共查询到20条相似文献,搜索用时 11 毫秒
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Raschperger E Neve EP Wernerson A Hultenby K Pettersson RF Majumdar A 《Developmental biology》2008,313(1):455-464
The coxsackie and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily and a component of vertebrate tight junctions. CAR protein is widely expressed in fish and mammals in organs of epithelial origin suggesting possible functions in epithelial biology. In order to gain insight into its function, we knocked the CAR gene down in zebrafish using antisense morpholinos. We identified a requirement for CAR in the terminal differentiation of glomerular podocytes and pronephric tubular epithelia. Podocytes differentiate in CAR morphants but are not able to elaborate a regularly patterned architecture of foot processes. In the tubules, CAR was required for the apposition of plasma membranes from adjacent epithelial cells but did not appear to be necessary for the formation of tight junctions. Additionally, tubular epithelia lacking CAR were not able to elaborate apical brush border microvilli. These results establish a requirement for CAR in the terminal differentiation of renal glomerular and tubular cell types. 相似文献
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Hauptmann G Belting HG Wolke U Lunde K Söll I Abdelilah-Seyfried S Prince V Driever W 《Development (Cambridge, England)》2002,129(7):1645-1655
Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories. 相似文献
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Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis 总被引:6,自引:0,他引:6
Kramer-Zucker AG Olale F Haycraft CJ Yoder BK Schier AF Drummond IA 《Development (Cambridge, England)》2005,132(8):1907-1921
Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be non-motile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates. 相似文献
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Ruffert K Himmel B Lall D Bamann C Bamberg E Betz H Eulenburg V 《Biochemical and biophysical research communications》2011,(4):86-743
Channelrhodopsin 2 (ChR2) is a microbial-type rhodopsin with a putative heptahelical structure that binds all-trans-retinal. Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions of these residues significantly altered ChR2 function as revealed by two-electrode voltage-clamp recordings of light-induced currents from Xenopus laevis oocytes expressing the respective mutant proteins. Specifically, replacement of E90 by lysine or alanine resulted in differential effects on H+- and Na+-mediated currents. Our results are consistent with this glutamate side chain within the proposed TM2 contributing to ion flux through and the cation selectivity of ChR2. 相似文献
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Xi Lu Xinde Hu Lingzhen Song Lei An Minghui Duan Shulin Chen Shanting Zhao 《BMB reports》2015,48(2):97-102
Neurons in the developing brain form the cortical plate (CP) in an inside-out manner, in which the late-born neurons are located more superficially than the early-born neurons. Fyn, a member of the Src family kinases, plays an important role in neuronal migration by binding to many substrates. However, the role of the Src-homology 2 (SH2) domain in function of Fyn in neuronal migration remains poorly understood. Here, we demonstrate that the SH2 domain is essential for the action of Fyn in neuronal migration and cortical lamination. A point mutation in the Fyn SH2 domain (FynR176A) impaired neuronal migration and their final location in the cerebral cortex, by inducing neuronal aggregation and branching. Thus, we provide the first evidence of the Fyn SH2 domain contributing to neuronal migration and neuronal morphogenesis. [BMB Reports 2015; 48(2): 97-102] 相似文献
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Zheng L Stathopulos PB Li GY Ikura M 《Biochemical and biophysical research communications》2008,369(1):240-246
Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER)-membrane associated Ca(2+) sensor which activates store-operated Ca(2+) entry (SOCE). The homologue, STIM2 possesses a high sequence identity to STIM1 ( approximately 61%), while its role in SOCE seems to be distinct from that of STIM1. In order to understand the underlying mechanism for the functional differences between STIM1 and STIM2, we investigated the biophysical properties of the luminal Ca(2+)-binding region which contains an EF-hand motif and a sterile alpha-motif (SAM) domain (hereafter called EF-SAM; residues 58-201 in STIM1 and 149-292 in STIM2). STIM2 EF-SAM has a low apparent Ca(2+)-binding affinity (K(d) approximately 0.5mM), which is similar to that reported for STIM1 EF-SAM. In the presence of Ca(2+), STIM2 EF-SAM is monomeric and well-folded, analogous to what was previously observed for STIM1 EF-SAM. In contrast to apo STIM1 EF-SAM, apo STIM2 EF-SAM is more structurally stable and does not readily aggregate. Our circular dichroism (CD) data demonstrate the existence of a long-lived, well-folded monomeric state for apo STIM2 EF-SAM, together with a less alpha-helical/partially unfolded aggregated state which is detectable only at higher protein concentrations and higher temperatures. Our biophysical studies reveal a structural stability difference in the EF-SAM region between STIM1 and STIM2, which may account for their different biological functions. 相似文献
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The calcium-binding EF-hand in polycystin-L is not a domain for channel activation and ensuing inactivation 总被引:4,自引:0,他引:4
Polycystin-L (PCL) shares high homology with polycystin-2, the product of polycystic kidney disease gene-2. It was previously shown that the PCL forms a non-selective cation channel activated by calcium influx. However, it remains unclear whether calcium activates/inactivates PCL by binding to the EF-hand motif located on the cytoplasmic carboxyl-terminus. Here we obtained two PCL splice variants from liver (PCL-LV, lacking the EF-hand) and testis (PCL-TS, lacking 45 amino acids on the carboxyl tail) using PCR-based approaches. When expressed in Xenopus oocytes and studied using electrophysiology both splice variants exhibited basal cation channel activity and calcium-induced channel activation. While PCL-TS displayed similar activation to PCL, PCL-LV exhibited a three-fold increased activation. All five PCL C-terminal artificial truncation mutants also exhibited basal and calcium-activated channel activities, in particular the mutant T622X lacking the EF-hand was associated with increased activation. Our data demonstrate that the EF-hand and other parts of the carboxyl tail of PCL are not determinants of channel activation/inactivation although the EF-hand seems to be involved in the modulation of these processes. 相似文献
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The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment. 相似文献
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Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for amino acid activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore amino acid activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS. 相似文献
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Mark D Allen Seema Qamar Murali K Vadivelu Richard N Sandford Mark Bycroft 《Protein science : a publication of the Protein Society》2014,23(9):1301-1308
Autosomal dominant polycystic kidney disease (ADPKD) affects over 1:1000 of the worldwide population and is caused by mutations in two genes, PKD1 and PKD2. PKD2 encodes a 968-amino acid membrane spanning protein, Polycystin-2 (PC-2), which is a member of the TRP ion channel family. The C-terminal cytoplasmic tail contains an EF-hand motif followed by a short coiled-coil domain. We have determined the structure of the EF-hand region of PC-2 using NMR spectroscopy. The use of different boundaries, compared with those used in previous studies, have enabled us to determine a high resolution structure and show that the EF hand motif forms a standard calcium-binding pocket. The affinity of this pocket for calcium has been measured and mutants that both decrease and increase its affinity for the metal ion have been created. 相似文献
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Apolipoprotein H (apoH, protein; APOH, gene) binds to negatively charged phospholipids, which triggers the production of a subset of autoantibodies against phospholipid in patients with autoimmune diseases. We have demonstrated that two naturally occurring missense mutations in the fifth domain of apoH, Trp316Ser and Cys306Gly, disrupt the binding of native apoH to phosphatidylserine [Sanghera, D. K., Wagenknecht, D. R., McIntyre, J. A. & Kamboh, M. I. (1997) Hum. Mol. Genet. 6, 311-316]. To confirm whether these are functional mutations, we mutagenized APOH cDNAs and transiently expressed them in COS-1 cells. The cardiolipin ELISA of wild-type and mutant recombinant apoH confirmed that the Gly306 and Ser316 mutations are responsible for abolishing the binding of recombinant apoH to cardiolipin. These mutations, however, had no effect on the levels of expression or secretion of recombinant apoH in transfected COS-1 cells. While the Cys306Gly mutation disrupts a disulfide bond between Cys306 and Cys281, which appears to be critical for clustering positively charged amino acids, the Trp316Ser mutation affects the integrity of an evolutionarily conserved hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp), which is hypothesized to interact with anionic phospholipid. To test this hypothesis, we exchanged the remaining three hydrophobic amino acids with neutral amino acids by site-directed mutagenesis (Leu313Gly, Ala314Ser and Phe315Ser). Binding of the Leu313Gly and Phe315Ser mutants to cardiolipin was significantly reduced to 25% and 13%, respectively, of that of the wild-type. On the other hand, the Ala314Ser mutation showed normal cardiolipin binding. Taken together with our previous findings, these results strongly suggest that the configuration of the fifth domain of apoH, as well as the integrity of the highly conserved hydrophobic amino acids at positions 313-316, is essential for the binding of apoH to anionic phospholipid. 相似文献
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Sylvain Brunet Todd Scheuer William A. Catterall 《The Journal of general physiology》2009,134(2):81-94
L-type Ca2+ currents conducted by Cav1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg2+ (Mgi) inhibits the ionic current of Cav1.2 channels. Because Mgi is altered in ischemia and heart failure, its regulation of Cav1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mgi on voltage-dependent inactivation (VDI) of Cav1.2 channels using Na+ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca2+-dependent inactivation process. We confirmed that increased Mgi reduces peak ionic currents and increases VDI of Cav1.2 channels in ventricular myocytes and in transfected cells when measured with Na+ as permeant ion. The increased rate and extent of VDI caused by increased Mgi were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mgi to the EF-hand (KD ≈ 0.9 mM) near the resting level of Mgi in ventricular myocytes. VDI was more rapid for L-type Ca2+ currents in ventricular myocytes than for Cav1.2 channels in transfected cells. Coexpression of Cavβ2b subunits and formation of an autoinhibitory complex of truncated Cav1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Cav1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mgi binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Cav1.2 channels by Mgi, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism. 相似文献
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Matei E Miron S Blouquit Y Duchambon P Durussel I Cox JA Craescu CT 《Biochemistry》2003,42(6):1439-1450
Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding. 相似文献
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Perera HD Ma YQ Yang J Hirbawi J Plow EF Qin J 《Structure (London, England : 1993)》2011,19(11):1664-1671
Kindlin-2 belongs to an emerging class of regulators for heterodimeric (α/β) integrin adhesion receptors. By binding to integrin β cytoplasmic tail via its C-terminal FERM-like domain, kindlin-2 promotes integrin activation. Intriguingly, this activation process depends on the N terminus of kindlin-2 (K2-N) that precedes the FERM domain. The molecular function of K2-N is unclear. We present the solution structure of K2-N, which displays a ubiquitin fold similar to that observed in kindlin-1. Using chemical shift mapping and mutagenesis, we found that K2-N contains a conserved positively charged surface that binds to membrane enriched with negatively charged phosphatidylinositol-(4,5)-bisphosphate. We show that while wild-type kindlin-2 is capable of promoting integrin activation, such ability is significantly reduced for its membrane-binding defective mutant. These data suggest a membrane-binding function of the ubiquitin-like domain of kindlin-2, which is likely common for all kindlins to promote their localization to the plasma membrane and control integrin activation. 相似文献
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CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function 总被引:2,自引:0,他引:2
The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function. 相似文献
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Shifley ET Vanhorn KM Perez-Balaguer A Franklin JD Weinstein M Cole SE 《Development (Cambridge, England)》2008,135(5):899-908
The Notch pathway plays multiple roles during vertebrate somitogenesis, functioning in the segmentation clock and during rostral/caudal (R/C) somite patterning. Lunatic fringe (Lfng) encodes a glycosyltransferase that modulates Notch signaling, and its expression patterns suggest roles in both of these processes. To dissect the roles played by Lfng during somitogenesis, a novel allele was established that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (Lfng(DeltaFCE1)). In the absence of oscillatory Lfng expression, Notch activation is ubiquitous in the PSM of Lfng(DeltaFCE1) embryos. Lfng(DeltaFCE1) mice exhibit severe segmentation phenotypes in the thoracic and lumbar skeleton. However, the sacral and tail vertebrae are only minimally affected in Lfng(DeltaFCE1) mice, suggesting that oscillatory Lfng expression and cyclic Notch activation are important in the segmentation of the thoracic and lumbar axial skeleton (primary body formation), but are largely dispensable for the development of sacral and tail vertebrae (secondary body formation). Furthermore, we find that the loss of cyclic Lfng has distinct effects on the expression of other clock genes during these two stages of development. Finally, we find that Lfng(DeltaFCE1) embryos undergo relatively normal R/C somite patterning, confirming that Lfng roles in the segmentation clock are distinct from its functions in somite patterning. These results suggest that the segmentation clock may employ varied regulatory mechanisms during distinct stages of anterior/posterior axis development, and uncover previously unappreciated connections between the segmentation clock, and the processes of primary and secondary body formation. 相似文献