Rapid Proteasomal Degradation of Posttranscriptional Regulators of the TIS11/Tristetraprolin Family Is Induced by an Intrinsically Unstructured Region Independently of Ubiquitination

The TIS11/tristetraprolin (TTP) CCCH tandem zinc finger proteins are major effectors in the destabilization of mRNAs bearing AU-rich elements (ARE) in their 3′ untranslated regions. In this report, we demonstrate that the Drosophila melanogaster dTIS11 protein is short-lived due to its rapid ubiquitin-independent degradation by the proteasome. Our data indicate that this mechanism is tightly associated with the intrinsically unstructured, disordered N- and C-terminal domains of the protein. Furthermore, we show that TTP, the mammalian TIS11/TTP protein prototype, shares the same three-dimensional characteristics and is degraded by the same proteolytic pathway as dTIS11, thereby indicating that this mechanism has been conserved across evolution. Finally, we observed a phosphorylation-dependent inhibition of dTIS11 and TTP degradation by the proteasome in vitro, raising the possibility that such modifications directly affect proteasomal recognition for these proteins. As a group, RNA-binding proteins (RNA-BPs) have been described as enriched in intrinsically disordered regions, thus raising the possibility that the mechanism that we uncovered for TIS11/TTP turnover is widespread among other RNA-BPs.     

A Mammalian Homolog of the Bacterial Monomeric Sarcosine Oxidases Maps to Mouse Chromosome 11, Close toCryba1

We have cloned a cDNA from a mouse gene,Pso(peroxisomal sarcosine oxidase).Psoappears to encode a homolog of the single-subunit (40 kDa) bacterial sarcosine oxidases. The mousePsogene product would contain a peroxisomal localization sequence, like that of the recently reported rabbit enzyme. MousePsolies between 20 and 50 kb upstream of the promoter of theSez6gene, close toCryba1on chromosome 11.Psois expressed very strongly and specifically in liver and kidney. The gene appears to be present widely in eutherian mammals.     

Cloning and characterization of the rat TIS11 gene

Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid peroxidation. Pf has been shown to cause inhibit NADPH-dependent lipid peroxidation in sheep liver microsomes in a dose-dependent manner. The concentration of Pf required to cause 50% inhibition of lipid peroxidation was ~ 6 mM. Pf was found to be ineffective as a superoxide radical scavenger. Pf was also ineffective in decomposing H₂O₂ and chelating iron. In deoxyribose degradation assays, Pf was a potent scavenger of hydroxyl radicals with a rate constant of 5.4 × 10⁹ M^-1 sec^-1. EPR spectroscopy in combination with spin trapping techniques, using a Fenton type reaction and DMPO as a spin-trapping agent, Pf scavenged hydroxyl radicals in a dose-dependent manner. The concentration of Pf required to inhibit 50% signal height was ~ 2.5 mM. Because iron was used in the Fenton reaction, the ability of Pf in chelating iron was verified in a fluorescent competitive assay using calcein as the fluorescent probe. Pf up to 10 mM concentration was ineffective in chelating either Fe²⁺ or Fe³⁺ in this system. We propose that Pf exerts its beneficial effects, at least in part, through its ability to scavenge toxic hydroxyl radicals.     

Drosophila RISC Component VIG and Its Homolog Vig2 Impact Heterochromatin Formation

Heterochromatin formation plays an important role in gene regulation and the maintenance of genome integrity. Here we present results from a study of the D. melanogaster gene vig, encoding an RNAi complex component and its homolog vig2 (CG11844) that support their involvement in heterochromatin formation and/or maintenance. Protein null mutations vig^EP812 and vig2^PL470 act as modifiers of Position Effect Variegation (PEV). VIG and Vig2 are present in polytene chromosomes and partially overlap with HP1. Quantitative immunoblots show depletion of HP1 and HP2 (large isoform) in isolated nuclei from the vig^EP812 mutant. The vig2^PL470 mutant strain demonstrates a decreased level of H3K9me2. Pull-down experiments using antibodies specific to HP1 recovered both VIG and Vig2. The association between HP1 and both VIG and Vig2 proteins depends on an RNA component. The above data and the developmental profiles of the two genes suggest that Vig2 may be involved in heterochromatin targeting and establishment early in development, while VIG may have a role in stabilizing HP1/HP2 chromatin binding during later stages.     

Mammalian Homolog of Drosophila Tumor Suppressor Lethal (2) Giant Larvae Interacts with Basolateral Exocytic Machinery in Madin-Darby Canine Kidney Cells

The Drosophila tumor suppressor protein lethal (2) giant larvae [l(2)gl] is involved in the establishment of epithelial cell polarity during development. Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion. Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain. These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis.     

A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee . We named the allele chickadee^bin , in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadee^{wc57 and WF57} (2). In addition, the egg chamber of chickadee^bin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.     

A Bacterial Homolog of a Eukaryotic Inositol Phosphate Signaling Enzyme Mediates Cross-kingdom Dialog in the Mammalian Gut

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果蝇心脏基因一个新人同源基因WNT-10A的研究初报

Ｗｇ基因是控制果蝇心脏前体细胞形成的一个关键基因,根据物种间同源异型基因结构上的保守性与功能上的相似性,我们运用计算机克隆的方法获得了一个新的人同源基因,命名为ＷＮＴ－１０Ａ。该基因有一富集ＧＣ碱基的启动子,ｍＲＮＡ全长约２．４ｋｂ,３′末端包含ＡＴＡＡＡ的加尾信号,编码一段长４１７个氨基酸的蛋白质,与小鼠Ｗｎｔ－１０ａ的编码蛋白高度相似。其心脏ＥＳＴ数目占正常组织ＥＳＴ总数的４５％,表明该基因在心脏组织高度表达,提示其可能与心脏发育有关。该基因与其基因家族成员具有相似的同源框序列,在肿瘤细胞中大量表达（占总ＥＳＴ的３１％）,表明该基因相似于其家族成员,可能与肿瘤的发生有关。     

A computational study of RNA binding and specificity in the tandem zinc finger domain of TIS11d

TIS11d is a member of the CCCH-type family of tandem zinc finger (TZF) proteins; the TZF domain of TIS11d (residues 151–220) is sufficient to bind and destabilize its target mRNAs with high specificity. In this study, the TZF domain of TIS11d is simulated in an aqueous environment in both the free and RNA-bound states. Multiple nanosecond timescale molecular dynamics trajectories of TIS11d wild-type and E157R/E195K mutant with different RNA sequences were performed to investigate the molecular basis for RNA binding specificities of this TZF domain. A variety of measures of the protein structure, fluctuations, and dynamics were used to analyze the trajectories. The results of this study support the following conclusions: (1) the structure of the two fingers is maintained in the free state but a global reorientation occurs to yield a more compact structure; (2) mutation of the glutamate residues at positions 157 and 195 to arginine and lysine, respectively, affects the RNA recognition by this TIS11d mutant in agreement with the findings of Pagano et al. (J Biol Chem 2007; 282:8883–8894); and (3) we predict that the E157R/E195K mutant will present a more relaxed RNA binding specificity relative to wild-type TIS11d based on the more favorable nonsequence-specific Coulomb interaction of the two positively charged residues at positions 157 and 195 with the RNA backbone, which compensates for a partial loss of the stacking interaction of aromatic side chains with the RNA bases.     

The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division

During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the G alpha binding partner Pins, but how the Pins-G alphai complex interacts with the mitotic spindle is unclear. Here, we show that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of NuMA. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. In the absence of Mud, mitotic spindles in Drosophila neuroblasts fail to align with the polarity axis. This can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the mis-specification of daughter cell fates and tumor-like over proliferation in the Drosophila nervous system. Our data suggest a model in which asymmetrically localized Pins-G alphai complexes regulate spindle orientation by directly binding to Mud.     

Maintenance of Interphase Chromosome Compaction and Homolog Pairing in Drosophila Is Regulated by the Condensin Cap-H2 and Its Partner Mrg15

Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein–protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.     

A Cytohesin Homolog in Dictyostelium Amoebae

Background

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.

Principal Findings

Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG⁻ cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced.

Significance

The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.     

Mammalian DNMTs in the male germ line DNA of Drosophila

It is controversial whether DNA methylation plays a functional role in Drosophila. We have studied testis DNA of Drosophila melanogaster Meigen, 1830 with antisera against 5-methylcytosine (5mC) and found no evidence for the presence of significant amounts of 5mC. Reactions occur only with 1 of 3 5mC antisera, but they are restricted to nuclear regions without detectable amounts of DNA. The antisera apparently cross-react with other nuclear components. If the murine de novo DNA methyltransferases, DNMT3A and DNMT3B, are expressed under the control of the spermatocyte-specific beta2-tubulin promoter in testes, DNA methylation is not increased and no effects on the fertility of the fly are seen. DNA methylation has, therefore, no functional relevance in the male germ line of Drosophila.     

苹果α-法尼烯合酶基因的启动子序列及同源基因

本文通过设计引物进行PCR扩增α-法尼烯合酶(AFS)基因的5'端区段并测序,获得510bp的‘国光’苹果AFS基因启动子和5'端非翻译区(5'UTR)序列,已在GenBank注册(登录号FJ263961)。序列分析结果表明,该序列具有典型的启动子特征,在转录起始点上游-46bp处有一个TATA盒,-93bp处有一个CAAT盒,-84bp处有一个W盒和-436bp处有一个热胁迫反应顺式作用元件GAAATTTTTT。与‘皇家嘎拉’苹果的AFS基因启动子序列(GenBank登录号AY786553.1)比对,本研究发现‘国光’苹果AFS基因启动子序列中有6个碱基(-186T,-207T,-283C,-301A,-413A和-433A)发生变异,‘皇家嘎拉’苹果AFS基因启动子序列相应位置的碱基分别为碱基缺失、-206C、-282G、-300G、-412G和-432G。重要的是,其中‘国光’苹果-413A碱基变异为G发生在一个热胁迫反应顺式作用元件GAAATTTTTT中,苹果虎皮病发生和AFS基因的转录表达是否受到这一碱基变异的影响值得进一步探讨。本研究结果还表明,在苹果AFS基因启动子和5'UTR序列中存在一个正向重复序列1(R9+IR18+R9),重复单元R9长度9bp,转录起始点位于其长度18bp的IR18区段。有趣的是,本研究新发现了一个与AFS基因启动子和5'UTR序列高度同源的450bp基因片段(GenBank注册登录号FJ469631),该同源片段缺失AFS基因中的27bp序列(R9+IR18,包含转录起始点)。据我们所知,这是果树中存在AFS基因启动子同源序列的首次报道。     

The Expression of Tristetraprolin and Its Relationship with Urinary Proteins in Patients with Diabetic Nephropathy

Objective

Tristetraprolin (TTP), also known as zinc finger protein 36, is an RNA binding protein that has a significant role in regulating the expression of mRNAs containing AU-rich elements. We postulated that TTP might regulate interleukin (IL)-6 and IL-18 expression in diabetes. This study aimed to test the hypothesis that the levels of TTP are correlated with nephropathy in patients with type 2 diabetes.

Methods

Eighty-seven patients (61.3±9.6 years old) who had been diagnosed with type 2 diabetes mellitus and 41 age and sex matched healthy control subjects were enrolled. The diabetes patients were classified into those without proteinuria, with microalbuminuria, and with clinical proteinuria groups according to the ratio of urinary excretion of albumin/creatinine (ACR).

Results

Serum and urinary levels of IL-6 and IL-18 were significantly elevated, but those of TTP were significantly decreased in patients with diabetes as compared with control subjects. In addition, serum and urinary levels of IL-6 and IL-18 were significantly higher, but those of TTP were significantly lower in patients with proteinuria than in patients without proteinuria or with microalbuminuria. There was a significant correlation between serum TTP and IL-6/IL-18 (correlation coefficients of -0.572 and -0.685, P < 0.05).

Conclusion

These results show that diabetes with clinical proteinuria is accompanied by decreased urinary and serum level of TTP and increased levels of IL-6 and IL-18. Decreased TTP expression might occur prior to the increase in IL-6 and IL-18, and decrease of TTP might provide an earlier marker for glomerular dysfunction than IL-6 and IL-18.