101 research outputs found
Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits
The collagen prolyl 4-hydroxylases (P4Hs) are
essential for proper extracellular matrix
formation in multicellular organisms. The
vertebrate enzymes are α2β2 tetramers, in
which the β subunits are identical to protein
disulfide isomerase (PDI). Unique P4H forms
have been shown to assemble from the
<i>Caenorhabditis</i> <i>elegans</i> catalytic α subunit
isoforms PHY-1 and PHY-2 and the β subunit
PDI-2. A mixed PHY-1/PHY-2/(PDI-2)<sub>2</sub>
tetramer is the major form, while PHY-1/PDI-
2 and PHY-2/PDI-2 dimers are also assembled
but less efficiently. Cloning and
characterization of the orthologous subunits
from the closely related nematode
<i>Caenorhabditis</i> <i>briggsae</i> revealed distinct
differences in the assembly of active P4H
forms in spite of the extremely high amino
acid sequence identity (92-97%) between the
<i>C. briggsae</i> and <i>C. elegans</i> subunits. In
addition to a PHY-1/PHY-2(PDI-2)<sub>2</sub> tetramer
and a PHY-1/PDI-2 dimer, an active (PHY-
2)<sub>2</sub>(PDI-2)<sub>2</sub> tetramer was formed in <i>C.
briggsae</i> instead of a PHY-2/PDI-2 dimer.
Site-directed mutagenesis studies and
generation of inter-species hybrid polypeptides
showed that the N-terminal halves of the
<i>Caenorhabditis</i> PHY-2 polypeptides
determine their assembly properties. Genetic
disruption of <i>C. briggsae phy-1</i> (<i>Cb-dpy-18</i>)
via a <i>Mos1</i> insertion resulted a small (short)
phenotype that is less severe than the dumpy
(short and fat) phenotype of the corresponding
<i>C. elegans</i> mutants (<i>Ce-dpy-18</i>). <i>C. briggsae</i>
<i>phy-2</i> RNA interference produced no visible
phenotype in the wild type nematodes but
produced a severe dumpy phenotype and larval
arrest in <i>phy-1</i> mutants. Genetic
complementation of the <i>C. briggsae</i> and <i>C.
elegans</i> <i>phy-1</i> mutants was achieved by
injection of a wild type <i>phy-1</i> gene from either
species
Prolyl 4-hydroxlase activity is essential for development and cuticle formation in the human infective parasitic nematode Brugia malayi
Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the triple helix. C-P4Hs are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here we characterise this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, while biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible, inter-subunit cross-link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control
Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase
Background: We describe a method for specific, quantitative and quick detection of human collagen prolyl 4- hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.
Results: We applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase®) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.
Conclusions: Here we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits
Structure of transmembrane prolyl 4-hydroxylase reveals unique organization of EF and dioxygenase domains
Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a third P4H—the poorly characterized endoplasmic reticulum–localized transmembrane prolyl 4-hydroxylase (P4H-TM)—is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome, but how these variants might contribute to disease is unknown. Here, we explored this question in a structural and functional analysis of soluble human P4H-TM. The crystal structure revealed an EF domain with two Ca2+-binding motifs inserted within the catalytic domain. A substrate-binding groove was formed between the EF domain and the conserved core of the catalytic domain. The proximity of the EF domain to the active site suggests that Ca2+ binding is relevant to the catalytic activity. Functional analysis demonstrated that Ca2+-binding affinity of P4H-TM is within the range of physiological Ca2+ concentration in the endoplasmic reticulum. P4H-TM was found both as a monomer and a dimer in the solution, but the monomer–dimer equilibrium was not regulated by Ca2+. The catalytic site contained bound Fe2+ and N-oxalylglycine, which is an analogue of the cosubstrate 2-oxoglutarate. Comparison with homologous P4H structures complexed with peptide substrates showed that the substrate-interacting residues and the lid structure that folds over the substrate are conserved in P4H-TM, whereas the extensive loop structures that surround the substrate-binding groove, generating a negative surface potential, are different. Analysis of the structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with other P4Hs while having a unique EF domain.publishedVersio
Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system
<p>Abstract</p> <p>Background</p> <p>Here we describe a new technical solution for optimization of <it>Pichia pastoris </it>shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in <it>P. pastoris </it>is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in <it>P. pastoris </it>is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.</p> <p>Results</p> <p>By applying on-line pO<sub>2 </sub>monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles.</p> <p>By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.</p> <p>Conclusion</p> <p>The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.</p
BAMBI is a novel HIF1-dependent modulator of TGF beta-mediated disruption of cell polarity during hypoxia
Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of similar to 300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation. Here, we report that hypoxia disrupts polarized epithelial morphogenesis of MDCK cells in a HIF1 alpha-dependent manner by modulating the transforming growth factor-beta (TGF beta) signaling pathway. Analysis of potential HIF1 targets in the TGF beta pathway identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transmembrane glycoprotein related to the type I receptors of the TGF beta family, whose expression was essentially lost in HIF1-depleted cells. Similar to what was observed in HIF1-deficient cells, BAMBI-depleted cells failed to efficiently activate TGF beta signaling and retained epithelial polarity during hypoxia. Taken together, we show that hypoxic conditions promote TGF beta signaling in a HIF1-dependent manner and BAMBI is identified in this pathway as a novel HIF1-regulated gene that contributes to hypoxia-induced loss of epithelial polarity.Peer reviewe
Inactivation of mouse transmembrane prolyl 4-hydroxylase increases blood brain barrier permeability and ischemia-induced cerebral neuroinflammation
Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of > 300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A trans membrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm(-/- )mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflam-matory microgliosis and neutrophil infiltration was observed in the P4htm-/- cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm(-/-) mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.Peer reviewe
HIF-P4H-2 inhibition enhances intestinal fructose metabolism and induces thermogenesis protecting against NAFLD
Non-alcoholic fatty liver disease (NAFLD) parallels the global obesity epidemic with unmet therapeutic needs. We investigated whether inhibition of hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2), a key cellular oxygen sensor whose inhibition stabilizes HIF, would protect from NAFLD by subjecting HIF-P4H-2-deficient (Hif-p4h-2(gt/gt)) mice to a high-fat, high-fructose (HFHF) or high-fat, methionine-choline-deficient (HF-MCD) diet. On both diets, the Hif-p4h-2(gt/gt) mice gained less weight and had less white adipose tissue (WAT) and its inflammation, lower serum cholesterol levels, and lighter livers with less steatosis and lower serum ALT levels than the wild type (WT). The intake of fructose in majority of the Hif-p4h-2(gt/gt) tissues, including the liver, was 15-35% less than in the WT. We found upregulation of the key fructose transporter and metabolizing enzyme mRNAs, Slc2a2, Khka, and Khkc, and higher ketohexokinase activity in the Hif-p4h-2(gt/gt) small intestine relative to the WT, suggesting enhanced metabolism of fructose in the former. On the HF-MCD diet, the Hif-p4h-2(gt/gt) mice showed more browning of the WAT and increased thermogenesis. A pharmacological pan-HIF-P4H inhibitor protected WT mice on both diets against obesity, metabolic dysfunction, and liver damage. These data suggest that HIF-P4H-2 inhibition could be studied as a novel, comprehensive treatment strategy for NAFLD. Key messages center dot HIF-P4H-2 inhibition enhances intestinal fructose metabolism protecting the liver. center dot HIF-P4H-2 inhibition downregulates hepatic lipogenesis. center dot Induced browning of WAT and increased thermogenesis can also mediate protection. center dot HIF-P4H-2 inhibition offers a novel, comprehensive treatment strategy for NAFLD.Peer reviewe
Inactivation of mouse transmembrane prolyl 4-hydroxylase increases blood brain barrier permeability and ischemia-induced cerebral neuroinflammation
Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of > 300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A trans membrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm(-/- )mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflam-matory microgliosis and neutrophil infiltration was observed in the P4htm-/- cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm(-/-) mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.Peer reviewe
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