Laura Tonachini

CRTAP Is Required for Prolyl 3- Hydroxylation and Mutations Cause Recessive Osteogenesis Imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

cDNA cloning, characterization and chromosome mapping of Crtap encoding the mouse cartilage associated protein.

Recently we have isolated and characterized a cDNA coding for a novel developmentally regulated chick embryo protein, cartilage associated protein (CASP). Here we describe the isolation and characterization of the cDNAs coding for the mouse CASP. Comparison of the mammalian putative protein sequence with the chick sequence shows a very high identity overall (51%); in particular the chick protein is homologous to the half amino terminus of the mouse protein. Furthermore, the comparison of the CASP cDNA sequence with sequences of the genebank database confirms our hypothesis that the CASP genes belong to a novel family that also includes genes encoding for some nuclear antigens. In all mouse tissues examined three CASP mRNAs species are detected, whereas in chick tissues a single mRNA is present. Immunohistochemistry studies show that the protein is expressed in all mouse embryonic cartilages. The mouse cartilage associated protein gene (Crtap) was assigned to chromosome 9F3-F4 by fluorescence in situ hybridization.

Gene expression profile of human bone marrow stromal cells determined by restriction fragment differential display analysis.

Using an in vitro osteogenic culture system, we carried out a restriction fragment differential display (RFDD‐PCR) to identify genes expressed by these cells in their undifferentiated stage and not expressed, or expressed at a lower level, in a closely related but distinct cell type: bone marrow stromal cells (BMSC)‐derived osteoblasts (BDO). Forty‐seven candidate regulated genes, selected by RFDD, were analyzed by RT‐PCR analysis in three cell clones and in primary cultures from seven different donors. A subset of three genes were confirmed as upregulated in BMSC relative to BDO in every primary culture and cloned population examined: βIG‐h3, IGFbp3, and LOXL2. Their differential expression was confirmed by Northern analysis and the corresponding proteins were detected by immunolocalization in BMSC.

cDNA cloning, characterization and chromosome mapping of the gene encoding human cartilage associated protein (CRTAP).

We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.

The Nuclear Genes Mtfr1 and Dufd1 Regulate Mitochondrial Dynamic and Cellular Respiration

Dufd1 (DUF729 domain containing 1) is related to Mtfr1 (mitochondrial fission regulator 1), a gene involved in the regulation of antioxidant activity in the mouse testis. The present study was undertaken to better understand their role in regulating mitochondrial architecture and function in the mouse. We show that Dufd1 is expressed as a 2 kb mRNA and has a more specific tissue pattern compared to Mtfr1, with highest level of expression in testes, lower level in spleen, and negligible levels in other organs and/or tissues. In the male gonad, Dufd1 mRNA expression increases during postnatal development, similarly to Mtfr1. In situ hybridization and real‐time PCR analyses show that Dufd1 is expressed in the seminiferous tubules by middle‐late pachytene spermatocytes and spermatids. In transfected cells, the Dufd1‐tagged protein is located in mitochondria, associated with the tips of mitochondrial tubules and to tubules constrictions, and induces mitochondrial fission although with a lesser efficiency than Mtfr1. We also found that both endogenous Dufd1 and Mtfr1 proteins are associated with membrane‐enriched subcellular fractions, including mitochondria. Inhibition of Mtfr1 and/or Dufd1 expression, in a testicular germ cells line, severely impairs O2 consumption and indicates that both genes are required for mitochondrial respiration. Accordingly, analysis of testes mitochondria from Mtfr1‐deficient mice reveals severely reduced O2 consumption and ATP synthesis compared to wt animals. These data show that, in murine testis, Dufd1 and Mtfr1 have redundant functions related to mitochondrial physiology and represent genes with a potential role in testicular function. J. Cell. Physiol. 225: 767–776, 2010.

Chondrocyte protein with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes mitochondrial fission

We have recently identified a chondrocyte protein with a poly‐proline region, referred to as CHPPR, and showed that this protein is expressed intracellularly in chick embryo chondrocytes. Conventional fluorescence and confocal localization of CHPPR shows that CHPPR is sorted to mitochondria. Furthermore, immunoelectron microscopy of CHPPR transfected cells demonstrates that this protein is mostly associated with the mitochondrial inner membranes. Careful analysis of CHPPR expressing cells reveals, instead of the regular mitochondrial tubular network, the presence of a number of small spheroid mitochondria. Here we show that the domain responsible for network–spheroid transition spans amino acid residues 182–309 including the poly‐proline region. Functional analyses of mitochondrial activity rule out the possibility of mitochondrial damage in CHPPR transfected cells. Since cartilage expresses high levels of CHPPR mRNA when compared to other tissues and because CHPPR is associated with late stages of chondrocyte differentiation, we have investigated mitochondrial morphology in hypertrophic chondrocytes by MitoTracker Orange labeling. Confocal microscopy shows that these cells have spheroid mitochondria. Our data demonstrate that CHPPR is able to promote mitochondrial fission with a sequence specific mechanism suggesting that this event may be relevant to late stage of chondrocyte differentiation.

Chondrocyte protein with a poly-proline region is a novel protein expressed by chondrocytes in vitro and in vivo.

Chondrogenic differentiation is a multistep process entailing the sequential activation and inhibition of the expression of a number of genes. To identify genes preferentially expressed at the hypertrophic stage rather than early differentiation stages of chicken chondrocyte differentiation, a subtracted cDNA library was generated. Here we describe the characterization of a cDNA isolated from this library and that of the encoded protein referred to as Chondrocyte Protein with a Poly-proline Region (CHPPR). The cDNA coding for CHPPR hybridizes with a 3.0-kb mRNA expressed at extremely low levels in dedifferentiated chondrocytes, cultured in adherent conditions, at low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes in suspension culture. The Parathyroid Hormone peptide [PTH (1-34)] enhances accumulation of CHPPR mRNA in cultured chondrocytes. This 3.0-kb mRNA is also detectable in several chick embryo tissues but at a lower extent when compared to that present in cartilage and in hypertrophic chondrocytes. The CHPPR cDNA has a complete open reading frame coding for a polypeptide with a calculated mass of 35.6 kDa containing a proline-rich region with a PPLP motif (single-letter amino acid code). We demonstrate by Western blot analysis that two CHPPR isoforms are detected in the cell lysates from cultured chondrocytes when they are not in the culture medium; furthermore, we find that the CHPPR gene is expressed in vivo by chick embryo chondrocytes at higher levels in the prehypertrophic and hypertrophic zones.