Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Proximal spinal muscular atrophy (SMA) is a common motor neuron disease caused by homozygous loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy of the gene and present in all SMA patients, fails to provide protection from SMA, due to the disruption of an exonic splicing enhancer (ESE) by a single translationally silent nucleotide exchange, which causes alternative splicing of SMN2 exon 7. Identification of splicing factors that stimulate exon 7 inclusion and thereby produce sufficient amounts of full-length transcripts from the SMN2 gene is of great importance for therapy approaches. Here, by use of in vivo splicing assays, we identified the protein hnRNP-G and its paralogue RBM as two novel splicing factors that promote the inclusion of SMN2 exon 7. Moreover, hnRNP-G and RBM non-specifically bind RNA, but directly and specifically bind Htra2-beta1, an SR-like splicing factor which we have previously shown to stimulate inclusion of exon 7 through a direct interaction with the AG-rich ESE in SMN2 exon 7 pre-mRNA. By using deletion mutants of hnRNP-G, we show that the specific protein-protein interaction of hnRNP-G with Htra2-beta1 mediates the inclusion of SMN2 exon 7 rather than the non-specific interaction of hnRNP-G with SMN pre-mRNA. Additionally, we show for the first time that recombinant trans-acting splicing factors such as hnRNP-G and Htra2-beta1 are also effective on endogenous SMN2 transcripts and increase the endogenous SMN protein level. Finally, we suggest a model of how the exon 7 mRNA processing is regulated by the splicing factors identified so far.
Hum Mol Genet 2002 Aug 15
PMID:hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1. 1216 65

Infantile spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN)1 gene. We investigated the role of human (h) SMN protein on cell death in PC12 and Rat-1 cells. hSMN prolonged cell survival in PC12 cells deprived of trophic support and in Rat-1 cells induced to die by activation of the proto-oncogene c-Myc, to similar magnitude as Bcl-2 or IAP-2. While hSMN was ineffective in inhibiting apoptosis induced by ultraviolet light (UV) or etoposide treatment in proliferating PC12 or Rat-1 cells, a protective effect was observed in terminally NGF/dBcAMP-differentiated PC12 cells. hSMN inhibited the onset of apoptosis in NGF/dBcAMP-deprived or UV-treated co-differentiated PC12 cells by preventing cytochrome c release and caspase-3 activation, indicating that its effects are through suppression of the mitochondrial apoptotic pathway. Expressing hSMN deleted for exon 7 (Delta7) or for exons 6 and 7 (Delta6/7), or with the SMA point mutant Y272C, resulted in loss of survival function. Moreover, these mutants also exhibited pro-apoptotic effects in Rat-1 cells. The localization pattern of full-length hSMN in PC12 and Rat-1 cells was similar to that of endogenous SMN: granular labelling in the cytoplasm and discrete fluorescence spots in the nucleus, some of which co-localized with p80 coilin, the characteristic marker of Cajal bodies. However, cytoplasmic and nuclear aggregates were often seen with hSMNDelta7, whereas the hSMNDelta6/7 mutant showed homogenous nuclear labelling that excluded the nucleolus. Thus, our results show that the C-terminal region is critical in suppression of apoptosis by SMN.
Hum Mol Genet 2002 Oct 15
PMID:Involvement of survival motor neuron (SMN) protein in cell death. 1237 65

Approximately 94% of patients with spinal muscular atrophy lack both copies of SMN1 exon 7, and most carriers have only one copy of SMN1 exon 7. We described previously the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is a multiplex quantitative PCR assay to determine the copy numbers of SMN1 and SMN2 using DraI digestion to differentiate SMN2 from SMN1. We describe herein the quantification of PCR bias between SMN1 exon 7 and SMN2 exon 7, which differ by only one nucleotide that is not present in either primer binding site. Using samples from 272 individuals with various SMN genotypes, we found that the amplification efficiency of SMN2 was consistent only approximately 80% that of SMN1. Thus, even a single nucleotide polymorphism, not in primer binding sites, can cause reproducible PCR bias. The precision and accuracy of our SMN gene dosage analysis are high because our assay design and controls take advantage of the consistency of the PCR bias. As additional clinically significant single nucleotide polymorphisms (SNPs) are discovered, assessment of PCR bias, and judicious selection of standards and controls, will be increasingly important for quantitative PCR assays.
J Mol Diagn 2002 Nov
PMID:Quantification of PCR bias caused by a single nucleotide polymorphism in SMN gene dosage analysis. 1241 85

Myofibroblasts, the hallmark of fibrotic disease, contribute to the pathology of fibrosis by secreting large amounts of extracellular matrix and contributing to alveolar contraction. Myofibroblasts are characterized by the expression of alpha-smooth muscle actin (alpha-SMA), a contractile protein normally associated with smooth muscle cells. Transforming growth factor-beta1 (TGF-beta1) is a well characterized profibrotic cytokine that induces myofibroblast transformation both in vitro and in vivo. We report here that the lipid mediator prostaglandin E2 (PGE2) inhibits TGF-beta1-induced expression of alpha-SMA in primary fetal and adult lung fibroblasts. This inhibition of alpha-SMA expression is associated with a reduction in the expression of collagen I. Inhibitory actions of PGE2 are mediated via E prostanoid receptor 2 (EP2) signaling, but not by EP3 signaling, and increases in cyclic adenosine monophosphate production. The inhibitory effects of PGE2 on TGF-beta1-induced alpha-SMA expression are mimicked by an EP2 selective agonist, butaprost, and by forskolin-induced direct activation of adenyl cyclase. An EP2 antagonist blocks the inhibitory effects of PGE2, and an EP3 agonist does not inhibit TGF-beta1-mediated increases in alpha-SMA expression. Our results demonstrate that PGE2 inhibits transition of fibroblasts to myofibroblasts by an EP2 receptor-activated pathway. Augmenting this pathway may serve as a potent antifibrotic therapeutic strategy.
Am J Respir Cell Mol Biol 2003 Nov
PMID:Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation. 1273 87

Spinal muscular atrophy (SMA) is a recessive autosomal disorder characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron gene (SMN1). No curative treatment is known so far. Mutant mice carrying homozygous deletion of Smn exon 7 directed to neurons display skeletal muscle denervation, moderate loss of motor neuron cell bodies and severe axonal degeneration. These features, similar to those found in human SMA, strongly suggest the involvement of a dying back process of motor neurons and led us to test whether neurotrophic factors might have a protective role in SMA. We report here the therapeutic benefits of systemic delivery of cardiotrophin-1 (CT-1), a neurotrophic factor belonging to the IL-6 cytokine family. Intra-muscular injection of adenoviral vector expressing CT-1, even at very low dose, improves median survival, delays motor defect of mutant mice and exerts protective effect against loss of proximal motor axons and aberrant cytoskeletal organization of motor synaptic terminals. In spite of the severity of SMA phenotype in mutant mice, CT-1 is able to slow down disease progression. Neuroprotection could be regarded as valuable therapeutic approach in SMA.
Hum Mol Genet 2003 Jun 01
PMID:Therapeutic benefits of cardiotrophin-1 gene transfer in a mouse model of spinal muscular atrophy. 1276 Oct 38

Autosomal recessive spinal muscular atrophy (SMA) is linked to mutations in the survival motor neuron (SMN) gene. The SMN protein has been implicated at several levels of mRNA biogenesis and is expressed ubiquitously. Studies in various model organisms have shown that the loss of function of the SMN gene leads to embryonic lethality. The human contains two genes encoding for SMN protein and in patients one of these is disrupted. It is thought the remaining low levels of protein produced by the second SMN gene do not suffice and result in the observed specific loss of lower motor neurons and muscle wasting. The early lethality in the animal mutants has made it difficult to understand why primarily these tissues are affected. We have isolated a Drosophila smn mutant. The fly alleles contain point mutations in smn similar to those found in SMA patients. We find that zygotic smn mutant animals show abnormal motor behavior and that smn gene activity is required in both neurons and muscle to alleviate this phenotype. Physiological experiments on the fly smn mutants show that excitatory post-synaptic currents are reduced while synaptic motor neuron boutons are disorganized, indicating defects at the neuromuscular junction. Clustering of a neurotransmitter receptor subunit in the muscle at the neuromuscular junction is severely reduced. This new Drosophila model for SMA thus proposes a functional role for SMN at the neuromuscular junction in the generation of neuromuscular defects.
Hum Mol Genet 2003 Jun 15
PMID:Neuromuscular defects in a Drosophila survival motor neuron gene mutant. 1278 45

Cultured myofibroblasts are characterized by stress fibers, containing alpha-smooth muscle actin (alpha-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of alpha-SMA-negative fibroblasts. We have investigated the role of alpha-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of alpha-SMA-negative fibroblasts. Then, myofibroblasts incorporate alpha-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When alpha-SMA expression is induced in 5 d culture by TGFbeta or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with alpha-SMA fusion peptide (SMA-FP), which inhibits alpha-SMA-mediated contractile activity, reduces their adhesion to the level of alpha-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and beta3 integrin-containing FAs; alpha5 integrin distribution is not affected. FRAP of beta3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high alpha-SMA-mediated contractile activity of myofibroblast stress fibers.
Mol Biol Cell 2003 Jun
PMID:Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts. 1280 47

Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in half of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10% of full-length protein identical to SMN1, whereas the majority of SMN2 transcripts is aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-beta 1. We show that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5-500 microM) of valproic acid (VPA), the level of full-length SMN2 mRNA/protein increased 2- to 4-fold. Importantly, this up-regulation of SMN could be most likely attributed to increased levels of Htra2-beta 1 which facilitates the correct splicing of SMN2 RNA as well as to an SMN gene transcription activation. Especially at low VPA concentrations, the restored SMN level depended on the number of SMN2 copies. Moreover, VPA was able to increase SMN protein levels through transcription activation in organotypic hippocampal brain slices from rats. Finally, VPA also increased the expression of further SR proteins, which may have important implications for other disorders affected by alternative splicing. Since VPA is a drug highly successfully used in long-term epilepsy therapy, our findings open the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing.
Hum Mol Genet 2003 Oct 01
PMID:Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy. 1291 51

The survival motor neuron (SMN) gene is the spinal muscular atrophy (SMA) determining gene. Here we report that the SMN protein product interacts in vitro and in vivo with the arginine/glycine (RG)-rich RNA binding protein and transcription factor, Ewing's sarcoma (EWS). Recently, the SMN encoded Tudor domain (exon 3) and the YG-motifs (exon 6) have been shown to be involved in binding to RG-rich proteins. Here, we demonstrate that the Tudor domain encoded by SMN exon 3 is independently sufficient to mediate the interaction with EWS. Synthetic mutations within the Tudor domain, as well as a SMA patient-derived mutation within exon 3, reduced the levels of the SMN/EWS interaction. Carboxyl-terminal SMN mutations that prevent formation of SMN oligomers also indirectly reduced EWS binding. A role for arginine methylation has been observed in some RG-containing SMN-interacting proteins. Here we demonstrate that SMN interacts with non-methylated EWS and an EWS-derived RG-containing peptide. In contrast to previously reported results, symmetrical dimethylation of the EWS-derived RG-peptide results in a quantitative increase in the dissociation rate between SMN and the symmetrical dimethylated EWS RG-peptide. Consistent with the interaction data, endogenous and transiently expressed SMN co-localizes with endogenous EWS in a number of cultured cell lines, as well as rat primary neuron cultures. Anti-sense RNA experiments, however, demonstrate that EWS does not mediate the nuclear distribution of SMN or other Cajal body components.
Brain Res Mol Brain Res 2003 Nov 06
PMID:The Ewing's sarcoma protein interacts with the Tudor domain of the survival motor neuron protein. 1459 28

Five affected siblings were referred with a probable diagnosis of proximal adult-type spinal muscular atrophy (SMA) based on lower motor neuron signs (muscle weakness and atrophy, hypotony, hypoactive or absent reflexes, and fasciculations), normal or borderline serum creatine kinase levels, and a neurogenic pattern on electromyography, compatible with motor neuron disease, in one patient. No exon 7-8 deletion in the survival motor neuron (SMN) gene was found. Linkage analysis excluded the SMN and all known autosomal recessive limb girdle muscular dystrophy loci, with the exception of LGMD-2A. A homozygous R769Q mutation in the calpain-3 gene and absence of muscle calpain-3 protein confirmed a calpainopathy. This family suggests that the clinical spectrum of calpainopathy might be broader and that this diagnosis might be considered in patients with an atypical motor neuron disease.
J Mol Neurosci 2003
PMID:Calpainopathy: how broad is the spectrum of clinical variability? 1464 90


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