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991.
We previously identified six single gene disruptions in Saccharomyces cerevisiae that allow enhanced immunoreactive insulin secretion primarily because of defective Kex2p-mediated endoproteolytic processing. Five eis mutants disrupted established VPS (vacuolar protein sorting) genes, The sixth, LTE1, is a Low Temperature (<15 degrees C) Essential gene encoding a large protein with potential guanine nucleotide exchange (GEF) domains. Lte1p functions as a positive regulator of the mitotic GTPase Tem1p, and overexpression of Tem1p suppresses the low temperature mitotic defect of lte1. By sequence analysis, Tem1p has highest similarity to Vps21p (yeast homolog of mammalian Rab5). Unlike TEM1, LTE1 is not restricted to mitosis but is expressed throughout the cell cycle. Lte1p function in interphase cells is largely unknown. Here we confirm the eis phenotype of lte1 mutant cells and demonstrate a defect in proalpha factor processing that is rescued by expression of full-length Lte1p but not a C-terminally truncated Lte1p lacking its GEF homology domain. Neither overexpression of Tem1p nor 13 other structurally related GTPases can suppress the secretory proprotein processing defect. However, overexpression of Vps21p selectively restores proprotein processing in a manner dependent upon the active GTP-bound form of the GTPase. By contrast, a vps21 mutant produces a synthetic defect with lte1 in proprotein processing, as well as a synthetic growth defect. Together, the data underscore a link between the mitotic regulator, Lte1p, and protein processing and trafficking in the secretory/endosomal system. 相似文献
992.
Yang XO Panopoulos AD Nurieva R Chang SH Wang D Watowich SS Dong C 《The Journal of biological chemistry》2007,282(13):9358-9363
993.
Wu H Kao SC Barrientos T Baldwin SH Olson EN Crabtree GR Zhou B Chang CP 《The Journal of biological chemistry》2007,282(42):30673-30679
994.
G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT(4b) receptor, a GPCR with high constitutive G(s) signaling and strong ligand-induced G-protein activation of the G(s) and G(s/q) pathways. The first receptor in this series, 5-HT(4)-D(100)A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G(s) signaling, but only a few (e.g., zacopride) also induced signaling via the G(q) pathway. Zacopride-induced G(q) signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT(2C) receptor. Additional point mutations (D(66)A and D(66)N) blocked constitutive G(s) signaling and lowered ligand-induced G(q) signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT(1A) conferred ligand-mediated G(i) signaling. This G(i)-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G(s) or G(q) pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection. 相似文献
995.
Background
Pten functionally acts as a tumor suppressor gene. Lately, tissue-specific ablation of Pten gene in mice has elucidated the role of Pten in different tumor progression models. However, a temporally controlled Pten loss in all adult tissues to examine susceptibility of various tissues to Pten-deficient tumorigenesis has not been addressed yet. Our goal was to explore the genesis of Pten-deficient malignancies in multiple tissue lineages of the adult mouse.Methods and Findings
We utilized an inducible Cre/loxP system to delete Pten exon 5 in the systemic organs of ROSA26 (R26)-CreERT;Ptenfx/fx mice. On reaching 45 weeks 4OHT-induced Pten loss, we found that the R26-CreERT;Ptenfx/fx mice developed a variety of malignancies. Overall tumor mean latency was 17 weeks in the Pten-deficient mice. Interestingly, mutant females developed malignancies more quickly at 10∼11 weeks compared with a tumor latency of 21 weeks for mutant males. Lymphoma incidence (76.9% in females; 40.0% in males) was higher than the other malignancies found in the mutant mice. Mutant males developed prostate (20.0%), intestinal cancer (35.0%) and squamous cell carcinoma (10.0%), whereas the mutant females developed squamous cell carcinoma (15.4%) and endometrial cancer (46.1%) in addition to lymphomas. Furthermore, we tested the pharmacological inhibition of the PTEN downstream effectors using on Pten-deficient prostate hyperplasia. Our data revealed that, indeed, the prostate hyperplasia resulting from the induced Pten loss was significantly suppressed by LY294002 (p = 0.007). LY294002Conclusions
Through monitoring a variety of Pten-deficient tumor formation, our results revealed that the lymphoid lineages and the epithelium of the prostate, endometrium, intestine and epidermis are highly susceptible to tumorigenesis after the Pten gene is excised. Therefore, this R26-CreERT; Ptenfx/fx mouse model may provide an entry point for understanding the role of Pten in the tumorigenesis of different organs and extend the search for potential therapeutic approaches to prevent Pten-deficient malignancies. 相似文献996.
Small ubiquitin-related protein modifiers (SUMO) modification is an important mechanism for posttranslational regulation of protein function. However, it is largely unknown how the sumoylation pathway is regulated. Here, we report that nitric oxide (NO) causes global hyposumoylation in mammalian cells. Both SUMO E2 conjugating enzyme Ubc9 and E3 ligase protein inhibitor of activated STAT3 (Pias3) were targets for S-nitrosation. S-nitrosation did not interfere with the SUMO conjugating activity of Ubc9, but promoted Pias3 degradation by facilitating its interaction with tripartite motif-containing 32 (Trim32), a ubiquitin E3 ligase. On the one hand, NO promoted Trim32-mediated Pias3 ubiquitination. On the other hand, NO enhanced the stimulatory effect of Pias3 on Trim32 autoubiquitination. The residue Cys459 of Pias3 was identified as a target site for S-nitrosation. Mutation of Cys459 abolished the stimulatory effect of NO on the Pias3-Trim32 interaction, indicating a requirement of S-nitrosation at Cys459 for positive regulation of the Pias3-Trim32 interplay. This study reveals a novel crosstalk between S-nitrosation, ubiquitination, and sumoylation, which may be crucial for NO-related physiological and pathological processes. 相似文献
997.
998.
999.
Dong A Xu X Edwards AM;Midwest Center for Structural Genomics;Structural Genomics Consortium Chang C Chruszcz M Cuff M Cymborowski M Di Leo R Egorova O Evdokimova E Filippova E Gu J Guthrie J Ignatchenko A Joachimiak A Klostermann N Kim Y Korniyenko Y Minor W Que Q Savchenko A Skarina T Tan K Yakunin A Yee A Yim V Zhang R Zheng H Akutsu M Arrowsmith C Avvakumov GV Bochkarev A Dahlgren LG Dhe-Paganon S Dimov S Dombrovski L Finerty P Flodin S Flores A Gräslund S Hammerström M Herman MD Hong BS 《Nature methods》2007,4(12):1019-1021
We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain. 相似文献
1000.
An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests. 相似文献