Prodynorphin mutations cause the neurodegenerative disorder spinocerebellar ataxia type 23

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (～0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.     

Endothelial LGALS9 splice variant expression in endothelial cell biology and angiogenesis

Galectins are carbohydrate binding proteins with versatile functions in tumor progression. Galectin-9, encoded by LGALS9, has been associated with metastasis and immunosuppression. We previously reported on regulation of LGALS9 expression during endothelial cell activation. Here, we show increased galectin-9 protein levels in the endothelium of different tumors, including carcinomas of the lung, liver, breast and kidney. Endothelial cells were found to express five LGALS9 splice variants, two of which have not been reported before. Splicing was found to be confined to exons 5, 6 and 10. Transfection of human microvascular endothelial cells (HMEC) with galectin-9∆5, a specific LGALS9 splice variant, induced a small but significant increase of proliferation, while migration was not affected by any LGALS9 splice variant. Application of recombinant galectin-9∆5 protein dose-dependently reduced proliferation and migration of HMEC as well as human umbilical vein endothelial cells in vitro. Enhanced sprouting and migration of human umbilical vein endothelial cell (HUVEC) towards a galectin-9∆5 gradient were observed. Interestingly, galectin-9∆5 was found to induce a small inhibitory effect on angiogenesis in vivo. Collectively, these data show that endothelial cells regulate the expression and splicing of LGALS9 during angiogenesis. The function of the dominant splice variant, i.e. galectin-9∆5, in endothelial cell biology depends on the concentration and environmental context in which it is presented to the cells.     

Identification of a cDNA encoding a novel small secretory protein,neurosecretory protein GL,in the chicken hypothalamic infundibulum

To find novel neuropeptide and/or peptide hormone precursors in the avian brain, we performed a cDNA subtractive screen of the chicken hypothalamic infundibulum, which contains one of the feeding and neuroendocrine centers. After sequencing 596 clones, we identified a novel cDNA encoding a previously unknown protein. The deduced precursor protein consisted of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. This small protein was flanked at the N-terminus by a signal peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the small protein were Gly-Leu-NH₂, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated specific expression of the NPGL precursor mRNA in the hypothalamic infundibulum. Furthermore, the mRNA levels in the hypothalamic infundibulum increased during post-hatching development. In situ hybridization analysis showed that the cells containing the NPGL precursor mRNA were localized in the medial mammillary nucleus and infundibular nucleus within the hypothalamic infundibulum of 8- and 15-day-old chicks. Subcutaneous infusion of NPGL in chicks increased body weight gain without affecting food intake. To our knowledge, this is the first report to describe the identification and localization of the NPGL precursor mRNA and the function of its translated product in animals. Our findings indicate that NPGL may participate in the growth process in chicks.     

SLM2 Is A Novel Cardiac Splicing Factor Involved in Heart Failure due to Dilated Cardiomyopathy

Alternative mRNA splicing is a fundamental process to increase the versatility of the genome.In humans,cardiac mRNA splicing is involved in the pathophysiology of heart failure.Mutations in the splicing factor RNA binding motif protein 20(RBM20) cause severe forms of cardiomyopathy.To identify novel cardiomyopathy-associated splicing factors,RNA-seq and tissue-enrichment analyses were performed,which identified up-regulated expression of Sam68-Like mammalian protein 2(SLM2) in the left ventricle...     

Disruption of the human COQ5-containing protein complex is associated with diminished coenzyme Q10 levels under two different conditions of mitochondrial energy deficiency

BackgroundThe Coq protein complex assembled from several Coq proteins is critical for coenzyme Q₆ (CoQ₆) biosynthesis in yeast. Secondary CoQ₁₀ deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ₁₀ levels and COQ5 protein maturation in human 143B cells.MethodsThis study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined.ResultsA high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ₁₀ levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids.ConclusionsA novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ₁₀ levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation.General significanceThe findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ₁₀ deficiency in human cells.     

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

The mRNA maturation of the tripartite chloroplast psaA gene from the green alga Chlamydomonas reinhardtii depends on various nucleus-encoded factors that participate in trans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the psaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the trans-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized trans-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential trans-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated trans-splicing mutants. Our assay revealed that the trans-splicing defect of mutant F19 was restored by a new factor we named RAA8; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first psaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring psaA mRNA trans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying trans-splicing factors.     

Differential proteomic analysis on the effects of 2-methoxy-1,4-naphthoquinone towards MDA-MB-231 cell line

BackgroundWe have previously reported the anti-metastatic effects of 2-methoxy-1,4-naphthoquinone (MNQ) against MDA-MB-231 cell line.PurposeTo investigate the molecular mechanism underlying the anti-metastatic effects of MNQ towards MDA-MB-231 cell line via the comparative proteomic approach.Study design/methodsDifferentially expressed proteins in MNQ-treated MDA-MB-231 cells were identified by using two-dimensional gel electrophoresis coupled with tandem mass spectrometry. Proteins and signalling pathways associated with the identified MNQ-altered proteins were studied by using Western blotting.ResultsSignificant modulation of MDA-MB-231 cell proteome was observed upon treatment with MNQ in which the expressions of 19 proteins were found to be downregulated whereas another eight were upregulated (>1.5 fold, p < 0.05). The altered proteins were mainly related to cytoskeletal functions and regulations, mRNA processing, protein modifications and oxidative stress response. Notably, two of the downregulated proteins, protein S100-A4 (S100A4) and laminin-binding protein (RPSA) are known to play key roles in driving metastasis and were verified using Western blotting. Further investigation using Western blotting also revealed that MNQ decreased the activations of pro-metastatic ERK1/2 and NF-κB signalling pathways. Moreover, MNQ was shown to stimulate the expression of the metastatic suppressor, E-cadherin.ConclusionThis study reports a proposed mechanism by which MNQ exerts its anti-metastatic effects against MDA-MB-231 cell line. The findings from this study offer new insights on the potential of MNQ to be developed as a novel anti-metastatic agent.     

Molecular cloning and expression of two chicken invariant chain isoforms produced by alternative splicing

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.     

Characterization of Hypothalamic Neurons Expressing a Neuropeptide Receptor, GALR2, Using Combined in Situ Hybridization–Immunohistochemistry

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system.     

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe–S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N–S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.