Taina Pihlajaniemi

Molecular Biology of Prolyl 4‐Hydroxylase

Prolyl 4-hydroxylase (EC I. 14.1 1.2) catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of proline residues in -X-Pro-Glysequences. The enzyme plays a central role in collagen synthesis, as the hydroxyl groups of the 4-hydroxyproline residues are essential for the folding of the newly synthesized procollagen polypeptide chains into triple-helical molecules at body temperature. This crucial function of 4-hydroxyproline in collagens makes prolyl 4-hydroxylase a potential target for pharmacological modulation of the excessive collagen formation found in patients with various fibrotic diseases and has prompted numerous studies on the enzyme (for reviews on prolyl 4-hydroxylase, see Refs. 1-4). Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, 0,, and ascorbate. The 2oxoglutarate is stoichiometrically decarboxylated during hydroxylation, with one atom of the 0, molecule being incorporated into the succinate while the other is incorporated into the hydroxyl group formed on the proline residue.14 Ascorbate is a highly specific requirement, but it is not consumed stoichiometrically, and the enzyme can catalyze its reaction for a number of catalytic cycles in the absence of ascorbate. The reaction requiring ascorbate is probably an uncoupled decarboxylation of 2-oxoglutarate-that is, decarboxylation without subsequent hydroxylation of a proline re~idue?~ Prolyl4hydroxylase catalyzes such uncoupled decarboxylation cycles even in the presence of a saturating concentration of its peptide substrate, and hence the main biological function of ascorbate in the prolyl 4-hydroxylase reaction seems to be to act as an alternative oxygen acceptor in the uncoupled catalytic The active prolyl4-hydroxylase in vertebrates is a tetramer (I@,) with a molecular weight of about 240,000 and consisting of two different types of inactive monomer with molecular weights of about 64,000 (a subunit) and 60,000 (p subunit). The enzyme tetramer has two catalytic sites-one catalytic site per pair of dissimilar subunit^.^.^ Binding studies with different suicide inactivators of prolyl 4-hydroxylase7-’ and photoaffinity labeling studies’”’ with analogs of 2-oxoglutarate and the peptide substrate indicate that the 2-0xoglutarate’.~ and peptide’.’’ binding sites of the enzyme are located on the a subunit, whereas the ascorbate binding sites” may be built up of

Type XIII Collagen Strongly Affects Bone Formation in Transgenic Mice

To characterize the function of type XIII collagen, a transmembrane protein occurring at cell adhesion sites, we generated transgenic mice overexpressing it. High transgene expression was detected in cartilage and bone. The overexpression mice developed an unexpected skeletal phenotype marked by a massive increase in bone mass caused by increased bone formation rather than impaired resorption.

Alport syndrome caused by a 5′ deletion within the COL4A5 gene

SummaryFourteen Italian patients affected with X-linked Alport syndrome were analyzed by Southern blotting, using cDNA probes of the COL4A5 gene. One proband was shown to carry a large deletion (> 38 kb) that included the 5′ part of the gene.

Mutations in Osteogenesis Imperfecta Leading to the Synthesis of Abnormal Type I Procollagens

Osteogenesis imperfecta or “brittle bone disease” is of interest for several reasons. From the point of view of the physician or of medicine in general, it is of great interest to establish the molecular basis of this disease, or more correctly, group of diseases (for reviews see refs. 1-5). At the phenotypic level, the diseases show a broad spectrum of heterogeneity. Brittle bones are the hallmark of the disease and in the most severe forms, bones are so brittle that death occurs in utero. In the mildest forms, individuals who avoid trauma have minimal disability. Also, in some individuals the disease involves the teeth, skin, and sclerae of the eyes. In others it is confined to the major bony structures. It would obviously be extremely helpful in dealing with patients with these conditions to understand the molecular basis of the diseases and thereby to explain the large heterogeneity in the clinical manifestations. In addition, defining the molecular defects should make it possible to develop DNA tests for the prenatal diagnosis of the severe forms of osteogenesis imperfecta. Hence, at a practical level, it should be possible to diminish greatly the number of new cases. From the point of view of general biology, osteogenesis imperfecta is of interest because it presents a series of highly informative “experiments of Nature.” It is now established that many forms of osteogenesis imperfecta are produced by mutations in the genes for type I procollagen (see refs. 1, 3, and 5). The specific kinds of mutations that are found indicate directly the special vulnerabilities of the genes to mutations. Because the amino acid sequence of the protein is highly repetitive, the coding sequences of the genes are highly repetitive. Experiments in bacteria and other organisms demonstrate that repetitive DNA sequences are prone to undergo extensive recombination during either meiosis or mitosis. Therefore the mutations found in collagen

Discovery of Potent and Selective Nonplanar Tankyrase Inhibiting Nicotinamide Mimics.

Diphtheria toxin-like ADP-ribosyltransferases catalyse a posttranslational modification, ADP-ribosylation and form a protein family of 17 members in humans. Two of the family members, tankyrases 1 and 2, are involved in several cellular processes including mitosis and Wnt/β-catenin signalling pathway. They are often over-expressed in cancer cells and have been linked with the survival of cancer cells making them potential therapeutic targets. In this study, we identified nine tankyrase inhibitors through virtual and in vitro screening. Crystal structures of tankyrase 2 with the compounds showed that they bind to the nicotinamide binding site of the catalytic domain. Based on the co-crystal structures we designed and synthesized a series of tetrahydroquinazolin-4-one and pyridopyrimidin-4-one analogs and were subsequently able to improve the potency of a hit compound almost 100-fold (from 11 μM to 150 nM). The most potent compounds were selective towards tankyrases over a panel of other human ARTD enzymes. They also inhibited Wnt/β-catenin pathway in a cell-based reporter assay demonstrating the potential usefulness of the identified new scaffolds for further development.

Physical mapping of mouse collagen genes on Chromosome 10 by high-resolution FISH

Abstract. Fluorescence in situ hybridization (FISH) on mechanically stretched chromosomes (MSCs) and extended DNA fibers enables construction of high-resolution physical maps by accurate ordering and orienting genomic clones as well as by measuring physical lengths of gaps and overlaps between them. These high-resolution FISH targets have hitherto been used mainly in the study of the human genome. Here we have applied both MSCs and extended DNA fibers to the physical mapping of the mouse genome. At first, five mouse collagen genes were localized by metaphase-FISH: Col10a1 to chromosomal bands 10B1-B3; Col13a1 to 10B4; and Col6a1, Col6a2, and Col18a1 to 10B5-C1. The mutual order of the genes, centromere–Col10a1–Col13a1–Col6a2–Col6a1–Col18a1–telomere, was determined by FISH on metaphase chromosomes, MSCs, and extended DNA fibers. To our knowledge, this is the first time mouse metaphase chromosomes have been stretched and used as targets for FISH. We also used MSCs to determine the transcriptional orientations, telomere–5′→3′–centromere, of both Col13a1 and Col18a1. With fiber-FISH, Col18a1, Col6a1, and Col6a2 were shown to be in a head-to-tail configuration with respective intergenic distances of about 350 kb and 90 kb. Comparison of our physical mapping results with the homologous human data reveals both similarities and differences concerning the chromosomal distribution, order, transcriptional orientations, and intergenic distances of the collagen genes studied.

Experimental Tumour Models in Mice

The complex multistage processes of tumour initiation, progression and metastasis challenge the methods that are used in basic cancer biology research and anticancer drug development. Experimental tumour modelling in mice provides means for observing tumour development, identifying target molecules and pathways and designing and testing novel strategies for diagnosing and treating cancer in a manner that is not possible in in vitro systems or in human patients. To gain maximal benefit from the use of mouse tumour models one should be aware of the possibilities and limitations of each approach, and pay careful attention to selection of the model and planning of the experiments. We aim in this review to give the reader some basic information on experimental mouse tumour models that have evolved from simple chemical treatments to extremely complex genetic models, and to discuss their advantages and disadvantages. We discuss some species-specific differences between mice and humans, and also between the inbred mouse strains, that can affect the various processes of tumorigenesis. Finally, we try with a few examples of cancer studies involving the p53 and retinoblastoma tumour suppressors, and the extracellular matrix protein collagen XVIII and its antiangiogenic endostatin fragment to illustrate the importance of evaluating data from various tumour models in order to achieve a proper understanding of the function of a given molecule or pathway in tumour development.