Sox2基因3'非翻译区保守元件对基因表达的调控作用

转录因子Sox2是脊椎动物早期发育中最早表达的神经系统特异性基因之一,同时在干细胞的维持中也起着关键作用。通过生物信息学分析,作者发现在脊椎动物Sox2 mRNA 3'非翻译区中存在4段非常保守的富含AU的区域。将这些片段按照不同的组合克隆到GFP和荧光素酶两种报告基因载体中,在非洲爪蟾胚胎和培养细胞中检测了这些片段对报告基因表达的影响。结果显示,Sox2的3'UTR可影响报告基因的表达水平,特别是其中的保守片段2可显著提高报告基因的表达水平,表明Sox2 3'非翻译区有可能参与Sox2表达的转录后调控。     

The Translation Regulatory Subunit eIF3f Controls the Kinase-Dependent mTOR Signaling Required for Muscle Differentiation and Hypertrophy in Mouse

The mTORC1 pathway is required for both the terminal muscle differentiation and hypertrophy by controlling the mammalian translational machinery via phosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interacting with the eIF3 initiation complex. The regulatory subunit eIF3f plays a major role in muscle hypertrophy and is a key target that accounts for MAFbx function during atrophy. Here we present evidence that in MAFbx-induced atrophy the degradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutant insensitive to MAFbx polyubiquitination maintained persistent phosphorylation of S6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif in eIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation. Thus eIF3f plays a major role for proper activity of mTORC1 to regulate skeletal muscle size.     

hNOA1 Interacts with Complex I and DAP3 and Regulates Mitochondrial Respiration and Apoptosis

Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O₂ consumption ∼20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as γ-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.Emerging evidence indicates that mitochondrial metabolism, apoptosis, and dynamics (fission and fusion) are closely intertwined. Apoptosis and changes in metabolism are associated with morphological changes in mitochondria (1, 2). Conversely, when mitochondrial morphology is altered either by mutations or altered expression of mitochondrial fission or fusion proteins such as the dynamin like large G proteins Drp1 and Opa1, the cell''s susceptibility to apoptotic agents (3) or ability to generate ATP (4, 5) is altered.Apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers), and mitochondria play central roles in both pathways (6). The apoptotic pathways involve a growing list of mitochondria-associated proteins, such as Bad, cytochrome c, Smac, AIF, Bcl-2, and others, most of which are located either on the outer mitochondrial membrane (OMM)³ or in the intermembrane space (IMS) (7). Recently, proteins of the mitochondrial matrix such as DAP3, have also been shown to be involved in apoptosis (8). DAP3 has been reported to be involved in both γ-interferon- (9) and tumor necrosis factor-α-induced (10) apoptosis as well as staurosporine-induced mitochondrial fragmentation (11), but the detailed mechanisms involved remain to be elucidated.Besides their role in apoptosis, much more is known about the functions of mitochondria in respiration and generation of ATP. The electron transport chain in the inner mitochondrial membrane (IMM) contains four major enzyme complexes (Complexes I, II, III, and IV) that are involved in transferring electrons from NADH (Complex I-linked) or FADH2 (Complex II-linked) to O₂ and in pumping protons out of the matrix to create an electrochemical proton gradient, which is harnessed by ATP synthase to make ATP (12).Despite the accumulating evidence showing intercommunication between mitochondrial metabolism, apoptosis, and dynamics, how these processes are coordinated remains to be elucidated. In this study we characterize hNOA1, the human homologue of Arabidopsis thaliana nitric oxide-associated protein, 1 (AtNOA1) (13). hNOA1 is a large G protein closely related to dynamin that is associated with the IMM. Perturbation of hNOA1 affects mitochondrial morphology, Complex I-linked O₂ consumption, and the cell''s susceptibility to apoptotic stimuli, possibly through interactions with proteins such as Complex I and DAP3.     

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.     

转录因子Oct4、Sox2和Nanog在早期胚胎发育过程中的表达调控

胚胎发育的分子机理是现今胚胎工程及发育生物学研究迫切需要了解的。由于胚胎发育的早期阶段就是胚胎干细胞阶段,因此两者在研究上有很好的重合性。通过对调节胚胎干细胞基因转录的三个具有代表性的转录因子Oct4、Sox2和Nanog的综述,展现出胚胎发育分子机理研究概况。     

High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.     

Molecular Mechanism for SHP2 in Promoting HER2-induced Signaling and Transformation

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.The Src homology phosphotyrosyl phosphatase 2 (SHP2)² functions as a positive effector of cell growth and survival (1–4), migration and invasion (5–8), and morphogenesis and transformation (9–11). In receptor-tyrosine kinase signaling (12–14), SHP2 positively transduces the Ras-extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the phosphoinositide-3-kinase-Akt (or protein kinase B) signaling pathways. SHP2 also promotes cell transformation induced by the constitutively active form of fibroblast growth factor receptor 3 and v-Src (9, 11). The discovery of germline-activating SHP2 mutations in Noonan and LEOPARD syndrome patients (15–18) and the subsequent experimental demonstration of these phenotypes in knockin and transgenic mice expressing these mutants (19, 20) has led to the conclusion that disregulation of SHP2 is responsible for these disease states. Furthermore, somatic activating SHP2 mutations were discovered in juvenile myelomonocytic leukemia, acute myelogenous leukemia, and chronic myelomonocytic (18, 21) and are suggested to play a causative role.SHP2 possesses two Src homology 2 (SH2) domains in the N-terminal region that allow the protein to localize to substrate microdomains after tyrosyl phosphorylation of interacting proteins. The phosphotyrosyl phosphatase (PTP) domain in the C-terminal region is responsible for dephosphorylation of target substrates (13, 22). Mutation of the critical Cys residue in the active site of SHP2 abolishes its phosphatase activity, leading to the production of a dominant-negative protein (23). The activity of SHP2 is regulated by an intramolecular conformational switch. SHP2 assumes a “closed conformation” when inactive and an “open conformation” when active. In the closed conformation the N-SH2 domain interacts with the PTP domain, physically impeding the activity of the enzyme. Upon engagement of the SH2 domains with phosphotyrosine, the PTP domain is relieved of autoinhibition and dephosphorylates target substrates (23–26). Interaction between specific residues on the N-SH2 and the PTP domains mediates the closed conformation. Mutation of these residues leads to a constitutively active SHP2, and the occurrence of such mutations in humans causes the development of Noonan syndrome and associated leukemia (16–18).Recently, we have shown that inhibition of SHP2 in the HER2-positive breast cancer cell lines abolishes mitogenic and cell survival signaling and reverses transformation, leading to differentiation of malignant cells into a normal breast epithelial phenotype (27). Given the significance of HER2 in breast cancer, the finding that SHP2 plays a positive role was very interesting. We, thus, sought to investigate the molecular mechanism that underlies the positive role of SHP2 in HER2-induced signaling and transformation. To do so, it was first necessary to decipher the identity of SHP2 substrates whose dephosphorylation promotes the oncogenic functions of HER2. Using the recently developed substrate-trapping mutant of SHP2 as a reagent (28), we have identified HER2 itself as an SHP2 substrate. We have further shown that SHP2 dephosphorylates an autophosphorylation site on HER2 that serves as a docking site for the SH2 domains of the Ras GTPase-activating protein (Ras-GAP), the down-regulator of Ras. This effect of SHP2 increases the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation.     

d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene, and Purification and Characterization of the Recombinant Enzyme

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding d-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by dl-α-glycerophosphate or ethanol and destabilized by d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.As a participant in the oxidative pentose phosphate pathway, Ru5P epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P and Xu5P, is widely distributed throughout nature. Beyond its catabolic role, the epimerase is also vital anabolically to photosynthetic organisms in the regenerative phase of the reductive pentose phosphate pathway (the Calvin cycle). In this capacity, Ru5P epimerase directs Xu5P, formed in two distinct transketolase reactions of the cycle, to Ru5P. Phosphorylation of the latter regenerates d-ribulose-1,5-bisphosphate, the substrate for net CO₂ fixation. Because both the oxidative and reductive pentose phosphate pathways coexist in chloroplasts (Schnarrenberger et al., 1995), Ru5P epimerase and R5P isomerase facilitate partitioning of pentose phosphates between the two pathways, as dictated by the metabolic needs and redox status of the cell.Scant structural and mechanistic information about Ru5P epimerase is available despite its inherent importance and dual metabolic roles. This neglect may in part reflect the low natural abundance of the enzyme. For example, achievement of electrophoretic homogeneity required a 2000-fold purification from yeast (Bär et al., 1996) and spinach (Spinacia oleracea L.) chloroplasts (Teige et al., 1998) and 9000-fold purification from beef liver (Terada et al., 1985). Although low overall recoveries (<10%) further limited the availability of pure material, molecular sieving and denaturing electrophoresis established that the epimerases from mammals (Wood, 1979; Karmali et al., 1983; Terada et al., 1985) and yeast (Bär et al., 1996) are homodimers of approximately 23-kD subunits, whereas the enzyme from spinach chloroplasts may be an octamer of 23-kD subunits (Teige et al., 1998). DNA-deduced amino acid sequences of Ru5P epimerases from both photosynthetic and nonphotosynthetic sources, which confirm this estimated subunit size, show greater than 50% similarities among the most evolutionarily distant species examined (Kusian et al., 1992; Blattner et al., 1993; Falcone and Tabita, 1993; Lyngstadaas et al., 1995; Nowitzki et al., 1995; Teige et al., 1995).Although Ru5P epimerase has very recently been purified from a photosynthetic organism (spinach) for the first time (Teige et al., 1998), the low recovery (100 μg from 3.8 g of soluble chloroplast protein, representing an overall yield of 5%) imposes severe constraints on the directions of future experiments. Furthermore, despite successful cloning of cDNA fragments encoding Ru5P epimerase of several photosynthetic organisms (Kusian et al., 1992; Nowitzki et al., 1995; Teige et al., 1995), to our knowledge high-level heterologous expression and purification of enzymically active recombinant enzyme have not been achieved. Because of our interest in the regulation of photosynthetic carbon assimilation and the requisite need for ample supplies of the participant enzymes for use in mechanistic studies, we have attempted to optimize the heterologous expression of the spinach gene for Ru5P epimerase. In this paper we report cDNA clones that encode the mature chloroplastic enzyme or its cytoplasmic precursor. We also describe an efficient isolation procedure for the mature spinach enzyme synthesized in Escherichia coli and some of the properties of the purified enzyme. Contrasting features of the plant Ru5P epimerase, relative to the animal and yeast counterparts, include an octameric rather than a dimeric structure (also see Teige et al., 1998) and striking instability under routine laboratory conditions.     

Expression and role of Oct3/4, Nanog and Sox2 in regeneration of rat tracheal epithelium

Objectives:  To explore the role of Oct3/4, Nanog and Sox2 in regeneration of rat tracheal epithelium.
Materials and methods:  An ex vivo model of rat tracheal epithelial regeneration using 5-fluorouracil (5-FU) was developed, to induce injury. Expression levels of Oct3/4, Nanog and Sox2 were examined using Western blot analysis, RT-PCR or microscopically observed immunofluorescence, and cell morphological changes were observed using HE staining, during the recovery process.
Results:  Oct3/4, Nanog and Sox2 were not detectable in normal tracheal epithelium. After treatment with 5-FU, the normally proliferating tracheal epithelium desquamated and only a few cells in G0 phase of the cell cycle were left on the basement membrane and Oct3/4, Nanog and Sox2 could be observed at this time. Thereafter, the number of Oct3/4-, Nanog- and Sox2-positive cells increased gradually. When the cells differentiated into ciliate cells, mucous cells or basal cells, and restored pseudostratified mucociliary epithelium, the number of Oct3/4-, Nanog- and Sox2-positive cells decreased and gradually disappeared.
Conclusions:  G0 phase cells with resistance to 5-FU damage expressed Oct3/4, Nanog and Sox2. This indicated that these cells were undifferentiated, but had the ability to terminally differentiate into downstream-type cells. They possessed stem cell properties. The results are consistent with Oct3/4, Nanog and Sox2-expressing cells being considered as tracheal stem cells.