雌激素受体2（ESR2）mRNA 5′非翻译区内部核糖体进入位点活性的鉴定与分析

真核生物除了传统的帽依赖型翻译机制外,还存在内部核糖体进入位点（internal ribosome entry site, IRES）介导的翻译机制。雌激素受体2（estrogen receptor 2, ESR2）是雌激素受体家族成员之一,其编码的蛋白质在许多肿瘤中发挥重要的作用。ESR2蛋白的异常表达会导致众多肿瘤的发生,但其蛋白质翻译水平的调控机制至今仍不清楚。研究发现,在药物刺激的条件下,乳腺癌细胞MCF7/WT中ESR2蛋白的表达提高,但是其转录水平基本未见发生改变。猜测ESR2 mRNA 5′非翻译区（5′ untranslated region, 5′ UTR）具有IRES活性。为了验证ESR2 mRNA 5′ UTR是否具有IRES元件,将ESR2 mRNA 5′ UTR插入到双顺反子报告基因载体（pRF）中,构建pRL-ESR2-FL重组质粒载体,将其瞬时转染到HEK293细胞。结果发现,ESR2 mRNA 5′ UTR有假定的IRES活性。并且通过3个排除实验验证了ESR2 IRES活性与其5′ UTR中的内部潜在启动子（P<0.0001）、内部剪切位点以及核糖体通读无关。进一步对其序列进行截短研究发现,ESR2 IRES活性发挥的关键区域是3′端的439～468 nt,且ESR2 IRES最大活性的发挥依赖于5′ UTR序列的完整性。并且发现,ESR2 IRES活性的发挥不但需要特定的一级核酸序列,还要有稳定的二级茎环结构。此研究有望为ESR2蛋白调控的相关疾病提供新的药物治疗靶点。     

Mutational analysis of the interaction between insulin receptor and IGF-I receptor with c-Crk and Crk-L in a yeast two-hybrid system

The SH2/SH3 adapter proteins of the Crk family are potent signal transducers after receptor tyrosine kinase stimulation with insulin or IGF-1. We have employed a yeast two-hybrid approach and mutational analysis to dissect the capabilities of the insulin receptor and the IGF-I receptor to directly associate with Crk isoforms. Insulin receptor stably recruits full length Crk by association with its SH2 domain in an auto-phosphorylation dependent manner. In contrast, interaction of the IGF-I receptor with the Crk-IISH2 domain was only detectable when Crk-II was truncated in its C-terminal part, indicating the transient nature of this interaction. From these data it can be concluded that members of the insulin receptor family activate Crk proteins in a differential manner.     

Local requirement of the Drosophila insulin binding protein imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions.     

The variation in Fas localization and the changes in Fas expression level upon stimulation with growth factors

Although Fas (APO-1/CD95) is well known as a death receptor, its stimulation occasionally fails to induce apoptosis in malignant cells. On the contrary, Fas is reported to advance the cell cycle in cancer cells. Therefore, we investigated roles of Fas in cell growth and apoptosis using human lung cancer cell lines. Fas was localized in the cytoplasm in exponentially growing cells, whereas only confluent cells expressed Fas on the cell membrane. A stimulation of confluent cells by either of EGF, IGF-I or VEGF induced once a decrease in Fas expression level and its sequential recovery. Fas expression levels in confluent cells were negatively correlated with cell doubling times (r=0.757, p=0.0088). Fas remained on the cell membrane of IgM-treated cells even after the growth factor stimulation, leading to apoptosis with abnormal mitosis, whereas the same stimulation induced Fas internalization in IgG(1)-treated cells. From these results, we suggest that Fas remaining on the cell membrane amplifies to induce apoptosis. Conversely, Fas internalization may enable cancer cells to escape from apoptosis. Our results suggest that growth factor may contribute to the resistance of cancer cells to Fas-mediated apoptosis in an autocrine or paracrine fashion.     

Substrate preference of transglutaminase 2 revealed by logistic regression analysis and intrinsic disorder examination

Tissue transglutaminase (TG2) catalyzes the Ca²⁺-dependent posttranslational modification of proteins via formation of isopeptide bonds between their glutamine and lysine residues. Although substrate specificity of TG2 has been studied repeatedly at the sequence level, no clear consensus sequences have been determined so far. With the use of the extensive structural information on TG2 substrate proteins listed in TRANSDAB Wiki database†, a slight preference of TG2 for glutamine and lysine residues situated in turns could be observed. When the spatial environment of the favored glutamine and lysine residues was analyzed with logistic regression, the presence of specific amino acid patterns was identified. By using the occurrence of the predictor amino acids as selection criteria, several polypeptides were predicted and later identified as novel in vitro substrates for TG2. By studying the sequence of TG2 substrate proteins lacking available crystal structure, the strong favorable influence on substrate selection of the presence of substrate glutamine and lysine residues in intrinsically disordered regions could also be revealed. The collected structural data have provided novel understanding of how this versatile enzyme selects its substrates in various cell compartments and tissues.     

Molecular Analysis of Two Novel Missense Mutations in the GDF5 Proregion That Reduce Protein Activity and Are Associated with Brachydactyly Type C

Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5^T201P and proGDF5^L263P revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.