Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:010955 (mda-7)
 464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine the active site cysteine residue in aldehyde dehydrogenase, we mutated amino acid residues 49, 162, and 302 of recombinantly expressed rat liver mitochondrial (class 2) aldehyde dehydrogenase. The C49A and C162A mutants were fully active tetrameric enzymes, although the C162A mutant was found to be highly unstable. The C302A mutant was also a tetramer and bound coenzyme, but lacked both dehydrogenase and esterase activities. To test for the role of cysteine 302 as a nucleophile, the residue was mutated to a serine, a poor nucleophile. this C302S mutant was active but was a much poorer catalyst, with a kcat/Km value 7 x 10(5) times lower than that of the recombinant native enzyme. Unlike with native enzyme where deacylation is rate limiting, formation of the serine hemiacetal intermediate appeared to be the rate-limiting step. Cysteine 302 is the only strictly conserved cysteine residue among all the available sequences of the aldehyde dehydrogenase superfamily, supporting the role of this residue as the active site nucleophile of aldehyde dehydrogenase.
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PMID:Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis. 787 40

We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants-C9A, C49A, C87A, C104A, C207S, C254A, and C267L-retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca(2+)-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca(2+) ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.
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PMID:Role of simian virus 40 Vp1 cysteines in virion infectivity. 1107 39

The median survival of metastatic renal cell carcinoma (RCC) is 12 months, and the majority of treatment options are palliative. MDA-7 (interleukin-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound antiproliferative and cytotoxic effects in a wide variety of tumor cells but not in nontransformed cells. The studies in this study examined the impact of MDA-7 on RCC proliferation and survival. RCC lines (A498 and UOK121N), but not primary renal epithelial cells, were resistant to adenoviral infection that correlated with a lack of coxsackievirus and adenovirus receptor expression. Additional studies were performed using purified preparations of bacterially synthesized glutathione S-transferase (GST)-MDA-7 protein. GST-MDA-7, but not GST, caused a dose-dependent inhibition of RCC proliferation but not of primary renal epithelial cells. Clinically achievable concentrations of the novel therapeutic agent arsenic trioxide (0.5-1 micro M) were found to have little effect on RCC growth. However, the combination of GST-MDA-7 and arsenic trioxide resulted in a greater than additive reduction in cell growth that correlated with a large increase in tumor cell death. The free radical scavenger N-acetyl cysteine abolished the potentiating effect of arsenic trioxide. Although pro-caspase 3, poly(ADP-ribose) polymerase, and Bcl-(XL) levels, as well as nucleosomal DNA integrity, were reduced by combined treatment, cell killing was predominantly nonapoptotic. Combined treatment of RCC lines with GST-MDA-7 and arsenic trioxide also resulted in a substantial reduction in clonogenic survival compared with either treatment individually. Collectively, these findings demonstrate that MDA-7 protein, in combination with agents that generate free radicals, may have potential in the treatment of RCC.
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PMID:MDA-7 (interleukin-24) inhibits the proliferation of renal carcinoma cells and interacts with free radicals to promote cell death and loss of reproductive capacity. 1288 35

Mda-7/IL-24 (Ad.mda-7) is a novel cytokine gene belonging to the interleukin (IL) 10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 causes growth suppression and apoptosis in a wide spectrum of cancer cells, including prostate, without harming normal cells. We now demonstrate that Ad.mda-7 selectively induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and reactive oxygen species (ROS) production. Antioxidants (N-acetyl-L-cysteine and Tiron) and inhibitors of mitochondrial permeability transition (cyclosporine A and bongkrekic acid) inhibit Ad.mda-7-induced mitochondrial dysfunction and apoptosis. Conversely, agents augmenting ROS production (arsenic trioxide, NSC656240, and PK11195) facilitate Ad.mda-7-induced apoptosis. Ectopic expression of Bcl-2 and Bcl-x(L) inhibits mitochondrial changes, ROS production, and apoptosis providing additional support for an association between mitochondrial dysfunction and Ad.mda-7 action. These studies present definitive evidence that changes in mitochondrial function and ROS production are key components associated with selective killing of prostate cancer cells by mda-7/IL-24.
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PMID:Melanoma differentiation associated gene-7, mda-7/interleukin-24, induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and inducing reactive oxygen species. 1467 67

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH ( p -aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys(335/379/427/434)-->Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.
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PMID:Cysteine residues in the organic anion transporter mOAT1. 1497 72

We examined the impact of purified bacterially synthesized GST-MDA-7 (IL-24) and ionizing radiation on the proliferation and survival of nonestablished human glioblastoma multiforme (GBM) cells. Glioma cell types expressing mutated PTEN and p53 molecules, activated ERBB1VIII, overexpressing wild type ERBB1 or without receptor overexpression were selected. In MTT assays, GST-MDA-7 caused a dose-dependent reduction in the proliferation of nonestablished glioma cells; however only at higher concentrations did GST-MDA-7 reduce cell viability. The anti-proliferative and cytotoxic effects of GST-MDA-7 were enhanced by radiation in a greater than additive fashion that correlated with JNK1/2/3 activation. The reduction in cell growth and enhancement in cell killing by the combination of GST-MDA-7 and radiation were blocked by an ROS scavenger, N-acetyl cysteine (NAC), a JNK1/2/3 inhibitor SP600125, a pan-caspase inhibitor (zVAD) and by an inhibitor of caspase 9 (LEHD), but not by an inhibitor of caspase 8 (IETD). Low concentrations of either GST-MDA-7 or radiation reduced clonogenic survival, however colony formation ability was significantly further decreased when the two treatments were combined, which was also blocked by inhibition of caspase 9 function. In general agreement with activation of the intrinsic caspase pathway, cell death correlated with reduced BCL-XL expression and with increased levels of the pro-apoptotic proteins BAD and BAX. Inhibition of caspase 9 after combination treatment blunted neither JNK1/2/3 activation nor the enhanced expression of BAD and BAX, but did block caspase 3 cleavage, reduced expression of BCL-XL and inhibition of ERK1/2 activity. In contrast, incubation with NAC blocked JNK1/2/3 activation and cell killing, but not the increases in BAD and BAX expression. These findings argue that after combination treatment JNK1/2/3 activation is a primary pro-apoptotic event and loss of BCL-XL expression and ERK1/2 activity are secondary caspase-dependent processes. This data also argues that GST- MDA-7 induces two parallel pro-apoptotic pathways via ROS-dependent and -independent mechanisms. Infection of primary human astrocytes with a recombinant adenovirus to express MDA-7, Ad.mda-7, but not infection with either Ad.cmv or Ad.mda-7SP- lacking MDA-7 secretion, resulted in the suppression of GBM cell colony formation in soft agar overlay assays, an effect that was enhanced in a greater than additive fashion by radiation. Collectively, our findings demonstrate that MDA-7 reduces proliferation and enhances the radiosensitivity of nonestablished human GBM cells in vitro, and when grown in 3 dimensions, and that sensitization occurs independently of basal EGFR/ERK1/2/AKT activity or the functions of PTEN and p53.
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PMID:MDA-7 regulates cell growth and radiosensitivity in vitro of primary (non-established) human glioma cells. 1532 89

Pancreatic cancer is exceptionally aggressive with no long-term effective therapy. Current interventional approaches, including surgery, radiation and/or chemotherapy, have done little to quell the mortality associated with this malignancy. Subtraction hybridization identified a cancer-specific apoptosis-inducing cytokine gene, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), with a broad range of selective antitumor activity in diverse cancers both in vitro and in vivo in nude mice and recently in patients with advanced carcinomas and melanomas. Unlike most neoplasms, pancreatic cancers display innate resistance to mda-7/IL-24-induced apoptosis, which correlates with a diminished capacity to convert mda-7/IL-24 mRNA into protein. We presently demonstrate that this translational block can be reversed by treatment with agents that elevate reactive oxygen species (ROS). Induction of apoptosis in vitro and suppression of tumorigenesis in vivo in nude mice are induced in pancreatic cancers, irrespective of the status of their K-ras gene, only when tumor cells simultaneously express mda-7/IL-24 and are treated with a ROS-inducer, such as arsenic trioxide (ARS), N-(4-hydroxyphenyl) retinamide (HPR) or dithiophene (NSC656240 (NSC)). In pancreatic cancer cells constitutively expressing mda-7/IL-24 mRNA, a single treatment with arsenic trioxide, HPR or NSC656240 induces apoptosis, which correlates with production of MDA-7/IL-24 protein. The specificity of this action is documented by the ability of ROS inhibitors, including N-acetyl-L-cysteine and Tiron, to block this killing effect. Of potential clinical significance, similar treatment of normal cells does not elicit significant changes in growth nor does it induce apoptosis. Analysis of signal transduction changes in pancreatic carcinoma cells infected with Ad.mda-7 in combination with a ROS-inducer indicate that cell death correlates with modulation of discrete cassettes of multiple signaling pathways in a pancreatic cancer cell-specific manner, supporting global signaling dysregulation as a potential mediator of apoptosis induction. These findings suggest a promising combinatorial approach for safely promoting cell death in pancreatic tumors that provides a rational framework for developing a selective and effective therapy for this invariably fatal cancer.
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PMID:Induction of reactive oxygen species renders mutant and wild-type K-ras pancreatic carcinoma cells susceptible to Ad.mda-7-induced apoptosis. 1558 Mar 5

Effective therapies for most solid cancers, especially those that have progressed to metastasis, remain elusive because of inherent and acquired resistance of tumor cells to conventional treatments. Additionally, the effective therapeutic window for many protocols can be very narrow, frequently resulting in toxicity. The present study explores an anticancer strategy that effectively eliminates resistant cancer cells without exerting deleterious effects on normal cells. This approach employs melanoma differentiation-induced gene-7/interleukin-24 (mda-7/IL-24), a cancer-specific, apoptosis-inducing cytokine, in combination with nontoxic doses of a chemical compound from the endoperoxide class that decomposes in water generating singlet oxygen. This combinatorial regimen specifically induced in vitro apoptosis in prostate carcinoma cells, with innate resistance to chemotherapy or engineered resistance to mda-7/IL-24, as well as pancreatic carcinoma cells inherently resistant to any treatment modality, including mda-7/IL-24. Apoptosis induction correlated with increased cellular reactive oxygen species production and was prevented by general antioxidants, such as N-acetyl-l-cysteine or Tiron. Induction of apoptosis in combination-treated cancer cells correlated with a reduction in the antiapoptotic protein BCL-x(L). In contrast, both normal prostate and pancreatic epithelial cells were unaffected by the single or combination treatment. These provocative findings suggest that this combinatorial strategy might provide a platform for developing effective treatments for therapy-resistant cancers.
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PMID:Strategy for reversing resistance to a single anticancer agent in human prostate and pancreatic carcinomas. 1736 Jun 70

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k(F)(H2O)=72,600 s(-1)), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.
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PMID:The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond. 1849 59

Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the wild-type (WT) protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/visible, Cu and S K-edge X-ray absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form, which is shown by extended X-ray absorption fine structure to be composed of a novel two-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49, H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans, which facilitates the formation of the highly stable two-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at approximately 2.24 A corresponding to the two thiolate-copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at approximately 2.16 A. S K-edge data give a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than that observed for nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, from which it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron-transfer reactivity.
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PMID:Anatomy of a red copper center: spectroscopic identification and reactivity of the copper centers of Bacillus subtilis Sco and its Cys-to-Ala variants. 2023 70


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