Molecular determinants of the interactions between SRC-1 and LXR/RXR heterodimers

Liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers have been shown to perform critical functions in cholesterol and lipid metabolism. Here, we have conducted a comparative analysis of the contributions of LXR and RXR binding to steroid receptor coactivator-1 (SRC-1), which contains three copies of the NR box. We demonstrated that the coactivator-binding surface of LXR, but not that of RXR, is critically important for physical and functional interactions with SRC-1, thereby confirming that RXR functions as an allosteric activator of SRC-1-LXR interaction. Notably, we identified NR box-2 and -3 as the essential binding targets for the SRC-1-induced stimulation of LXR transactivity, and observed the competitive in vitro binding of NR box-2 and -3 to LXR.

Structured summary

MINT-7986678, MINT-7986639, MINT-7986700, MINT-7986720, MINT-7986736, MINT-7986760, MINT-7986787: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) and RXR (uniprotkb:P19793) by pull down (MI:0096)MINT-7986596, MINT-7986621: SRC1 (uniprotkb:Q15788) physically interacts (MI:0915) with LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986555, MINT-7986575: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) by two hybrid (MI:0018)MINT-7986808, MINT-7986907, MINT-7986890: SRC1 (uniprotkb:Q15788) binds (MI:0407) to LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986822, MINT-7986848, MINT-7986865: SRC1 (uniprotkb:Q15788) binds (MI:0407) to RXR (uniprotkb:P19793) by pull down (MI:0096)     

HIV-1 Protease Has a Genetic T-Cell Adjuvant Effect Which Is Negatively Regulated by Proteolytic Activity

HIV protease (PR) mediates the processing of human immunodeficiency virus (HIV) polyproteins and is necessary for the viral production. Recently, HIV PR was shown to possess both cytotoxic and chaperonelike activity. We demonstrate here that HIV PR can serve as a genetic adjuvant that enhances the HIV Env and human papillomavirus (HPV) DNA vaccine-induced T-cell response in a dose-dependent manner, only when codelivered with DNA vaccine. Interestingly, the T-cell adjuvant effects of HIV PR were increased by introducing several mutations that inhibited its proteolytic activity, indicating that the adjuvant properties were inversely correlated with its proteolytic activity. Conversely, the introduction of a mutation in the flap region of HIV PR limiting the access to the core domain of HIV PR inhibited the T-cell adjuvant effect, suggesting that the HIV PR chaperonelike activity may play a role in mediating T-cell adjuvant properties. A similar adjuvant effect was also observed in adenovirus vaccine, indicating vaccine type independency. These findings suggest that HIV PR can modulate T-cell responses elicited by a gene-based vaccine positively by inherent chaperonelike activity and negatively by its proteolytic activity.Human immunodeficiency virus protease (HIV PR) is a typical aspartic protease required for processing HIV polyproteins such as Gag and Pol and is essential for HIV production (5). HIV PR consists of three domains: terminal, core, and flap domains, and each has a unique role in the proteolytic process (21). The substrate-binding site is found in the core domain that contains the catalytic triad (DTG). The terminal domain is required for dimerization, which is also important for its proteolytic activity, and the flap domain regulates substrate access (2).In addition to the above-described processes, HIV PR also cleaves cellular proteins important to cell survival including the antiapoptotic protein, Bcl-2 (26), and procaspase-8 (20), which mediates the apoptosis of HIV-infected cells or of cells transfected with DNA encoding HIV PR. The importance of HIV PR proteolytic activity in mediating cell death was highlighted in studies that demonstrated the inhibition of cell death after mutations to the DTG catalytic site (26) or after the treatment with protease inhibitors such as ritonavir or saquinavir (19).HIV PR was also shown to have inherent chaperonelike activity mediated by the core domain. The introduction of a mutation that removed the aspartic acid residue of the catalytic site or inhibition of dimerization necessary for proteolytic activity did not affect its chaperonelike activity in vitro, suggesting that the chaperonelike activity was independent of its proteolytic properties (8).Typical chaperones, such as heat shock protein 70 (Hsp70), Hsp90, and gp96, were reported to chaperone antigenic peptides and mediate cross-priming of cognate antigen-specific CD8 T cells in vivo (1). In addition, the minimal 136-amino-acid peptide binding domain of the mycobacterial Hsp70 efficiently generated CD8 T-cell responses against the complexed peptide. Hsps can also protect processed peptides from further proteosomal degradation and enhance targeting to dendritic cells via the interactions of Hsps with surface molecules, including CD91, TLR4, or CCR5, resulting in CD8 T-cell cross-priming (10).In the present study, we demonstrated that the codelivery of HIV PR could enhance the T-cell response, but not the humoral response, elicited by DNA and adenoviral vaccines and that the T-cell response was further augmented by HIV PR mutations that inhibited proteolytic activity. Interestingly, the T-cell adjuvant effect of the catalytic mutant was reduced by the introduction of a point mutation that stabilized the flap domain into a closed position and not by a mutation that inhibited dimerization. These data suggested that HIV PR has a T-cell adjuvant effect presumably due to the intrinsic chaperonelike activity which is veiled by its proteolytic activity.     

Cremanthodium latilobum sp. nov. and C. medogense sp. nov. (Asteraceae) from Chinese eastern Himalaya

Cremanthodium latilobum Y. S. Chen and C. medogense Y. S. Chen, two new species from Chinese eastern Himalaya are described and illustrated. A diagnostic key to the species of Cremanthodium section Pinnatinervia Y. Ling & S. W. Liu series Cuneata Y. Ling & S. W. Liu is provided.     

Ectopic overexpression of <Emphasis Type="Italic">Arabidopsis AtmiR393a</Emphasis> gene changes auxin sensitivity and enhances salt resistance in tobacco

To characterize the biological function of microRNA miR393 in tobacco, AtmiR393a gene was isolated from Arabidopsis using PCR and fused downstream to CaMV 35S promoter to make a plant expression construct 35S::AtmiR393a. The resultant construct was then introduced into tobacco with Agrobacterium-mediated transformation. Transgenic tobacco lines ectopically overexpressing AtmiR393a were successfully obtained. Transgenic lines L1 (a weak line), L2 (a middle line), and L3 (a strong line) were confirmed using stem-loop RT-PCRs and used to characterize the function of miR393 in tobacco. The results showed that L1, L2, and L3 exhibited reduced plant size and root length related to the WT control. In addition, seedling growth was less sensitive to IAA treatment and NaCl stress in three transgenic lines than the non-transgenic WT control. Furthermore, L1, L2, and L3 showed reduced phototropism relative to WT. Therefore, the biological function of miR393 is conserved in tobacco, just like in Arabidopsis. It regulates plant growth and development as well as the responses to environmental cues by influencing auxin sensitivity.     

Colorimetric pH measurement of animal cell culture media

Most animal cell culture media can be buffered using bicarbonate and high pressure CO₂ in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO₂ pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.     

Gaegurin-6 stimulates insulin secretion through calcium influx in pancreatic β Rin5mf cells

Gaegurin-6, an antimicrobial peptide that belongs to the alpha-helix family, was isolated from the skin of Rana rugosa. Gaegurin-6 contains a hydrophobic motif at the N-terminus and a helical region at the C-terminus. Although gaegurin-6 has been implicated in cell signaling, the precise role in insulin secretion is currently unknown. We have attempted to determine whether gaegurin-6 affects insulin secretion and tried to elucidate the relationship between the structural motifs and biological activity. In this study, we have shown that gaegurin-6 stimulates insulin secretion and also increases the intracellular calcium concentration in pancreatic β Rin5mf cells. Moreover, a corollary study revealed that both the hydrophobicity of the N-terminus and the disulfide bridge of the C-terminus of gaegurin-6 are critical for its effects on insulin secretion. Membrane pore-forming ability is also observed in gaegurin-6, but not in the linear form or the N-terminus hydrophobic amino acid-deleted form. We further showed that these regions of gaegurin-6 are responsible for calcium influx in pancreatic β Rin5mf cells. Taken together, these results indicate that gaegurin-6 can affect insulin secretion in pancreatic β cells through the modulation of calcium influx.     

Molecular characterization,expression pattern,and functional analysis of the <Emphasis Type="Italic">OsIRL</Emphasis> gene family encoding intracellular Ras-group-related LRR proteins in rice

Leucine-rich repeat proteins constitute a large gene family and play important roles in plant growth and development. Among them, Arabidopsis PIRL is a plant-specific class of intracellular Ras-group-related leucine-rich repeat proteins. In this study, we identified eight homologues of PIRLs in rice and designated them as OsIRL proteins. We described the gene structures, chromosome localizations, protein motifs, and phylogenetic relationships of the OsIRL gene family. The expression profiles of OsIRL genes were analyzed throughout the entire rice life cycle, along with light and three hormone stress conditions, using quantitative RT-PCR and microarray data. All OsIRL genes were expressed in at least one experimental stage and exhibited divergent expression patterns, with several genes showing preferential expression at specific stages. OsIRL4 and OsIRL5 showed higher expression levels under light compared to dark. OsIRL4 and OsIRL7 exhibited significant differential expression in response to hormone treatments. Six T-DNA or Tos17 insertion lines for five individual OsIRL genes were identified and examined morphologically. The comprehensive expression profile elucidated in this investigation together with the characterized insertion lines will provide a solid foundation for in-depth dissection of OsIRL functions.     

Signaling through Tyr985 of Leptin Receptor as an Age/Diet-Dependent Switch in the Regulation of Energy Balance

Leptin regulates energy homeostasis through central activation of multiple signaling pathways mediated by Ob-Rb, the long form of leptin receptor. Leptin resistance underlies the pathogenic development of obesity, which is closely associated with environmental factors. To further understand the physiological function of leptin signaling mechanisms, we generated a knock-in line of mice (Y985F) expressing a mutant Ob-Rb with a phenylalanine substitution for Tyr⁹⁸⁵, one of the three intracellular tyrosines that mediate leptin''s signaling actions. Surprisingly, whereas young homozygous Y985F animals were slightly leaner, they exhibit adult-onset or diet-induced obesity. Importantly, both age-dependent and diet-induced deterioration of energy balance was paralleled with pronounced leptin resistance, which was largely attributable to attenuation of leptin-responsive hypothalamic STAT3 activation as well as prominently elevated expression of hypothalamic SOCS3, a key negative regulator of leptin signaling. Thus, these results unmask distinct binary roles for Try⁹⁸⁵-mediated signaling in energy metabolism, acting as an age/diet-dependent regulatory switch to counteract age-associated or diet-induced obesity.As a component of the metabolic syndrome, obesity is closely associated with increased risk for the development of type 2 diabetes and cardiovascular disorders (16). Arising from a chronic imbalance between energy intake and expenditure, the pathogenic progression of obesity is attributable to the complex interactions between genetic factors and environmental influences. In mammals, energy balance is maintained through multiple homeostatic mechanisms that operate coordinately in response to hormonal and nutritional cues. Leptin is an adipose-secreted hormone (43) that plays a pivotal role in the regulation of energy metabolism. Acting through its active-form receptor Ob-Rb in distinct classes of leptin-responsive neurons (11, 14, 34), leptin activates multiple signaling pathways in the hypothalamus to regulate food intake and energy expenditure. Mice with deficiency in leptin (ob/ob) or its functional receptor (db/db) develop morbid obesity, hyperphagia, and diabetes (20). Impaired leptin responsiveness, i.e., leptin resistance (33), is a key characteristic of the metabolic defects that are responsible for disrupted energy control, presumably underlying the pathogenic development of human obesity (29). Although diminished leptin signaling has been found to occur in association with aging (21, 39) or feeding of a high-fat diet (HFD) (17, 18), the exact physiological mechanisms linking the environmental factors to the impairment in leptin-mediated regulation of energy metabolism remain largely elusive.Leptin binds to Ob-Rb and elicits an array of subsequent intracellular signaling cascades (7, 22) via Jak2 phosphorylation. The mouse Ob-Rb comprises three cytoplasmic tyrosine residues, Tyr⁹⁸⁵, Tyr¹⁰⁷⁷, and Tyr¹¹³⁸, which are known to be phosphorylated and mediate leptin''s physiological functions (22, 26). The phosphorylated Tyr¹¹³⁸ is thought to recruit STAT3 (1), thereby activating the JAK2-STAT3 pathway, which has been shown to play an important role in the control of energy balance (2), whereas our recent investigation has demonstrated crucial actions in vivo for Ob-Rb tyrosine-dependent as well as tyrosine-independent mechanisms in the regulation of energy and glucose homeostasis (26). Our earlier studies in vitro as well as observations from other laboratories have also documented that phosphorylation at Tyr⁹⁸⁵ leads to recruitment of SH2-containing protein tyrosine phosphatase 2 (SHP2) (28, 41) and activation of extracellular signal-regulated kinase (ERK) (9). On the other hand, phosphorylated Tyr⁹⁸⁵ has been postulated to serve as a docking site for SOCS3, thereby exerting an antagonizing effect on Tyr¹¹³⁸-mediated STAT3 activation (8). Consistent with this, a leptin-activated autoinhibitory action in vivo has recently been suggested for Tyr⁹⁸⁵ in the l/l mice expressing a mutant leptin receptor where Tyr⁹⁸⁵ was replaced with leucine (10). However, whereas elevated hypothalamic expression of SOCS3 has been reported to occur in aged rodents (36) or in mice with diet-induced obesity (18, 42), it has yet to be understood whether Ob-Rb Tyr⁹⁸⁵-mediated mechanisms are physiologically connected to altered SOCS3 expression, particularly in the face of aging or high dietary fat intake. Moreover, direct in vivo evidence also has been lacking with respect to whether there exist potential interplays between Ob-Rb Tyr⁹⁸⁵ signaling and other Ob-Rb tyrosine-dependent mechanisms, which act to influence the homeostatic control of energy balance.To gain further insight into the roles of Ob-Rb intracellular tyrosine phosphorylation in mediating leptin''s physiological functions in vivo, we previously generated two lines of mice expressing mutant leptin receptors with phenylalanine substitution for all three tyrosines or for Tyr¹¹³⁸ alone, revealing the metabolic contribution of both tyrosine-dependent and -independent actions in energy homeostasis (26). Here we investigated the physiological consequences of abrogation of signaling through Ob-Rb Tyr⁹⁸⁵ via characterization of the knock-in mice generated by introducing a phenylalanine substitution mutation at this site. We examined the impact of deficiency in Tyr⁹⁸⁵-mediated signaling upon the susceptibility of mice to age-associated and diet-induced energy imbalance, attempting to explore the potential mechanistic links between leptin resistance and environmental influences such as aging and overnutrition.     

蓝花侧金盏化学成分的研究

从藏药蓝花侧金盏的乙醇溶液中分离得到8个化合物,通过波谱学方法分别鉴定为三十一烷醇(1)、对甲酰基肉桂酸(2)、芹菜素(3)、木犀草素(4)、荭草苷(5)、木犀草素-7-O-β-D-葡萄糖苷(6)、异荭草苷(7)及侧金盏醇(8)。化合物1、2、4、6、7均首次从该种植物中分离得到。