Cupins are a large superfamily of enzymatic and non-enzymatic members that contain a conserved β-barrel domain, or double-stranded β-helix (DSBH) fold. The cupin superfamily is one of the most functionally diverse groups of proteins known to exist. The vast majority of cupins contain a mononuclear metal binding site at the core of the DSBH fold capable of binding different metal ions. One of the largest cupin subfamilies is known as the Fe(II)/α-ketoglutarate (αKG)-dependent dioxygenases. Prolyl 4-hydroxylases (P4Hs) belong to the group of Fe(II)/αKG-dependent dioxygenases and catalyze the formation of 4R-hydroxyproline (Hyp) from various proline-containing substrates. The formation of Hyp is an important post-translational modification to many different proteins involved in essential biochemical pathways. Abnormalities in these pathways can lead to diseases such as cancer, fibrosis, respiratory issues, scurvy, and stroke. An Fe(II)/αKG-dependent prolyl hydroxylase from Bacillus anthracis (BaP4H) was investigated to understand its substrate recognition ability and catalytic properties. Novel crystal structures were solved that revealed conformational changes upon substrate binding and key interactions of various ligands in the active site for different catalytic steps. Although the majority of cupin family enzymes catalyze a reaction using iron as an essential cofactor, other metal cofactors can allow the diverse biological transformations carried out by this group of enzymes. A class of enzymes known as dimethylsulfoniopropionate (DMSP) lyases uses different metal ions to catalyze the formation of acrylate and dimethylsulfide (DMS) from DMSP. DMSP is one of the most prevalent and significant molecules to the life and biogeochemistry of the oceans. The products DMS and acrylate are environmentally significant and industrially valuable. DMSP is predominantly catabolized by marine bacteria and can serve different functions. One of the most abundant bacteria in the ocean, Pelagibacter, was determined to contain a DMSP lyase DddK. The DddK catalyzed DMSP lyase activity in the presence of different metal ions has shown that it catalytically prefers Ni(II) compared to other transition metal ions examined. Spectroscopic, site-directed mutagenesis, and crystallographic studies illustrate central residues responsible for metal ion binding and possible roles in transition state stabilization. A greater mechanistic understanding of DMSP lyases will lead to more impactful information about global environmental climate regulation.

Abstract An efficient system for expressing recombinant human collagens is expected to have numerous scientific and medical applications, but this is difficult to achieve because most systems do not have sufficient levels of activity of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. A recombinant form of human type II collagen, the main structural component of cartilage, was produced here in insect cells by coinfecting them with two baculoviruses, one coding for the proα chains of human type II procollagen, and the other for both the α and β subunits of human prolyl 4-hydroxylase. The amino acid composition of the recombinant form was very similar to that of the non-recombinant protein, with the exception that the hydroxylysine content was very low. The highest expression levels obtained in suspension cultures were 50 mg/l. An additional baculovirus coding for human lysyl hydroxylase was used to express type II collagen with a high hydroxylysine content. Marked differences in the rate of fibril formation in vitro and the morphology of the resulting fibrils were found between the recombinant type II collagens having 2 and 19 hydroxylysine residues/1000 amino acids, the maximal turbidity of the former being reached within 5 min, whereas the absorbance of the latter increased up to about 10 h. In addition, the latter collagen formed thin fibrils, whereas the former produced thick fibrils on a background of thin ones. The data indicate that regulation of the extent of lysine hydroxylation, and consequently of the amounts of hydroxylysine-linked carbohydrate units, may have major effects on collagen fibril formation. In order to study the expression of recombinant human collagens in yeasts, cDNAs for the proα chains of procollagens of type I, II and III were transformed into a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase subunits. All the P. pastoris strains obtained produced full-length proα chains. Cells coexpressing the proα1(I) chains and prolyl 4-hydroxylase produced homotrimeric type I procollagen molecules, whereas cells coexpressing the proα1(I) and proα2(I) chains and prolyl 4-hydroxylase produced heterotrimeric molecules with the correct 2:1 chain ratio. pCα1(I) and pCα2(I) chains lacking the N propeptides assembled into pCcollagen molecules and yielded correctly folded and fully hydroxylated collagen molecules upon pepsinization. The Tm values of recombinant type I-III collagens produced in shaker flasks were about 38°C and the degree of hydroxylation of proline residues was lower than that in the corresponding non-recombinant collagens. When the recombinant collagens were produced in a 2-litre fermentor equipped with an O2 supply system, the expression levels increased markedly to 0.2–0.6 g/l. In addition, all these collagens were identical in 4-hydroxyproline content to the corresponding non-recombinant proteins, and all of them formed native-type fibrils.

Abstract Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline residues in collagens. The vertebrate enzymes are α2β2 tetramers in which the β subunit is identical to protein disulphide isomerase (PDI). Two isoforms of the catalytic α subunit have been identified in vertebrates, forming type I [α(I)]2β2 and type II [α(II)]2β2 collagen prolyl 4-hydroxylase tetramers. This thesis reports on the cloning and characterization of a third vertebrate α subunit isoform, α(III). The recombinant human α(III) isoform associates with PDI to form an active type III collagen prolyl 4-hydroxylase tetramer, and its Km values for the cosubstrates are very similar to those of the type I and II enzymes, those for a peptide substrate and an inhibitor being found to lie between the two. The α(III) mRNA is expressed in all tissues studied but at much lower levels than the α(I) mRNA. A novel mixed tetramer PHY-1/PHY-2/(PDI-2)2 was found to be the main collagen prolyl 4-hydroxylase form produced in the nematode Caenorhabditis elegans in vivo and in vitro. However, mutant nematodes can compensate for the lack of the mixed tetramer by increasing the assembly of PHY-1/PDI-2 and PHY-2/PDI-2 dimers, these forms also being unique. The catalytic properties of the recombinant mixed tetramer were characterized, and it was shown by the analysis of mutant worms that PHY-1 and PHY-2 represent the only catalytic subunits needed for the hydroxylation of cuticular collagens. The roles of the two catalytic sites in a collagen prolyl 4-hydroxylase tetramer were studied by using the C. elegans mixed tetramer and a hybrid C. elegans PHY-1/human PDI dimer. An increase in the chain length of the peptide substrate led to an identical decrease in the Km values in both enzyme forms. It is thus clear that two catalytic sites are not required for efficient hydroxylation of long peptides, and their low Km values most probably result from more effective binding to the peptide-substrate-binding domain. Inactivation of one catalytic site in the mixed tetramer reduced the activity by more than 50%, indicating that the remaining wild-type subunit cannot function fully independently.

Abstract Protein disulfide bonds are covalent links formed between the thiol groups of cysteine residues. In many proteins, they have an important role in stabilizing the three-dimensional conformation of the polypeptide chain. Usually proteins are physiologically active and functional only when they are correctly folded. Protein folding takes place very soon after the synthesis of a new polypeptide chain. Proteins which are to be secreted from the cell fold in a specialized compartment, the endoplasmic reticulum (ER). Folding and disulfide bond formation in the ER does not happen spontaneously, there are proteins which are specialized in assisting in these processes. Protein disulfide isomerase (PDI) is a multifunctional protein, which is capable of catalysing both of disulfide bond formation and folding of a protein. In addition, it has other functions: it is an essential part of two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein. C-P4H is an enzyme essential in the formation of collagens, proteins found in connective tissue. The function of C-P4H is to catalyse the hydroxylation of prolines, which is essential for the structural stability of collagens. C-P4H is a tetramer, formed of two catalytic α subunits and two β subunits, which are identical to PDI. The function of PDI in C-P4H is apparently to keep it in a soluble, functionally active conformation. In mammals there are several proteins similar to PDI, together forming a PDI family of proteins. They share both structural and functional similarities. One of these proteins is ERp57. It is specialized in assisting in the folding and disulfide bond formation of glycoproteins. PDI consists of four domains, two of which contain a catalytic site for disulfide bond formation. One domain is the main site of interaction with other proteins and one domain is of unknown function. In this study, the role of these domains in the activities of PDI was investigated. The peptide-binding domain was characterized in detail. In addition, structural similarities of PDI and ERp57 were studied by formation of hybrid proteins containing domains of both and comparing the activities of these recombinant proteins to those of PDI.

No description available.

The ability of cells to sense oxygen is a highly evolved process that facilitates adaptations to the local oxygen environment and is critical to energy homeostasis. In vertebrates, this process is largely controlled by three intracellular prolyl-4-hydroxylases (PHD 1-3). These related enzymes share the ability to hydroxylate the hypoxia-inducible transcription factor (HIF), and therefore control the transcription of genes involved in metabolism and vascular recruitment. However, it is becoming increasingly apparent that proline-4-hydroxylation controls much more than HIF signaling, with PHD3 emerging as the most unique and functionally diverse of the PHD isoforms. In fact, PHD3-mediated hydroxylation has recently been purported to function in such diverse roles as sympathetic neuronal and muscle development, sepsis, glycolytic metabolism, and cell fate. PHD3 expression is also highly distinct from that of the other PHD enzymes, and varies considerably between different cell types and oxygen concentrations. This thesis will specifically examine the role of PHD3 expression in cancer cells, with a focus on the mechanisms of PHD3 gene silencing. In the final chapters, I will examine the consequences of this silencing in cancer, and discuss the discovery of a novel role for PHD3 in epithelial-to-mesenchymal-like transition and cell migration.

myotubesProper muscle function depends upon the fine tuning of the different molecular components of the neuromuscular junction (NMJ). Synaptic acetylcholinesterase (AChE) is responsible for rapidly terminating neurotransmission. Neuroscientists in the field have elucidated many aspects of synaptic AChE structure, function, and localization during the last 75 years. Nevertheless, how the enzyme is regulated and targeted to the NMJ is not completely understood. In skeletal muscle the synaptic AChE form derives from two separate genes encoding the catalytic and the collagenic tail (ColQ) subunits respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place remains poorly understood. We found that over or down-regulation of ColQ is sufficient to change the levels of AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. Furthermore, when peptides containing the Proline Rich Attachment Domain (PRAD), the region of ColQ that interacts with the AChE, are fed to muscle cells or cell lines expressing AChE, they are taken up by the cells and retrogradely transported to the endoplasmic reticulum (ER)/Golgi network where they induce assembly of newly synthesize AChE into tetramers. This results in an increase, as a consequence, in total cell associated AChE activity and active tetramer secretion, making synthetic PRAD peptides potential candidates for the treatment of organophosphate pesticides and nerve gas poisoning. To study the developmental regulation of ColQ-AChE we determined the levels of ColQ and ColQ mRNA in primary quail muscle cells in culture and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of ColQ from its assembly into ColQ-AChE indicating the importance of posttranslational controls in the regulation of AChE folding and assembly. Furthermore, we found that the vast majority of the ColQ molecules in QMCs are not assembled into ColQ-AChE, suggesting that they can have alternative function(s). Finally, we found that the levels of ER molecular chaperones calnexin, calreticulin, and particularly protein disulfide isomerase are regulated by muscle activity and they correlate with the levels of ColQ-AChE. More importantly, our results suggest that newly synthesized proteins compete for chaperone assistance during the folding process.

Abstract The collagens are a family of extracellular matrix proteins with a widespread tissue distribution. Collagen biosynthesis requires the hydroxylation of a number of proline residues by prolyl 4-hydroxylase. This posttranslational modification is essential for the synthesis of all collagens, as 4-hydroxyproline deficient collagens cannot form stable triple helices at body temperature. The genes for the human and mouse prolyl 4-hydroxylase α(II) subunits were cloned and characterized in this study. The human and mouse genes are 34.6 and 30.3 kb in size, respectively, consisting of 16 exons and 15 introns. The intron sizes vary from 48-49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but there are several motifs that may act as transcription factor binding sites. A novel mutually exclusively spliced exon 12a was identified in both genes. Both variants of the α(II) subunit were found to be expressed in a variety of tissues and both formed a fully active recombinant tetramer with the β subunit when expressed in insect cells. Tissue distribution of the type I and type II prolyl 4-hydroxylase isoenzymes was studied in developing, mature, and malignant cells and tissues by immunofluorescence and Western blotting. The results indicate that the type I isoenzyme is the main form in many cell types. Skeletal myocytes and smooth muscle cells appeared to have the type I isoenzyme as their only prolyl 4-hydroxylase form, whereas the type II isoenzyme was clearly the main form in chondrocytes. A strong signal for the type II enzyme was detected in cultured umbilical and capillary endothelial cells, whereas the type I isoenzyme could not be detected in these cells by immunostaining or Western blotting. Similar studies on primary chondro- and osteosarcomas and benign bone tumours indicated that the type I isoenzyme is the predominant form in both types of bone sarcoma, whereas the type II isoenzyme was more abundantly expressed in benign tumours. In chondrosarcomas, the type II isoenzyme was expressed in the nonmalignant chondrocytes, whereas their malignant counterparts switched their expression pattern to that of the type I isoenzyme. Two isoforms of the catalytic prolyl 4-hydroxylase α subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. This study reports the cloning and characterization of a third C. elegans α subunit isoform, PHY-3, which is much shorter than the previously characterized vertebrate and C. elegans α subunits. Nematodes homozygous for a phy-3 deletion were phenotypically wild type and fertile, but the 4-hydroxyproline content of their early embryos was reduced by about 90%. The expression of PHY-3 was found to be restricted to spermatheca of late larvae and adult nematode, indicating that PHY-3 is likely to be involved in the synthesis of collagens of the early embryo egg shells.

Abstract Collagen prolyl 4-hydroxylases (C-P4Hs) and related enzymes catalyze the hydroxylation of certain proline residues in animal collagens and plant hydroxyproline-rich proteins, respectively. Animal C-P4Hs and their isoenzymes have been characterized to date from humans, rodents, insects and nematodes. Most of the animal C-P4Hs are α2β2 tetramers in which protein disulphide isomerase (PDI) serves as the β subunit, but the nematode C-P4Hs characterized so far have unique molecular compositions. Two P4Hs have been cloned from the plant Arabidopsis thaliana and one from the Paramecium bursaria Chlorella virus-1, these being monomeric enzymes. This thesis reports on the identification of a large P4H family in the green alga Chlamydomonas reinhardtii and the cloning and characterization of one member, Cr-P4H-1. This is a soluble monomer that hydroxylates in vitro several peptides representing sequences found in C. reinhardtii cell wall proteins. Lack of its activity led to a defective cell wall structure, indicating that Cr-P4H-1 is essential for proper cell wall assembly and that the other P4Hs cannot compensate for the lack of its activity. Two C. elegans genes, Y43F8B.4 and C14E2.4, predicted to code for C-P4H α subunit-like polypeptides were analyzed. Three transcripts were generated from Y43F8B.4, one of them coding for a functional C-P4H α subunit named PHY-4.1. C14E2.4 turned out not to be a C-P4H α subunit gene, as a frame-shift led to the omission of codons for two catalytically critical residues. PHY-4.1 formed active tetramers and dimers with PDI-2 and had unique substrate requirements in that it hydroxylated certain other proline-rich sequences besides collagen-like peptides. Inactivation of the Y43F8B.4 gene led to no obvious morphological abnormalities. Spatial expression of the phy-4.1 transcript and PHY-4.1 polypeptide was localized to the pharynx and the excretory duct. Taken together, these data indicate that PHY-4.1 is not involved in the hydroxylation of cuticular collagens but is likely to have other substrates in vivo. Cloning and characterization of the PHY-1 and PHY-2 subunits from the closely related nematode Caenorhabditis briggsae revealed distinct differences in assembly properties between the C. elegans and C. briggsae PHY-2 subunits in spite of their high amino acid sequence identity. Genetic disruption of C. briggsae phy-1 resulted in a less severe phenotype than that observed in C. elegans, evidently on account of its more efficient assembly of the C. briggsae PHY-2 to an active C-P4H explaining the milder phenotype. Rescue of C. elegans and C. briggsae phy-1 mutants was achieved by injection of a wild-type phy-1 gene from either species.

Abstract Collagen molecules consist of three polypeptide chains that are coiled around each other to form a triple-helical structure. The formation of stable collagen triple helices requires the hydroxylation of proline residues catalyzed by collagen prolyl 4-hydroxylases (C-P4H). Vertebrate C-P4H is an ER-resident enzyme that consists of two catalytically active α subunits and two β subunits. Production of recombinant human collagen and gelatin could have numerous medical and industrial applications, but most recombinant systems lack the C-P4H activity. The yeast Pichia pastoris has been successfully engineered to produce stable human collagens and gelatins by co-expression of the collagen polypeptide chains with the two C-P4H subunits. This study examined the effect of deletion of the C-propeptide, or its replacement by a trimerizing foldon domain, on the assembly of type I and III collagen triple helices in P. pastoris. It was observed that the absence of the C-propeptide leads to inefficient collagen chain assembly whereas the replacement of C-propeptide with a foldon domain increased the assembly up to 3-fold. Moreover, the co-expression of α1(I) and α2(I) chains fused with foldon yielded heterotrimeric type I collagen molecules with a typical chain ratio of 2:1. As the foldon domain contains no information for collagen chain recognition, the present data indicate that the chain assembly is defined not only by the C-propeptides but also by other determinants present in the α chains. Another aspect studied here was the expression and secretion of gelatin fragments of varying size and conformation in P. pastoris. It was discovered that gelatin fragment size affects its secretion as the 90 kDa fragment was less efficiently secreted than the 45 kDa fragment. Secretion was also dependent on the fragment conformation as induction of the triple helix formation by either C-propeptide or foldon led to the accumulation of the fragments inside the yeast cells despite the presence of an efficient secretory signal. C-P4H was long assumed to exist as one type only but the cloning of several C-P4H α subunits raised questions concerning the specific roles of the C-P4H isoenzymes. The generation of mice lacking the type I C-P4H, which is regarded as the major C-P4H isoenzyme, indicated that this isoenzyme is essential for the embryonic development of the mouse. The embryos lacking type I C-P4H died at an early stage of their development due to the disruption of basement membranes. It was found that the basement membranes of the homozygous null embryos lacked type IV collagen whereas the fibrillar collagens were synthesized, although with altered morphology. The data reported here also demonstrate that the other C-P4H isoenzymes cannot compensate for the lack of type I isoenzyme.