Johanna Myllyharju

Collagens and collagen-related diseases

The collagen superfamily of proteins plays a dominant role in maintaining the integrity of various tissues and also has a number of other important functions. The superfamily now includes more than 20 collagen types with altogether at least 38 distinct polypeptide chains, and more than 15 additional proteins that have collagen-like domains. Most collagens form polymeric assemblies, such as fibrils, networks and filaments, and the superfamily can be divided into several families based on these assemblies and other features. All collagens also contain noncollagenous domains, and many of these have important functions that are distinct from those of the collagen domains. Major interest has been focused on endostatin, a fragment released from type XVIII collagen, which potently inhibits angiogenesis and tumour growth. Collagen synthesis requires eight specific post-translational enzymes, some of which are attractive targets for the development of drugs to inhibit collagen accumulation in fibrotic diseases. The critical roles of collagens have been clearly illustrated by the wide spectrum of diseases caused by the more than 1000 mutations that have thus far been identified in 22 genes for 12 out of the more than 20 collagen types. These diseases include osteogenesis imperfecta, many chondrodysplasias, several subtypes of the Ehlers-Danlos syndrome, Alport syndrome, Bethlem myopathy, certain subtypes of epidermolysis bullosa, Knobloch syndrome and also some cases of osteoporosis, arterial aneurysms, osteoarthrosis, and intervertebral disc disease. The characterization of mutations in additional collagen genes will probably add further diseases to this list. Mice with genetically engineered collagen mutations have proved valuable for defining the functions of various collagens and for studying many aspects of the related diseases.

Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF

The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by two oxygen-dependent events that are catalyzed by three HIF prolyl 4-hydroxylases (HIF-P4Hs) and one HIF asparaginyl hydroxylase (FIH). We have studied possible links between metabolic pathways and HIF hydroxylases by analyzing the abilities of citric acid cycle intermediates to inhibit purified human HIF-P4Hs and FIH. Fumarate and succinate were identified as in vitro inhibitors of all three HIF-P4Hs, fumarate having Ki values of 50–80 μm and succinate 350–460 μm, whereas neither inhibited FIH. Oxaloacetate was an additional inhibitor of all three HIF-P4Hs with Ki values of 400–1000 μm and citrate of HIF-P4H-3, citrate being the most effective inhibitor of FIH with a Ki of 110 μm. Culturing of cells with fumarate diethyl or dimethyl ester, or a high concentration of monoethyl ester, stabilized HIF-1α and increased production of vascular endothelial growth factor and erythropoietin. Similar, although much smaller, changes were found in cultured fibroblasts from a patient with fumarate hydratase (FH) deficiency and upon silencing FH using small interfering RNA. No such effects were seen upon culturing of cells with succinate diethyl or dimethyl ester. As FIH was not inhibited by fumarate, our data indicate that the transcriptional activity of HIF is quite high even when binding of the coactivator p300 is prevented. Our data also support recent suggestions that the increased fumarate and succinate levels present in the FH and succinate dehydrogenase-deficient tumors, respectively, can inhibit the HIF-P4Hs with consequent stabilization of HIF-αs and effects on tumor pathology.

Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis.

The collagen prolyl 4-hydroxylases (P4Hs), enzymes residing within the endoplasmic reticulum, have a central role in the biosynthesis of collagens. In addition, cytoplasmic P4Hs play a critical role in the regulation of the hypoxia-inducible transcription factor HIFalpha. Collagen and HIF P4Hs constitute enzyme families as several isoenzymes have been identified. Two catalytic alpha subunit isoforms have been cloned and characterized for collagen P4Hs from vertebrates, both of them assembling into alpha(2)beta(2) P4H tetramers in which protein disulfide isomerase (PDI) acts as the beta subunit. The catalytic properties of the two isoenzymes are very similar, but distinct differences are found in the binding properties of peptide substrates and inhibitors, and major differences are seen in the expression patterns of the isoenzymes. The nematode Caenorhabditis elegans has five P4H alpha subunit isoforms, PHY1-PHY5. The C. elegans PHY1 and PHY2, together with PDI, are expressed in the collagen synthesizing hypodermal cells and three P4H forms are assembled from them, a PHY-1/PHY-2/PDI(2) mixed tetramer and PHY-1/PDI and PHY-2/PDI dimers. The mixed tetramer is the main P4H form in wild-type C. elegans. PHY-3 is much shorter than PHY-1 and PHY-2, has a unique expression pattern, and is most likely involved in the synthesis of collagens in early embryos. The genome of Drosophila melanogaster contains approximately 20 P4H alpha subunit-related genes, and that of Arabidopsis thaliana six. One A. thaliana P4H has been cloned and shown to be a soluble monomer with several unexpected properties. It effectively hydroxylates poly(L-proline), (Pro-Pro-Gly)(10) and many other proline-containing peptides.

Prolyl 4-hydroxylases and their protein disulfide isomerase subunit

Prolyl 4-hydroxylases (EC 1.14,11.2) catalyze the formation of 4-hydroxyproline in collagens and other proteins with collagen-like sequences. The vertebrate type I and type II enzymes are [alpha (I)]2 beta 2 and [alpha (II)]2 beta 2 tetramers, respectively, whereas the enzyme from the nematode Caenorhabditis elegans is an alpha beta dimer. The type I enzyme is the major form in most but not all vertebrate tissues. The catalytic properties of the various enzyme forms are highly similar, but there are distinct, although small, differences in K(m) values for various peptide substrates between the enzyme forms and major differences in Ki values for the competitive inhibitor, poly(L-proline). Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. Kinetic studies and theoretical considerations have led to elucidation of the reaction mechanism, and recent extensive site-directed mutagenesis studies have identified five critical residues at the cosubstrate binding sites. A number of compounds have been characterized that inhibit it competitively with respect to some of the cosubstrates, and three groups of suicide inactivators have also been identified. The beta subunit in all forms of prolyl 4-hydroxylase is identical to protein disulfide isomerase (PDI), a multifunctional polypeptide that also serves as a subunit in the microsomal triglyceride transfer protein, as a chaperone-like polypeptide that probably assists folding of a number of newly synthesized proteins, and in several other functions. The main role of the PDI polypeptide as a protein subunit is probably related to its chaperone function. Recent expression studies of recombinant human prolyl 4-hydroxylase subunits in a yeast have indicated that the formation of a stable enzyme tetramer in vivo requires coexpression of collagen polypeptide chains.

Prolyl 4-hydroxylation regulates Argonaute 2 stability

Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference. Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies. However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the α-(P4H-α(I)) and β-(P4H-β) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-α(I) or P4H-β by short hairpin RNA and P4H-α(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

Lysyl oxidases, a family comprising LOX and four LOX-like enzymes, catalyze crosslinking of elastin and collagens. Mouse Lox was recently shown to be crucial for development of the cardiovascular system because null mice died perinatally of aortic aneurysms and cardiovascular dysfunction. We show here that Lox is also essential for development of the respiratory system and the integrity of elastic and collagen fibers in the lungs and skin. The lungs of E18.5 Lox(-/-) embryos showed impaired development of the distal and proximal airways. Elastic fibers in E18.5 Lox(-/-) lungs were markedly less intensely stained and more disperse than in the wild type, especially in the mesenchyme surrounding the distal airways, bronchioles, bronchi, and trachea, and were fragmented in pulmonary arterial walls. The organization of individual collagen fibers into tight bundles was likewise abnormal. Similar elastic and collagen fiber abnormalities were seen in the skin. Lysyl oxidase activity in cultured Lox(-/-) skin fibroblasts and aortic smooth muscle cells was reduced by approximately 80%, indicating that Lox is the main isoenzyme in these cells. LOX abnormalities may thus be critical for the pathogenesis of several common diseases, including pulmonary, skin, and cardiovascular disorders.

Characterization of the iron‐ and 2‐oxoglutarate‐binding sites of human prolyl 4‐hydroxylase

Prolyl 4‐hydroxylase (EC 1.14.11.2), an α2β2 tetramer, catalyzes the formation of 4‐hydroxyproline in collagens. We converted 16 residues in the human α subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild‐type β subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the Km for Fe2+ 15‐fold and that for 2‐oxoglutarate 5‐fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the Km for 2‐oxoglutarate 15‐fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85‐95%; all these mutations increased the Km for 2‐oxoglutarate 2‐ to 3‐fold and enhanced the rate of uncoupled decarboxylation of 2‐oxoglutarate as a percentage of the rate of the complete reaction up to 12‐fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C‐5 carboxyl group of 2‐oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C‐1 carboxyl group of 2‐oxoglutarate and the decarboxylation of this cosubstrate.

Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway

Hypoxia‐inducible transcription factor (HIF) is regulated by two oxygen‐dependent events that are catalyzed by the HIF prolyl 4‐hydroxylases (HIF‐P4Hs) and HIF asparaginyl hydroxylase (FIH). We have purified the three recombinant human HIF‐P4Hs to near homogeneity and characterized their catalytic properties and inhibition and those of FIH. The specific activities of the HIF‐P4Hs were at least 40–50 mol/mol/min, and they and FIH catalyzed an uncoupled decarboxylation of 2‐oxoglutarate in the absence of any peptide substrate. The purified HIF‐P4Hs showed considerable activities even without added Fe2+, their apparent Km values for iron being markedly lower than that of FIH. Desferrioxamine and several metals were effective inhibitors of FIH, but surprisingly, ineffective inhibitors of the HIF‐P4Hs in vitro, especially of HIF‐P4H‐2. Desferrioxamine and cobalt were more effective in cultured insect cells synthesizing recombinant HIF‐P4H‐2, but complete inhibition was not achieved and most of the enzyme was inactivated irreversibly. Cobalt also rapidly inactivated HIF‐P4Hs during storage at 4°C. The well‐known stabilization of HIF‐α by cobalt and nickel is thus not due to a simple competitive inhibition of HIF‐P4Hs. The effective inhibition of FIH by these metals and zinc probably leads to full transcriptional activity of HIF‐α even in concentrations that produce no stabilization of HIF‐α.

Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase

Prolyl 4‐hydroxylase, the key enzyme of collagen synthesis, is an α2β2 tetramer, the β subunit of which is protein disulfide isomerase (PDI). Coexpression of the human α subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre‐pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10‐fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4‐hydroxyproline‐containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47‐like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4‐hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high‐level production of various recombinant collagens for numerous scientific and medical purposes.

Prolyl 4-hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets

Prolyl 4-hydroxylases (P4Hs) have central roles in the synthesis of collagens and the regulation of oxygen homeostasis. The 4-hydroxyproline residues generated by the endoplasmic reticulum (ER) luminal collagen P4Hs (C-P4Hs) are essential for the stability of the collagen triple helix. Vertebrate C-P4Hs are α2β2 tetramers with three isoenzymes differing in their catalytic α subunits. Another P4H family, the HIF-P4Hs, hydroxylates specific prolines in the α subunit of the hypoxia-inducible transcription factor (HIF), a master regulator of hypoxia-inducible genes, and controls its stability in an oxygen-dependent manner. The HIF-P4Hs are cytoplasmic and nuclear enzymes, likewise with three isoenzymes in vertebrates. A third vertebrate P4H type is an ER transmembrane protein that can act on HIF-α but not on collagens. All P4Hs require Fe2+, 2-oxoglutarate, O2, and ascorbate. C-P4Hs are regarded as attractive targets for pharmacological inhibition to control excessive collagen accumulation in fibrotic diseases and severe scarring, while HIF-P4H inhibitors are believed to have beneficial effects in the treatment of diseases such as myocardial infarction, stroke, peripheral vascular disease, diabetes, and severe anemias. Studies with P4H inhibitors in various animal models of fibrosis, anemia, and ischemia and ongoing clinical trials with HIF-P4H inhibitors support this hypothesis by demonstrating efficacy in many applications.