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991.
H.-T. Zhao S. Endo S. Ishikura R. Chung P. J. Hogg A. Hara O. El-Kabbani 《Cellular and molecular life sciences : CMLS》2009,66(9):1570-1579
l-Xylulose reductase (XR) is involved in water re-absorption and cellular osmoregulation. The crystal structure of human XR
complemented with site-directed mutagenesis (Cys138Ala) indicated that the disulfide bond in the active site between Cys138
and Cys150 is unstable and may affect the reactivity of the enzyme. The effects of reducing agents on the activities of the
wild-type and mutant enzymes indicated the reversibility of disulfide-bond formation, which resulted in three-fold decrease
in catalytic efficiency. Furthermore, the addition of cysteine (>2 mM) inactivated human XR and was accompanied by a 10-fold
decrease in catalytic efficiency. TOF-MS analysis of the inactivated enzyme showed the S-cysteinylation of Cys138 in the wild-type
and Cys150 in the mutant enzymes. Thus, the action of human XR may be regulated by cellular redox conditions through reversible
disulfide-bond formation and by S-cysteinylation.
Received 25 January 2009; received after revision 12 February 2009; accepted 16 February 2009
H.-T. Zhao, S. Endo: These two authors contribute equally to this work. 相似文献
992.
生物信息学分析显示,拟南芥基因At5g62390编码一种钙调素结合蛋白,在其钙调素结合结构域有一个BAG(Bcl-2-associated athnogene)结构域存在,与钙调素结构域部分重叠.为了从实验上进一步研究该蛋白的钙调素结合特征及BAG结构域在钙调素结合中可能的调节作用,通过聚合酶链式反应(PCR)扩增不同结构域编码区的cDNA序列,构建到原核表达载体pET32-a中.测序分析表明:目的序列已正确克隆到表达载体上,为进一步表达目的蛋白及其不同结构域用于生化功能鉴定打下了基础. 相似文献
993.
The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism
including splicing regulation, polyadenylation, 3′end formation, internal ribosomal entry site-mediated translation, RNA localization
and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural
information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray
scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as
a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights
into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation.
Received 16 August 2007; received after revision 18 September 2007; accepted 2 October 2007 相似文献
994.
Pozza A Perez-Victoria JM Sardo A Ahmed-Belkacem A Di Pietro A 《Cellular and molecular life sciences : CMLS》2006,63(16):1912-1922
Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate,
and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding
capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered
sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated
a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding
of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound
similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic
inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound
similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug
transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency
is not mediated by changes in drug binding.
Received 10 April 2006; received after revision 22 May 2006; accepted 12 June 2006
A. Pozza and J. M. Perez-Victoria contributed equally to this work 相似文献
995.
996.
Cowan-Jacob SW 《Cellular and molecular life sciences : CMLS》2006,63(22):2608-2625
Our current understanding of the structure, mechanism of action and modes of regulation of the protein tyrosine kinase family
owes a great deal to structural biology. Structures are now available for more than 20 different tyrosine kinase domains,
many of these in multiple conformational states. They form the basis for the design of experiments to further investigate
the role of different structural elements in the normal function and regulation of the protein and in the pathogenesis of
many human diseases. Once thought to be too similar to be specifically inhibited by a small molecule, structural differences
between kinases allow the design of compounds which inhibit only an acceptable few. This review gives a general overview of
protein tyrosine kinase structural biology, including a discussion of the strengths and limitations of the investigative methods
involved.
Received 2 May 2006; received after revision 21 June 2006; accepted 9 August 2006 相似文献
997.
Vastiau IM Anthonio EA Brams M Brees C Young SG Van de Velde S Wanders RJ Mannaerts GP Baes M Van Veldhoven PP Fransen M 《Cellular and molecular life sciences : CMLS》2006,63(14):1686-1699
Pex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs), and is essential for the formation of
functional peroxisomal membranes. Pex19p orthologues contain a C-terminal CAAX motif common to prenylated proteins. In addition, Saccharomyces cerevisiae and Chinese hamster Pex19p are at least partially farnesylated in vivo. Whether farnesylation of Pex19p plays an essential or merely ancillary role in peroxisome biogenesis is currently not clear.
Here, we show that (i) nonfarnesylated and farnesylated human Pex19p display a similar affinity towards a select set of PMPs,
(ii) a variant of Pex19p lacking a functional farnesylation motif is able to restore peroxisome biogenesis in Pex19p-deficient
cells, and (iii) peroxisome protein import is not affected in yeast and mammalian cells defective in one of the enzymes involved
in the farnesylation pathway. Summarized, these observations indicate that the CAAX box-mediated processing steps of Pex19p are dispensable for peroxisome biogenesis in yeast and mammalian cells.
Received 10 March 2006; received after revision 28 April 2006; accepted 30 May 2006 相似文献
998.
Endomannosidase processes oligosaccharides of α1-antitrypsin and its naturally occurring genetic variants in the Golgi apparatus 总被引:3,自引:0,他引:3
Torossi T Fan JY Sauter-Etter K Roth J Ziak M 《Cellular and molecular life sciences : CMLS》2006,63(16):1923-1932
Endomannosidase provides an alternate glucose-trimming pathway in the Golgi apparatus. However, it is unknown if the action
of endomannosidase is dependent on the conformation of the substrate. We have investigated the processing by endomannosidase
of the α1-antitrypsin oligosaccharides and its disease-causing misfolded Z and Hong Kong variants. Oligosaccharides of wild-type
and misfolded α1-antitrypsin expressed in castanospermine-treated hepatocytes or glucosidase II-deficient Phar 2.7 cells were
selectively processed by endomannosidase and subsequently converted to complex type oligosaccharides as indicated by Endo
H resistance and PNGase F sensitivity. Overexpression of endomannosidase in castanospermine-treated hepatocytes resulted in
processing of all oligosaccharides of wild-type and variants of α1-antitrypsin. Thus, endomannosidase does not discriminate
the folding state of the substrate and provides a back-up mechanism for completion of N-glycosylation of endoplasmic reticulum-escaped glucosylated glycoproteins. For exported misfolded glycoproteins, this would
provide a pathway for the formation of mature oligosaccharides important for their proper trafficking and correct functioning.
Received 18 April 2006; received after revision 12 June 2006; accepted 15 June 2006 相似文献
999.
Despite the current availability of several hundreds of thousands of amino acid sequences, more than 39% of the well-defined
enzyme activities (EC numbers) are not associated with any sequence in major public databases. This wide gap separating knowledge
of biochemical function and sequence information is found in nearly all classes of enzymes. Thus, there is an urgent need
to explore the 1525 orphan enzymes (EC numbers without associated sequences), in order to progressively bridge this unwanted
gap. Improving genome annotation could unveil a significant proportion of sequenceless enzymes. Peptide mass mapping and further
genome mining would be useful to identify proper sequence for enzymes found in species for which genetic tools are missing.
Finally, the whole community must help major public databases to begin addressing the problem of missing or incomplete information.
Received 31 October 2005; received after revision 8 December 2005; accepted 20 December 2005 相似文献
1000.
Sarramegn V Muller I Milon A Talmont F 《Cellular and molecular life sciences : CMLS》2006,63(10):1149-1164
G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes
and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target
GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular
mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated
messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution
three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are
not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents
and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the
perspective of structural biology experiments.
Received 21 November 2005; received after revision 20 January 2006; accepted 2 February 2006
An erratum to this article is available at . 相似文献