Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Increasing survival motor neuron 2 (SMN2) gene expression may be an effective strategy for the treatment of spinal muscular atrophy (SMA). Histone deacetylase (HDAC) inhibitors have been shown to increase SMN transcript and protein levels, but the specific role of histone acetylation in regulating SMN gene expression has not been explored. Using chromatin immunopreciptation, we investigated the levels of acetylated H3 and H4 histones and HDACs associated with different regions of the human and mouse SMN genes in both cultured cells and tissues. We show that the SMN gene has a reproducible pattern of histone acetylation that is largely conserved among different tissues and species. A limited region of the promoter surrounding the transcriptional start site has relatively high levels of histone acetylation, whereas regions further upstream or downstream have lower levels. After HDAC inhibitor treatment, acetylated histone levels increased, particularly at upstream regions, correlating with a 2-fold increase in promoter activity. During development in mouse tissues, histone acetylation levels decreased and associated HDAC2 levels increased at the region closest to the transcriptional start site, correlating with a 40-60% decrease in SMN transcript and protein levels. These data indicate that histone acetylation modulates SMN gene expression and that pharmacological manipulation of this epigenetic determinant is feasible. HDAC2, in particular, may be a future therapeutic target for SMA.
Hum Mol Genet 2005 May 01
PMID:The role of histone acetylation in SMN gene expression. 1577 88

Spinal muscular atrophy (SMA) is caused by homozygous loss of the survival motor neuron (SMN1) gene. In virtually all SMA patients, a nearly identical copy gene is present, SMN2. SMN2 cannot fully compensate for the loss of SMN1 because the majority of transcripts derived from SMN2 lack a critical exon (exon 7), resulting in a dysfunctional SMN protein. Therefore, the critical distinction between a functional and a dysfunctional SMN protein is the inclusion or the exclusion of the exon 7 encoded peptide. To determine the role of the 16 amino acids encoded by SMN exon 7, a panel of synthetic mutations were transiently expressed in SMA patient fibroblasts and HeLa cells. Consistent with previous reports, the protein encoded by SMN exons 1-6 was primarily restricted to the nucleus. However, a variety of heterologous sequences fused to the C-terminus of SMN exons 1-6 allowed mutant SMN proteins to properly distribute to the cytoplasm and to the nuclear gems. These data demonstrate that the SMN exon 7 sequence is not specifically required, rather this region functions as a non-specific 'tail' that facilitates proper localization. Therefore, a possible means to restore additional activity to the SMNDelta7 protein could be to induce a longer C-terminus by suppressing recognition of the native stop codon. To address this possibility, aminoglycosides were examined for their ability to restore detectable levels of SMN protein in SMA patient fibroblasts. Aminoglycosides can suppress the accurate identification of translation termination codons in eukaryotic cells. Consistent with this, treatment of SMA patient fibroblasts with tobramycin and amikacin resulted in a quantitative increase in SMN-positive gems and an overall increase in detectable SMN protein. Taken together, this work describes the role of the critical exon 7 region and identifies a possible alternative approach for therapeutic intervention.
Hum Mol Genet 2005 May 01
PMID:A non-sequence-specific requirement for SMN protein activity: the role of aminoglycosides in inducing elevated SMN protein levels. 1579 May 98

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in the telomeric copy of the survival motor neuron (SMN1) gene. Although the SMN protein has been implicated in the biogenesis of ribonucleoprotein complexes and RNA processing, it is not clear how these functions contribute to the pathogenesis of SMA. To gain a further understanding of SMN function, we have investigated its role in cell survival in skin fibroblasts derived from SMA patients and age-matched controls. SMA fibroblasts exposed to camptothecin, a specific inhibitor of DNA topoisomerase I, consistently showed cell death at a lower concentration than normal controls. Treatment with other cell death-inducing agents did not cause differences in survival of SMA fibroblasts as compared with control fibroblasts. Camptothecin treatment resulted in activation of caspase-3 with generation of the caspase-3 cleavage product, poly ADP-ribose polymerase (PARP). Depletion of SMN protein by RNA interference in control fibroblasts increased caspase-3 activity, whereas transfection of SMA fibroblasts with wild-type SMN decreased caspase-3 activity. Our data demonstrate that SMA fibroblasts are more prone to some, but not all, death-stimuli. Vulnerability to death-stimuli is associated with decreased levels of SMN protein and is mediated by activation of caspase-3.
Mol Genet Metab 2005 May
PMID:Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin-induced cell death. 1586 79

Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbetaRII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGFbetaRII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.
Exp Mol Med 2005 Apr 30
PMID:HCV core protein promotes liver fibrogenesis via up-regulation of CTGF with TGF-beta1. 1588 28

We have exploited the existence of a second copy of the human SMN gene (SMN2) to develop a high-throughput screening strategy to identify potential small molecule therapeutics for the genetic disease spinal muscular atrophy (SMA), which is caused by the loss of the SMN1 gene. Our screening process was designed to identify synthetic compounds that increase the total amount of full-length SMN messenger RNA and protein arising from the SMN2 gene, thereby suppressing the deleterious effects of losing SMN1. A cell-based bioassay was generated that detects SMN2 promoter activity, on which greater than 550,000 compounds was tested. This resulted in the identification of 17 distinct compounds with confirmed biological activity on the cellular primary assay, belonging to nine different structural families. Six of the nine scaffolds were chosen on the basis of their drug-like features to be tested for their ability to modulate SMN gene expression in SMA patient-derived fibroblasts. Five of the six compound classes altered SMN mRNA levels or mRNA splicing patterns in SMA patient-derived fibroblasts. Two of the compound classes, a quinazoline compound series and an indole compound, also increased SMN protein levels and nuclear gem/Cajal body numbers in patient-derived cells. In addition, these two distinct scaffolds showed additive effects when used in combination, suggesting that they may act on different molecular targets. The work described here has provided the foundation for a successful medicinal chemistry effort to further advance these compounds as potential small molecule therapeutics for SMA.
Hum Mol Genet 2005 Jul 15
PMID:Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy. 1594 1

Defective heme synthesis may cause acute porphyrias, which are associated with a wide array of neurological disturbances involving both the central and peripheral nervous systems. Thus, the understanding of the roles of heme in neuronal cell function may provide insights into the molecular events underlying the pathogenesis of neuropathies associated with defective heme synthesis. In this report, we use rat pheochromocytoma (PC12) clonal cells as a model system for studying the role of heme in neuronal cell survival. We examined the effects of inhibition of heme synthesis on signaling pathways and gene expression in nerve growth factor (NGF)-induced PC12 cells. We found that succinyl acetone-induced heme deficiency selectively caused apoptosis in NGF-induced PC12 cells. Further, we found that in succinyl acetone-treated, NGF-induced cells, the pro-survival Ras-ERK1/2 signaling pathway was inactivated and the pro-apoptotic JNK signaling pathway was activated. In these cells, the activation of caspase and the cleavage of nuclear poly (ADP-ribose) polymerase (PARP) were also evident. Importantly, microarray gene expression analysis showed that more than 20 key neuronal genes that were induced by NGF were suppressed by succinyl acetone. These genes include those encoding survival motor neuron protein, synaptic vesicle protein SVOP, and neural cell adhesion molecule NCAM. These results indicate that heme is important for neuronal cell signaling and the proper functioning of neuronal cells.
Brain Res Mol Brain Res 2005 Jun 13
PMID:Heme deficiency suppresses the expression of key neuronal genes and causes neuronal cell death. 1595 Jul 57

We describe here--presumably for the first time--a Cajal-like type of tubal interstitial cells (t-ICC), resembling the archetypal enteric ICC. t-ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene-blue, crystal-violet, Janus-Green B or MitoTracker-Green FM Probe vital stainings. Also, t-ICC were identified in fixed specimens by light microscopy (methylene-blue, Giemsa, trichrome stainings, Gomori silver-impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t-ICC was strengthened by immunohistochemistry (IHC; CD117/c-kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c-kit+ and other 7 antigens). The spatial density of t-ICC (ampullar-segment cryosections) was 100-150 cells/mm2. Non-conventional light microscopy (NCLM) of Epon semithin-sections revealed a network-like distribution of t-ICC in lamina propria and smooth muscle meshwork. t-ICC appeared located beneath of epithelium, in a 10-15 microm thick 'belt', where 18+/-2% of cells were t-ICC. In the whole lamina propria, t-ICC were about 9%, and in muscularis approximately 7%. In toto, t-ICC represent ~8% of subepithelial cells, as counted by NCLM. In vitro, t-ICC were 9.9+/-0.9% of total cell population. TEM showed that the diagnostic 'gold standard' (Huizinga et al., 1997) is fulfilled by 'our' t-ICC. However, we suggest a 'platinum standard', adding a new defining criterion- characteristic cytoplasmic processes (number: 1-5; length: tens of microm; thickness: < or =0.5 microm; aspect: moniliform; branching: dichotomous; organization: network, labyrinthic-system). Quantitatively, the ultrastructural architecture of t-ICC is: nucleus, 23.6+/-3.2% of cell volume, with heterochromatin 49.1+/-3.8%; mitochondria, 4.8+/-1.7%; rough and smooth endoplasmic-reticulum (1.1+/-0.6%, 1.0+/-0.2%, respectively); caveolae, 3.4+/-0.5%. We found more caveolae on the surface of cell processes versus cell body, as confirmed by IF for caveolins. Occasionally, the so-called 'Ca2+-release units' (subplasmalemmal close associations of caveolae+endoplasmic reticulum+mitochondria) were detected in the dilations of cell processes. Electrophysiological single unit recordings of t-ICC in primary cultures indicated sustained spontaneous electrical activity (amplitude of membrane potentials: 57.26+/-6.56 mV). Besides the CD117/c-kit marker, t-ICC expressed variously CD34, caveolins 1&2, alpha-SMA, S-100, vimentin, nestin, desmin, NK-1. t-ICC were negative for: CD68, CD1a, CD62P, NSE, GFAP, chromogranin-A, PGP9.5, but IHC showed the possible existence of (neuro)endocrine cells in tubal interstitium. We call them 'JF cells'. In conclusion, the identification of t-ICC might open the door for understanding some tubal functions, e.g. pace-making/peristaltism, secretion (auto-, juxta- and/or paracrine), regulation of neurotransmission (nitrergic/purinergic) and intercellular signaling, via the very long processes. Furthermore, t-ICC might even be uncommitted bipotential progenitor cells.
J Cell Mol Med
PMID:Novel type of interstitial cell (Cajal-like) in human fallopian tube. 1596 70

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.
Mol Biol Cell 2005 Sep
PMID:Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization. 1598 42

Spinal muscular atrophy has been classified into four groups based on the age of onset and clinical severity of the disease. Homozygous deletion in SMN1 gene causes the disease but the clinical severity may be modified by copy number of homologous gene SMN2 as well as the extent of deletion at SMN locus. In the view of scarcity of genotype and phenotype correlation data from India, this study has been undertaken to determine that correlation in SMA patients by using the SMN and NAIP genes and two polymorphic markers C212 and C272 located in this region. Two to four alleles of the markers C212 and C272 were observed in normal individuals. However, majority of Type I patients showed only one allele from both markers whereas in Type II and III patients, 2-3 alleles were observed. The SMN2 copy number in our type III patients showed that patients carry 3-5 copies of SMN2 gene. Our results suggest that extent of deletions encompassing H4F5, SMN1, NAIP and copy number of SMN2 gene can modify the SMA phenotype, thus accounting for the different clinical subtypes of the disease.
Exp Mol Med 2005 Jun 30
PMID:Genotype-phenotype correlation of SMN locus genes in spinal muscular atrophy patients from India. 1600 Aug 67

Spliceosomal Uridine-rich small ribonucleo protein (U snRNP) assembly is an active process mediated by the macromolecular survival motor neuron (SMN) complex. This complex contains the SMN protein and six additional proteins, named Gemin2-7, according to their localization to nuclear structures termed gems. Here, we provide biochemical evidence for the existence of another, yet atypical, SMN complex component, termed unr-interacting protein (unrip). This abundant factor has been previously shown to form a complex with unr, a protein implicated in cap-independent translation of cellular and viral mRNA. We show that unrip is integrated into a complex with unr or with the SMN complex in vivo in a mutually exclusive manner. In the latter case, unrip is recruited to the active SMN complex via a stable interaction with Gemin7. However, unlike SMN and Gemins, unrip localizes predominantly to the cytoplasm and is absent from gems/Cajal bodies. Interestingly, RNAi-induced reduction of unrip protein levels leads to enhanced accumulation of SMN in the nucleus as evident by the increased formation of nuclear gems/Cajal bodies. Our data identify unrip as the first component of the U snRNP assembly machinery that associates with the SMN complex in a compartment-specific way. We speculate that unrip plays a crucial role in the intracellular distribution of the SMN complex.
Hum Mol Genet 2005 Oct 15
PMID:Unrip, a factor implicated in cap-independent translation, associates with the cytosolic SMN complex and influences its intracellular localization. 1615 90


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