The two-stranded alpha-helical coiled-coil is an ideal model for studying protein stability and subunit interactions.

We have designed de novo a two-stranded alpha-helical coiled-coil which consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment. Their structure is stabilized by interchain hydrophobic interactions from hydrophobes at positions "a" and "d" of a repeating heptad sequence. The formation and stability of the coiled-coil is dependent on peptide concentration due to the monomer-dimer equilibrium. In contrast, that coiled-coil containing an inter-helical disulfide bond does not show any concentration dependence in the guanidine hydrochloride denaturation experiments as expected. Replacement of one large hydrophobic Leu residue in each chain with Ala significantly decreases coiled-coil stability in both the reduced and oxidized coiled-coils [decreases in transition midpoint of 1.6M (2.3-0.7) and 2.4M (5.3-2.9), respectively]. A large pH dependence on coiled-coil stability is observed over the pH range 4 to 7 (transition midpoints at pH 4, 5, 5.5, 6 and 7 were 3.8, 3.2, 2.0, 1.2 and 0.7M, respectively). The increasing stability with decreasing pH correlates with the protonation of the Glu acid side-chains and reduction of intrachain repulsions between Glu-Glu side-chains in positions i, i + 3 or i, i + 4 along each alpha-helix of the coiled-coil. In addition, coiled-coil stability increases with increasing ionic strength.     

Conformational interconversion in protein crystals.

We present evidence that the structure of carbonmonoxy myoglobin crystals can be altered by lowering the pH. This structural change is monitored by the characteristic Fe-CO Raman modes at 508 and 491 cm-1 and is thought to involve a localized distal pocket transition from a "closed" conformation at pH 7 to a more "open" conformation at pH 4. These changes take place in the crystal without loss of intensity of a conformationally sensitive Raman mode at 252 cm-1 that signals a partial unfolding of the globin structure in solution. Quantitative studies, which monitor the open and closed populations as a function of laser photolysis, demonstrate that the interconversion rates (k+/-) in solution at 298 K are fast compared to the photolysis and CO entry rates (i.e. k+/- much greater than 10(3) s-1), while in frozen samples the interconversion is much slower than the experimental time scale (minutes). Since the open conformation is a minority species at pH 7, rapid exchange in aqueous solution is a necessary condition for this species to play a functional role. In the crystal, the interconversion rates are slowed compared to solution and begin to approach the photolysis rate (i.e. k+/- approximately 10(3) to 10(4) s-1). This indicates that the barriers for conformational exchange are increased in the crystal environment, compared to the solution, apparently due to the packing forces of the surrounding molecules. X-ray and neutron diffraction studies of MbCO crystals at high and low pH are needed to characterize the details of the structural changes and to test the hypothesis that closed and open distal pocket structures are associated with the 508 and 491 cm-1 Fe-CO modes.     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.     

华东地区黑果蝇自然群体同工酶遗传多态的研究

我们用标准垂直板聚丙烯酰胺凝胶电泳和水平板琼脂糖凝胶电泳技术检测了黑果蝇(D.virilis)在合肥、芜湖、九江、南昌、福州、泉州和常州7个自然群体中Est-α、Est-β、Amy、Acph和α-Gpdh 5个座位的遗传变异,发现Est-α、Est-β和Amy 3个座位是高度多态的,Acph、α-Gpdh两个座位则是单态的。根据这5个座位等位基因的频率,我们计算了群体间的遗传距离。综合何朝珍报道的宁波、杭州、南京和洪泽4个群体的结果和我们的结果,我们作出系统树并发现泉州、福州两群体和其他群体在基因频率的分布和遗传距离方面有显著差异;分析显示这种差异与群体间地理隔离有关。     

Thioredoxin 1 supports colorectal cancer cell survival and promotes migration and invasion under glucose deprivation through interaction with G6PD

Overcoming energy stress is a critical step for cells in solid tumors. Under this stress microenvironment, cancer cells significantly alter their energy metabolism to maintain cell survival and even metastasis. Our previous studies have shown that thioredoxin-1 (Trx-1) expression is increased in colorectal cancer (CRC) and promotes cell proliferation. However, the exact role and mechanism of how Trx-1 is involved in energy stress are still unknown. Here, we observed that glucose deprivation of CRC cells led to cell death and promoted the migration and invasion, accompanied by upregulation of Trx-1. Increased Trx-1 supported CRC cell survival under glucose deprivation. Whereas knockdown of Trx-1 sensitized CRC cells to glucose deprivation-induced cell death and reversed glucose deprivation-induced migration, invasion, and epithelial-mesenchymal transition (EMT). Furthermore, we identified glucose-6-phosphate dehydrogenase (G6PD) interacting with Trx-1 by HuPortTM human protein chip, co-IP and co-localization. Trx-1 promoted G6PD protein expression and activity under glucose deprivation, thereby increasing nicotinamide adenine dinucleotide phosphate (NADPH) generation. Moreover, G6PD knockdown sensitized CRC cells to glucose deprivation-induced cell death and suppressed glucose deprivation-induced migration, invasion, and EMT. Inhibition of Trx-1 and G6PD, together with inhibition of glycolysis using 2-deoxy-D-glucose (2DG), resulted in significant anti-tumor effects in CRC xenografts in vivo. These findings demonstrate a novel mechanism and may represent a new effective therapeutic regimen for CRC.