Hydroxylation Of Proline Residues Research Articles

Effects of dietary collagen cofactors and hydroxyproline on the growth performance, textural properties and collagen deposition in swim bladder of Nibea coibor based on orthogonal array analysis

The global demand for collagen is continuously growing over years. Nutritional regulation of quality and collagen deposition in the swim bladder, an ideal collagen source of marine product, is a promising method for improving the economic value of fish. In this study, to enhance growth efficiency and collagen deposition in swim bladder of an important economic marine fish, the chu’s croaker (Nibea coibor), a L9(34) orthogonal array was designed with three levels for each factor, which are hydroxyproline (Hyp) and the key cofactors for proline hydroxylation during collagen post-translation modification, such as A1-A3 (Hyp): 0, 10 and 20 g/kg; B1-B3 (vitamin C, VC): 0, 0.20 and 0.40 g/kg; C1-C3 (Fe2+): 0, 0.20 and 0.40 g/kg; D1-D3 (α-ketoglutarate, AKG): 0, 10 and 20 g/kg, to formulate the trial feeds. An 8-week feed trial was carried out on 405 fish (initial weight 269.04 ± 3.18 g) which were randomly distributed into the nine treatment groups with three replicates of 15 fish per cage. Results indicated that appropriate increase of dietary Hyp and VC level is more beneficial to collagen deposition and textural properties in swim bladder, while Hyp and AKG exerted more significant effects on the growth performance. Gene expression analysis of collagen metabolism indicated that Hyp mainly improved the synthesis of type I collagen and inhibited collagen degradation, while VC and AKG mainly play an important role in hydroxylation of proline residues on collagen. The best combination for growth performance and collagen deposition in swim bladder were suggested to be A2B2C1D3 and A2B2C1D1, respectively. The results demonstrated that appropriate dietary supplementation of Hyp and cofactors can improve growth and collagen production of swim bladder. Our result would provide reference data for nutritional regulation of the species and optimization of the commercial feed to provide fish products with high quality.

Mechanism of HIF1-α-mediated regulation of Tribolium castaneum metabolism under high CO2 concentration elucidated

HIF1-α is an inducer that plays an important role in the regulation of hypoxia stress in insects in the way of regulating the corresponding enzyme activity which depends on the oxygen concentration through the hydroxylation of proline residues. In this study, we silenced HIF1-α expression in the model insect Tribolium castaneum under a hypoxic environment to explore its role in regulating metabolism in T. castaneum. We found that ATP and NAD + contents decreased significantly after HIF1-α interference at normoxic conditions, while activity of the soluble trehalase increased. The expression of TRE1-2, TRE1-3, TRE2, and TPS genes in the trehalose metabolism pathway were highly enhanced which are compared with normoxic conditions. Meanwhile, the expression of InR2 was significantly up-regulated, both of which play an important role in metabolism. We speculate that the enhancement of the insulin metabolic pathway may lead to enhanced glycolysis. We propose that the HIF1-α gene effectively regulates metabolism for T. castaneum to adapt to high CO2 concentrations in the environment. Our results provide a solid theoretical basis for controlling stored-grain pests incidence using high CO2 treatment.

Collagen prolyl 4-hydroxylases modify tumor progression.

Collagen is the main component of the extracellular matrix. Hydroxylation of proline residues on collagen, catalyzed by collagen prolyl 4-hydroxylase (C-P4H), is essential for the stability of the collagen triple helix. Vertebrate C-P4H is an α2β2 tetramer with three isoenzymes differing in the catalytic α-subunits, which are encoded by P4HA1, P4HA2, and P4HA3 genes. In contrast, β-subunit is encoded by a single gene P4HB. The expressions of P4HAs and P4HB are regulated by multiple cellular factors, including cytokines, transcription factors, and microRNAs. P4HAs and P4HB are highly expressed in many tumors and participate in cancer progression. Several inhibitors of P4HAs and P4HB have been confirmed to have anti-tumor effects, suggesting that targeting C-P4H is a feasible strategy for cancer treatment. Here, we summarize recent progresses on the function and expression of regulatory mechanisms of C-P4H in cancer progression and point out the potential development of therapeutic strategies in targeting C-P4H in the future.

Identification of Hydroxyproline-Containing Proteins and Hydroxylation of Proline Residues in Rice.

The hydroxyproline-containing proteins (HCPs) among secretory and vacuolar proteins play important roles in growth and development of higher plants. Many hydroxyproline-rich glycoproteins (HRGPs), including Arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs), are identified as HCPs by bioinformatics approaches. The experimental evidence for validation of novel proline hydroxylation sites is vital for understanding their functional roles. In this study, the 62 HCPs containing 114 hydroxyproline (O, Hyp) residues were identified, and it was found that hydroxylation of proline residues in the HCPs could either constitute attachment sites for glycans or have other biological function in rice. The glycomodules of AO, OA, OG, VO, LO, and OE were abundant in the 62 HCPs. Further analysis showed that the 22 of 62 HCPs contained both signal peptides and transmembrane domains, and the 19 HCPs only contained transmembrane domains, while 21 HCPs contained neither. This study indicated the feasibility of mass spectrometry-based proteomics combined with bioinformatics approaches for the large-scale characterization of Hyp sites from complex protein digest mixtures. Furthermore, the expression of AGPs in rice was detected by using β-GlcY reagent and JIM13 antibody. The results displayed that the AGPs were widely distributed in different tissues and organs of rice, especially expressed highly in lateral root, pollen and embryo. In conclusion, our study revealed that the HCPs and Hyp residues in rice were ubiquitous and that these Hyps could be candidates for linking to glycans, which laid the foundation for further studying the functions of HCPs and hydroxylation of proline residues in rice.

Intrabody against prolyl hydroxylase 2 ameliorates acetaminophen-induced acute liver injury in mice via concomitant promotion of angiogenesis and redox homeostasis

Acetaminophen (APAP) overdose has become the most common cause of drug-induced acute liver failure. Angiogenesis and redox homeostasis play an important role in liver protection and repair of APAP-induced acute liver injury (AILI). Hypoxia inducible factor-1 (HIF-1) is a transcription factor that plays a crucial role in regulating the expression of genes associated with angiogenesis, redox homeostasis and energy balance. Prolyl hydroxylase 2 (PHD2) predominantly hydroxylates proline residues in HIF-1α to promote its degradation. In our previous study, we reported an intrabody against PHD2 (ER-INP) that enhances angiogenesis by blocking PHD2 activity to increase HIF-1α abundance and activity. The present study was designed to explore the role and possible mechanisms of ER-INP in AILI in mice. Mice were pretreated intravenously with ER-INP before intraperitoneal injection of APAP to induce AILI. The results showed that pretreatment with ER-INP dramatically decreased the high ALT and AST activities and significantly ameliorated the centrilobular necrosis induced by APAP administration. ER-INP expression promoted angiogenesis in vivo by upregulating the mRNA and protein levels of HIF-1α target genes. Meanwhile, ER-INP pretreatment restored redox homeostasis, verified by reinforcement of PRDX4 activity and suppression of GSH depletion. This study demonstrated that ER-INP protects against AILI in part by increasing angiogenesis and maintaining redox homeostasis. These results indicate that ER-INP may provide a potential liver protection strategy against AILI in the future.

Identification and analysis of characteristic tryptic peptides from porcine gelatin extracted with multi-stage batch processing

The extraction process significantly affects the characteristics and identification of gelatins. Here, three porcine gelatins were extracted using multi-stage batch processing by three extraction stages with temperatures of 50, 70, and 90 °C, respectively. Liquid chromatography-high-resolution mass spectrometry was used to investigate differences in the characteristic tryptic peptides derived from three porcine gelatins. Identification by accurate mass and tandem mass spectrometry yielded 97, 88, and 58 characteristic tryptic peptides from porcine gelatin extracted in the first, second, and third stages, respectively. Among these, 46 characteristic tryptic peptides were detected in all porcine gelatin samples. A comparison was then made of gelatins extracted by one-stage processing and those from multi-stage batch processing. Two methods included different extraction times and condition sets, as well as produced various gelatins. These analyses yielded 35 characteristic tryptic peptides, including 8 unmodified peptides from all porcine gelatins recovered from the two extraction methods. Moreover, 27 characteristic peptides showed various modifications with hydroxylation of proline residues. The resultant marker peptides can be highly useful in the accurate identification of porcine gelatins in industrial settings.

Abstract 851: MicroRNA-124 modulated collagen-prolyl hydroxylase P4HA1 expression regulates colon cancer progression

Abstract In the US, colon adenocarcinoma is the third most common cause of cancer-related deaths. Since various genetic and epigenetic alterations contribute to progression of this disease, it is necessary to evaluate these molecular events as markers of progression and as potential druggable targets to develop therapeutics. Analysis of publicly available TCGA transcriptome sequencing data using UALCAN, a bioinformatics web portal (http://ualcan.path.uab.edu/), identified in colon adenocarcinoma, overexpression of collagen prolyl-4-hydroxylase α subunit 1 (P4HA1), an enzyme involved in collagen metabolism. P4HA1 changes the composition of the extra cellular matrix by hydroxylating proline residues in collagen. We validated this in silico finding by measuring P4HA1 RNA expression levels in paired normal (n=105) and tumor (n=105) frozen samples by qRT-PCR. Our investigations showed overexpression of P4HA1 in colon cancers compared to their paired normal colonic epithelial tissues. Expression of P4HA1 was independent of pathologic stage; tumor histologic type; and the race/ethnicity, age, and gender of patients. Furthermore, Western blotting and immunohistochemical analyses showed elevated expression of P4HA1 protein in colon tumor tissues as compared to normal colonic epithelial specimens. To assess the functional role of P4HA1 in colon cancer cells, we performed in vitro and in vivo analyses using RNA interference. The results showed that depletion of P4HA1 led to reduced cell growth and cell invasion, and to less tumor growth in xenograft models. Bioinformatics analysis indicated that microRNA miR-124, a tumor suppressor, could be targeting P4HA1. Forced overexpression of miR-124 in colon cancer cells reduced P4HA1 expression and diminished the malignant phenotypes of colon cancer cells, confirming that, in these cells, miR-124 targeted P4HA1. Further, treatment of the cells with diethyl-pythiDC (100 uM), a small molecule inhibitor of P4HA1, reduced their malignant phenotypes. In sum, the present investigations show that overexpression of P4HA1 is involved in progression of colon adenocarcinomas and that it is a potential therapeutic target that is amenable to small molecule inhibition. This work was partially supported by a NIH grant (3U54CA118948) and by the ELKUS foundation grant to Dr. Manne. Citation Format: Sumit Agarwal, Balabhadrapatruni V. Chakravarthi, Michael Behring, Hyung-Gyoon Kim, Kevin Hale, Abduljalil M. Alsubaie, Sameer Al Diffalha, Shajan P. Sugandha, Sooryanarayana Varambally, Upender Manne. MicroRNA-124 modulated collagen-prolyl hydroxylase P4HA1 expression regulates colon cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 851.

IHyd-PseAAC (EPSV): Identifying Hydroxylation Sites in Proteins by Extracting Enhanced Position and Sequence Variant Feature via Chou's 5-Step Rule and General Pseudo Amino Acid Composition.

BackgroundIn various biological processes and cell functions, Post Translational Modifications (PTMs) bear critical significance. Hydroxylation of proline residue is one kind of PTM, which occurs following protein synthesis. The experimental determination of hydroxyproline sites in an uncharacterized protein sequence requires extensive, time-consuming and expensive tests.MethodsWith the torrential slide of protein sequences produced in the post-genomic age, certain remarkable computational strategies are desired to overwhelm the issue. Keeping in view the composition and sequence order effect within polypeptide chains, an innovative in-silico> predictor via a mathematical model is proposed.ResultsLater, it was stringently verified using self-consistency, cross-validation and jackknife tests on benchmark datasets. It was established after a rigorous jackknife test that the new predictor values are superior to the values predicted by previous methodologies.ConclusionThis new mathematical technique is the most appropriate and encouraging as compared with the existing models.

Spider silk proteome provides insight into the structural characterization of Nephila clavipes flagelliform spidroin

The capture spiral of web from N. clavipes spider consists of a single type of spidroin - the flagelliform silk protein, a natural material representing a combination of strength and high elasticity. Flagelliform spider silk is the most extensible silk fibre produced by orb weaver spiders and the structure of this remarkable material is still largely unknown. In the present study we used a proteomic approach to elucidate the complete sequence and the post-translational modifications of flagelliform silk proteins. The long sequence of flagelliform silk protein presents 45 hydroxylated proline residues, which may contribute to explain the mechanoelastic property of these fibres, since they are located in the GPGGX motif. The 3D-structure of the protein was modelled considering the three domains together, i.e., the N- and C-terminal non-repetitive domains, and the central repetitive domain. In the resulting molecular model there is a predominance of random structures in the solid fibres of the silk protein. The N-terminal domain is composed of three α-helices and the C-terminal domain is composed of one small helical section. Proteomic data reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based spider polymers.

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha-L-iduronidase.

SummaryThe Brassica rapa hairy root based expression platform, a turnip hairy root based expression system, is able to produce human complex glycoproteins such as the alpha—L—iduronidase (IDUA) with an activity similar to the one produced by Chinese Hamster Ovary (CHO) cells. In this article, a particular attention has been paid to the N‐ and O‐glycosylation that characterize the alpha‐L‐iduronidase produced using this hairy root based system. This analysis showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility. Indeed, on each of the 6 N‐glycosylation sites of the IDUA, a single N‐glycan composed of a core Man3GlcNAc2 carrying one beta(1,2)‐xylose and one alpha(1,3)‐fucose epitope (M3XFGN2) was identified, highlighting the high homogeneity of the production system. Hydroxylation of proline residues and arabinosylation were identified during O‐glycosylation analysis, still with a remarkable reproducibility. This platform is thus positioned as an effective and consistent expression system for the production of human complex therapeutic proteins.

Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.

Understanding How Prolyl-4-hydroxylase Structure Steers a Ferryl Oxidant toward Scission of a Strong C-H Bond.

Prolyl-4-hydroxylase (P4H) is a non-heme iron hydroxylase that regio- and stereospecifically hydroxylates proline residues in a peptide chain into R-4-hydroxyproline, which is essential for collagen cross-linking purposes in the human body. Surprisingly, in P4H, a strong aliphatic C-H bond is activated, while thermodynamically much weaker aliphatic C-H groups, that is, at the C3 and C5 positions, are untouched. Little is known on the origins of the high regio- and stereoselectivity of P4H and many non-heme and heme enzymes in general, and insight into this matter may be relevant to Biotechnology as well as Drug Development. The active site of the protein contains two aromatic residues (Tyr140 and Trp243) that we expected to be crucial for guiding the regioselectivity of the reaction. We performed a detailed quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) study on wild-type and mutant structures. The work shows that Trp243 is involved in key protein loop-loop interactions that affect the shape and size of the substrate binding pocket and its mutation has major long-range effects. By contrast, the Tyr140 residue is shown to guide the regio- and stereoselectivity by holding the substrate and ferryl oxidant in a specific orientation through hydrogen bonding and π-stacking interactions. Compelling evidence is found that the Tyr140 residue is involved in expelling the product from the binding pocket after the reaction is complete. It is shown that mutations where the hydrogen bonding network that involves the Tyr140 and Trp243 residues is disrupted lead to major changes in folding of the protein and the size and shape of the substrate binding pocket. Specifically, the Trp243 residue positions the amino acid side chains of Arg161 and Glu127 in specific orientations with substrate. As such, the P4H enzyme is a carefully designed protein with a subtle and rigid secondary structure that enables the binding of substrate, guides the regioselectivity, and expels product efficiently.

Abstract 5942: Collagen proly-hydroxylase regulates breast cancer progression and chemoresistance

Abstract Collagen prolyl hydroxylation (P4H), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. Upregulation of collagen prolyl-4-hydroxylase in cancer cells is required for breast cancer promotion. P4HA1 mRNA levels were significantly upregulated in triple-negative breast cancer (TNBC) compared to other cancer subtypes. Increased mRNA levels of P4HA1 correlated with poor clinical outcome in breast cancer patients. Silencing P4HA1 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen. We found that activation of the P4HA1 in TNBC cells enhanced cancer cell stemness. We also found that knockdown of P4HA1 inhibited mammary tumor growth and metastasis to lungs in xenograft models. Inhibition of P4HA1 activity with the P4H inhibitor sensitized TNBC to the docetaxel treatment in the patient-derived organoid (PDO) and xenograft models. Therefore, these results suggest the crucial role of P4HA1 in breast cancer progression and that targeting collagen P4H is a promising strategy to inhibit tumor progression and to sensitize TNBC to chemotherapy. Citation Format: Gaofeng Xiong, Ren Xu. Collagen proly-hydroxylase regulates breast cancer progression and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5942. doi:10.1158/1538-7445.AM2017-5942

Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition

BackgroundIncreased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated.MethodsGene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis.ResultsUsing gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models.ConclusionThese results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression.

A Novel Prolyl Hydroxylase Inhibitor Protects Against Cell Death After Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.

Quantitative Targeted Absolute Proteomics-Based Large-Scale Quantification of Proline-Hydroxylated α-Fibrinogen in Plasma for Pancreatic Cancer Diagnosis

Pancreatic cancer is a devastating disease and early diagnosis and treatment are essential to improve the prognosis. We previously showed that α-fibrinogen containing hydroxylated proline residues at positions 530 and 565 is increased in plasma of pancreatic cancer patients. However, no antibody specific for hydroxylated proline-530 is available. Therefore, the purposes of this study were to develop a quantification method specific for both proline-hydroxylated α-fibrinogens by selected/multiple reaction monitoring (SRM/MRM), and to validate these modifications as pancreatic cancer markers. The target peptide for hydroxylated proline-530 contained methionine, and since variable partial oxidation of this residue would affect the quantification, hydrogen peroxide treatment was carried out to ensure complete oxidation. Quantification values of modified and unmodified α-fibrinogen were well correlated with those obtained by immunoblotting. Concentrations of modified and unmodified α-fibrinogen were quantified in 70 pancreatic cancer patients and 27 healthy controls. Percent hydroxylation of α-fibrinogen and concentration of hydroxylated α-fibrinogen were significantly greater in the plasma of patients. Furthermore, among 8 carbohydrate antigen 19-9 (CA19-9)-negative patients in stages I/II, 6 were positive for proline-hydroxylated α-fibrinogen. These results indicate that plasma concentration of proline-hydroxylated α-fibrinogen measured by SRM/MRM analysis may be a good pancreatic cancer marker, especially in CA19-9-negative patients.

HIF1α is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development

Collagen production is fundamental for the ontogeny and the phylogeny of all multicellular organisms. It depends on hydroxylation of proline residues, a reaction that uses molecular oxygen as a substrate. This dependency is expected to limit collagen production to oxygenated cells. However, during embryogenesis, cells in different tissues that develop under low oxygen levels must produce this essential protein. In this study, using the growth plate of developing bones as a model system, we identify the transcription factor hypoxia-inducible factor 1 α (HIF1α) as a central component in a mechanism that underlies collagen hydroxylation and secretion by hypoxic cells. We show that Hif1a loss of function in growth plate chondrocytes arrests the secretion of extracellular matrix proteins, including collagen type II. Reduced collagen hydroxylation and endoplasmic reticulum stress induction in Hif1a-depleted cells suggests that HIF1α regulates collagen secretion by mediating its hydroxylation and consequently its folding. We demonstrate in vivo the ability of Hif1α to drive the transcription of collagen prolyl 4-hydroxylase, which catalyzes collagen hydroxylation. We also show that, concurrently, HIF1α maintains cellular levels of oxygen, most likely by controlling the expression of pyruvate dehydrogenase kinase 1, an inhibitor of the tricarboxylic acid cycle. Through this two-armed mechanism, HIF1α acts as a central regulator of collagen production that allows chondrocytes to maintain their function as professional secretory cells in the hypoxic growth plate. As hypoxic conditions occur also during pathological conditions such as cancer, our findings may promote the understanding not only of embryogenesis, but also of pathological processes.

Topical prolyl hydroxylase domain-2 silencing improves diabetic murine wound closure.

Prolyl hydroxylase domain 2 (PHD2) has been implicated in several pathways of cell signaling, most notably in its regulation of hypoxia-inducible factor (HIF)-1α stability. In normoxia, PHD2 hydroxylates proline residues on HIF-1α, rendering it inactive. However, in hypoxia, PHD2 is inactive, HIF-1α is stabilized and downstream effectors such as vascular endothelial growth factor and fibroblast growth factor-2 are produced to promote angiogenesis. In the present study we utilize RNA interference to PHD2 to promote therapeutic angiogenesis in a diabetic wound model, presumably by the stabilization of HIF-1α. Stented wounds were created on the dorsum of diabetic Lepr db/db mice. Mice were treated with PHD2 small interfering RNA (siRNA) or nonsense siRNA. Wounds were measured photometrically on days 0-28. Wounds were harvested for histology, protein, and RNA analysis. Diabetic wounds treated with siRNA closed within 21±1.2 days; sham-treated closed in 28±1.5 days. By day 7, Western blot revealed near complete suppression of PHD protein and corresponding increased HIF-1α. Angiogenic mediators vascular endothelial growth factor and fibroblast growth factor-2 were elevated, corresponding to increased CD31 staining in the treated groups. siRNA-mediated silencing of PHD2 increases HIF-1α and several mediators of angiogenesis. This corresponded to improved time to closure in diabetic wounds compared with sham-treated wounds. These findings suggest that impaired wound healing in diabetes can be ameliorated with therapeutic angiogenesis.

Abstract LB-142: Regulation of MYCN stability by reactive oxygen species in neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in children. Amplification of the MYCN proto-oncogene is associated with older age, rapid tumor progression, and the worst outcome of this disease. As MYCN amplification leads to its over-expression, high-level expression of MYCN is thought to cause an aggressive behavior of MYCN amplified tumors. In fact, the forced reduction of MYCN protein expression by siRNA results in growth suppression and apoptosis of NB cells with MYCN amplification. We have identified several compounds that can rapidly destabilize the MYCN protein (within a few hours), in NB cells and cause growth suppression. The compounds include FCCP, OSU-03012, and Salinomycin. Our recent data suggest that a common effect of the above compounds appears to be the inhibition of mitochondrial functions. Moreover, ascorbic acid, an anti-oxidant, abolishes the effect of these compounds on MYCN protein stability. It is known that inhibition of mitochondrial oxidative phosphorylation increases the production of reactive oxygen species (ROS), which include superoxide (O2-), hydroxyl radical (•OH), and hydrogen peroxide (H2O2). Anti-oxidants are also known to quench ROS. Preliminary data showed that ROS was detected when the IMR5 NB cells were treated with OSU-03012 for three hours. In addition, we have found that forced over-expression of pVHL, an E3 ubiquitin ligase, in NB cell lines results in reduction of MYCN protein levels. We are currently testing whether ROS regulates MYCN stability by hydroxylation of proline residues on MYCN, and whether pVHL recognizes the ROS-modified MYCN, which is then subjected to rapid proteasome-dependent degradation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-142. doi:10.1158/1538-7445.AM2011-LB-142

Crystal Structure of the C-Terminal Domain of the Hypoxia Regulator Ofd1

Sre1, the fission yeast homologue of the mammalian sterol regulatory element binding protein, SREBP, is a hypoxic transcription factor required for sterol homeostasis and low oxygen growth. The level of the N-terminal transcription factor domain of Sre1, Sre1N, is regulated by Ofd1-Nro1 in an oxygen-dependent manner. Ofd1 is a putative prolyl hydroxylase of the 2-oxyoglutarate-Fe(II) dioxygenase protein superfamily. Unlike the prolyl hydroxylase PHD which hydroxylates proline residues in the hypoxia-inducible factor HIF, Ofd1 utilizes its N-terminal dioxygenase domain to control the interaction between itself and its inhibitor Nro1: in hypoxia, Nro1 binds to Ofd1 and this inhibits its C-terminal degradation domain (CTDD) function on Sre1N destabilization. As shown previously, Ofd1CTDD by itself is sufficient to promote Sre1N degradation and there's direct interaction between Nro1 and Ofd1CTDD. However, the molecular mechanism by which Ofd1CTDD accelerates Sre1N degradation remains unclear. Here we report the crystal structure of Ofd1CTDD at 2.0 Å resolution. Interestingly, Ofd1CTDD has a double-stranded beta helix (DBSH) fold like that of the 2-oxyoglutarate-Fe(II) dioxygenase protein superfamily but lacks the iron-binding motif characteristic of the superfamily. This structure may help elucidate how Ofd1CTDD promotes Sre1N turnover and how Ofd1-Nro1 as a whole functions as an oxygen-sensor.