ITD cooperatively induced AML in the mouse model. When only three out of the four mutant genes were transduced, the onset of AML was delayed in any combinations. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. Gene-expression analysis indicated that IDH2/R140Q and NPMc cooperatively activate Hoxa9/Meis1 and hypoxia pathways to maintain AML cells in vivo. These two pathways are likely to be important for the IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. DNMT3A/R882H further upregulated the expression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance of cells in an undifferentiated state. Previous studies have shown that FLT3/ITD promotes cell growth and survival. Taken together, our results suggest that multiple signaling pathways are activated in this IDH-mediated AML system.The tumor-associated mutant IDHs catalyze the formation of an oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-ketoglutarate-dependent dioxygenases that includes epigenetic regulators (TETs and KDM4A), hypoxic signaling molecule (EGLN) and others (collagen prolyl 4-hydroxylases). Because mutant IDHs act via a mechanism that is completely different from those of previously described oncogenes, it has attracted increasing attention as a new therapeutic target. Small molecules that potently and selectively inhibit the mutant IDHs induced differentiation of cancer cells in vitro. However, it remains unclear whether the mutant IDHs are valid targets for cancer therapy in vivo. Here we report that AML harboring IDH2/R140Q can be blocked by conditional deletion of IDH2/R140Q, even after leukemia has developed. Deletion of IDH2/R140Q blocked 2-HG production and the maintenance of Csf-1r+ and c-Kit+ leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Disclosures:No relevant conflicts of interest to declare.

The Structural Motifs for Substrate Binding and Dimerization of the α Subunit of Collagen Prolyl 4-Hydroxylase

Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the proline hydroxylation of procollagen, an essential modification in the maturation of collagens. C-P4H consists of two catalytic α subunits and two protein disulfide isomerase β subunits. The assembly of these subunits is unknown. The α subunit contains an N domain (1-143), a peptide-substrate-binding-domain (PSB, 144-244) and a catalytic domain (245-517). Here, we report the dimeric structure of the N-terminal region (1-244) of the α subunit. It is shown that the N domain has an important role in the assembly of the C-P4H tetramer, by forming an extended four-helix bundle that includes an antiparallel coiled-coil dimerization motif between the two α subunits. Complexes of this construct with a C-P4H inhibitor and substrate show the mode of peptide-binding to the PSB domain. Both peptides adopt a poly-(L)-proline-type-II helix conformation and bind in a curved, asymmetric groove lined by conserved tyrosines and an Arg-Asp salt bridge.

RETRACTED: Collagen prolyl hydroxylases are essential for breast cancer metastasis.

The presence of hypoxia and fibrosis within the primary tumor are two major risk factors for metastasis of human breast cancer. In this study, we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Finally, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies and show that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer.

Bioavailable affinity label for collagen prolyl 4-hydroxylase

Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the nonheme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases.

Abstract 3937: Collagen prolyl hydroxylases are essential for breast cancer metastasis.

Abstract Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies to treat cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced cancer cell invasion and metastasis to lymph nodes and lungs. We establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies and evaluate the novel use of ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, which decreases tumor fibrosis and metastasis in a mouse model of breast cancer. Citation Format: Daniele M. Gilkes, Pallavi Chaturvedi, Saumendra Bajpai, Maimon Hubbi, Denis Wirtz, Gregg Semenza. Collagen prolyl hydroxylases are essential for breast cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3937. doi:10.1158/1538-7445.AM2013-3937