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1.
Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine‐based signal (GYDQL) in its C‐terminal tail and a non‐classical motif in its fifth inter‐TM loop. Using the yeast two‐hybrid system, we showed that the GYDQL motif specifically interacted with the μ subunit of the adaptor protein complex 3 (AP‐3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP‐3‐depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP‐3 knockdown cells where it also accumulated in the trans‐Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63‐GYDQL chimeric protein were not increased when clathrin‐mediated endocytosis was impaired, our data show that the tyrosine‐based motif of cystinosin is a ‘strong’ AP‐3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway.   相似文献   
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A new homology model of human microsomal epoxide hydrolase was derived based on multiple templates. The model obtained was fully evaluated, including MD simulations and ensemble‐based docking, showing that the quality of the structure is better than that of only previously known model. Particularly, a catalytic triad was clearly identified, in agreement with the experimental information available. Analysis of intermediates in the enzymatic mechanism led to the identification of key residues for substrate binding, stereoselectivity, and intermediate stabilization during the reaction. In particular, we have confirmed the role of the oxyanion hole and the conserved motif (HGXP) in epoxide hydrolases, in excellent agreement with known experimental and computational data on similar systems. The model obtained is the first one that fully agrees with all the experimental observations on the system. Proteins 2017; 85:720–730. © 2016 Wiley Periodicals, Inc.  相似文献   
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It has been shown that probable portions which form contacts in a protein can be predicted by means of an average distance map (ADM) as well as regular structures (-helices and -turns) defined as short-range compact regions (Kikuchiet al., 1988a,c). In this paper, we analyze the occurrence of those portions and short-range compact regions on ADMs for various proteins regarding their folding types. We have found out that each folding type of proteins shows characteristic distribution of such parts on ADMS. We also discuss the possibility of the prediction of folding types of proteins by ADMs.  相似文献   
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曹媛  杨云  徐化全  刘洋  王丹阳 《植物学报》2018,53(1):104-109
T-DNA突变体是研究基因功能的重要资源。高效热不对称交错PCR(hiTAIL-PCR)是克隆突变体中T-DNA插入位点侧翼序列的常用方法。然而我们发现,利用hiTAIL-PCR克隆到的一些侧翼序列并不对应于宿主的染色体DNA序列,而是质粒的骨架DNA片段。通过设置1组RB-S4/AC1或者LB-A4/AC1对照反应,用PCR方法鉴定了hiTAIL-PCR扩增产物中位于T-DNA侧翼的质粒骨架片段。在后续分析中,通过排除这些片段,提高了利用hiTAIL-PCR获得宿主染色体DNA片段的效率。同时,通过调整反应程序,使得整个PCR的反应时间也大为缩短。在拟南芥(Arabidopsis thaliana)T-DNA突变体drf1侧翼序列的克隆实例中,对照反应的引入将hiTAIL-PCR中需鉴定的22条扩增产物降至4条,效率提高了81.8%。  相似文献   
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A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12 Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na+-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the β-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent.  相似文献   
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In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal structures of GluK1 in complex with related ligands, the compounds were designed to explore possible interactions with non-conserved residues outside the glutamate ligand binding site and challenge the water binding network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl-substituted urea (compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that this preference may be attributed to contacts between the NH of the urea substituent and non-conserved Ser741 and Ser761 residues.  相似文献   
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Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.  相似文献   
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