Cloning, structure, expression analysis, and assignment to mouse Chromosome 7 of the gene Zfp296 encoding a zinc finger protein

Received: 4 May 2000 / Accepted: 16 June 2000     

Molecular cloning and characterization of a novel mouse actin-binding protein Zfp185

Zinc finger protein (Zfp) 185 is a mouse protein containing a Lin-l1, Isl-1 and Mec-3 (LIM) domains at its C-terminus. It was recognized by comparing the genome sequence between humans and mice in 1997. In this study, we cloned the full-length Zfp185 by means of RACE and RT-PCR. Zfp185 may be closely associated with F-actin in cells as determined by a confocal microscopy. With a series of deletants of Zfp185 and GST-pull-down assay, we determined that N-terminus region (1–144) but not the LIM domain at C-terminus of Zfp185 protein was essential and sufficient to bind to F-actin cytoskeleton. Thus, our data offered evidence for the association of mouse Zfp185 with F-actin, which supports the potential role of Zfp185 in cell fundamental activity.     

Macrophage stimulating protein is a novel neurotrophic factor

Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are alpha/beta heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration.     

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.     

Mighty is a novel promyogenic factor in skeletal myogenesis

Genetic analysis has revealed an important function in myogenesis for Myostatin, a member of the TGF-beta superfamily. However, the cascade of genes that responds to Myostatin signalling to regulate myogenesis is not well understood. Thus, a suppressive subtraction hybridization to identify such genes was undertaken and here we report the cloning and characterization of a novel gene, Mighty. Mighty is expressed in a variety of different tissues but appears to be specifically regulated by Myostatin in skeletal muscle. Overexpression of Mighty in C2C12 cells results in early withdrawal of myoblasts from the cell cycle, enhanced and accelerated differentiation and hypertrophy of myotubes. Most importantly, Mighty overexpression leads to increased and earlier expression of MyoD and increased secretion of another known differentiation inducing factor, IGF-II. Furthermore, viral expression of Mighty in mdx mice resulted in an increase in the number of larger healthy muscle fibers. Given its role in myogenesis, we propose that Mighty is a critical promyogenic factor which plays a key role in the signalling pathway downstream of Myostatin.     

Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.     

Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly

DNA double-strand breaks (DSBs) occur at random upon genotoxic stresses and represent obligatory intermediates during physiological DNA rearrangement events such as the V(D)J recombination in the immune system. DSBs, which are among the most toxic DNA lesions, are preferentially repaired by the nonhomologous end-joining (NHEJ) pathway in higher eukaryotes. Failure to properly repair DSBs results in genetic instability, developmental delay, and various forms of immunodeficiency. Here we describe five patients with growth retardation, microcephaly, and immunodeficiency characterized by a profound T+B lymphocytopenia. An increased cellular sensitivity to ionizing radiation, a defective V(D)J recombination, and an impaired DNA-end ligation process both in vivo and in vitro are indicative of a general DNA repair defect in these patients. All five patients carry mutations in the Cernunnos gene, which was identified through cDNA functional complementation cloning. Cernunnos/XLF represents a novel DNA repair factor essential for the NHEJ pathway.     

Myodulin is a novel potential angiogenic factor in skeletal muscle

We examined the expression and function of a gene we previously cloned from its downregulation in a muscle atrophy model. The encoded protein was named myodulin because of sequence homologies with the cartilage-specific chondromodulin-I (ChM-I) protein, its restricted expression in skeletal muscle tissue, and its modulating properties on vascular endothelial cells described here. We investigated the expression of myodulin in muscle fibers and cultured muscle cells. Myodulin RNA messengers were found in muscle fibers and their tendon extensions. Overexpression of myodulin fused to a FLAG peptide showed evidence of a muscle cell surface protein. Myodulin functions were assessed from similarities with chondromodulin-I. Coculture experiments using C(2)C(12) mouse myoblasts or myotubes, which stably overexpress myodulin, with H5V mouse cardiac vascular endothelial cells revealed that myodulin had a very active role in the invasive action of endothelial cells, without any evidence of extracellular myodulin secretion. Our results suggest that myodulin may be a muscle angiogenic factor operating through direct cell-to-cell interactions. This role is consistent with the correlation between modulations in myodulin expression and modifications in muscle microvascularization associated with activity-dependent muscle mass variations.     

Myostatin is a novel tumoral factor that induces cancer cachexia

Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. Although several humoral proteins, namely TNFα (tumour necrosis factor α) and IL (interleukin)-6, have been shown to induce skeletal muscle wasting, there is a lack of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, in the present study, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In the present study, we show that myostatin, a procachectic TGFβ (transforming growth factor β) superfamily member, is abundantly secreted by C26 cells. Consistent with myostatin signalling during cachexia, treating differentiated C2C12 myotubes with C26 CM (conditioned medium) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), and enhanced activity of the ubiquitin-proteasome pathway. Furthermore, the C26 CM also activated ActRIIB (activin receptor type?II B)/Smad and NF-κB (nuclear factor κB) signalling, and reduced the activity of the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway, three salient molecular features of myostatin action in skeletal muscles. Antagonists to myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy-lysosome pathway and reduced the number of mitochondria in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumour-bearing mice.