Sorting nexin 17 (SNX17) is a member of the family of cytoplasmic sorting nexin adaptor proteins that regulate endosomal trafficking of cell surface proteins. SNX17 localizes to early endosomes where it directly binds NPX(Y/F) motifs in the cytoplasmic tails of its target receptors to mediate their rates of endocytic internalization, recycling, and/or degradation. SNX17 has also been implicated in mediating cell signaling and can interact with cytoplasmic proteins. KRIT1 (Krev interaction trapped 1), a cytoplasmic adaptor protein associated with cerebral cavernous malformations, has previously been shown to interact with SNX17. Here, we demonstrate that SNX17 indeed binds directly to KRIT1 and map the binding to the second Asn-Pro-Xaa-Tyr/Phe (NPX(Y/F)) motif in KRIT1. We further characterize the interaction as being mediated by the FERM domain of SNX17. We present the co-crystal structure of SNX17-FERM with the KRIT1-NPXF2 peptide to 3.0 Å resolution and demonstrate that the interaction is highly similar in structure and binding affinity to that between SNX17 and P-selectin. We verify the molecular details of the interaction by site-directed mutagenesis and pulldown assay and thereby confirm that the major binding site for SNX17 is confined to the NPXF2 motif in KRIT1. Taken together, our results verify a direct interaction between SNX17 and KRIT1 and classify KRIT1 as a SNX17 binding partner.
The in vivo and in vitro immunostimulating properties of chitosan oligosaccharide (COS) prepared by enzymatic hydrolysis of chitosan and the mechanisms mediating the effects were investigated. Our data showed that the highly active chitosanase isolated could hydrolyze chitosan to the polymerization degree of 3-8. The resulting COS was an efficient immunostimulator. COS markedly enhanced the proliferation and neutral red phagocytosis by RAW 264.7 macrophages. The production of nitric oxide (NO) and tumor necrosis factor alpha (TNF-?) by macrophages was significantly increased after incubation with COS. Oral administration of COS in mice could increase spleen index and serum immunoglobin G (IgG) contents. COS was labeled with FITC to study the pinocytosis by macrophages. Results showed that FITC-COS was phagocyted by macrophages and anti-murine TLR4 antibody completely blocked FITC-COS pinocytosis. RT-PCR indicated that COS treatment of macrophages significantly increased TLR4 and inducible nitric oxide synthase (iNOS) mRNA levels. When cells were pretreated with anti-murine TLR4 antibody, the effect of COS on TLR4 and iNOS mRNA induction was decreased. COS-induced NO secretion by macrophages was also markedly decreased by anti-murine TLR4 antibody pretreatment. In conclusion, the present study revealed that COS possesses potent immune-stimulating properties by activating TLR4 on macrophages.
The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.
Chitosanase is able to specifically cleave ?-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase-substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion.
The aim of this study was to examine symptoms of posttraumatic stress disorder (PTSD), depression, and anxiety among junior high school students in worst-hit areas 3 years after the Wenchuan earthquake. Analyses were carried out on 373 of the 377 students enrolled. In addition to obtaining demographic characteristics, the Impact of Event Scale-Revised, the Zung Self-rating Depression Scale, the Zung Self-rating Anxiety Scale, and an Earthquake exposure screening scale were administered. It was found that 29.6%, 44.8%, and 37.6% of participants reported clinical symptoms of PTSD, depression, and anxiety, respectively. PTSD, depression, and anxiety were highly comorbid. Having witnessed someone being killed, family members being killed, close friends seriously injured or being killed, and felt scared remained as significant predictors for PTSD. Having witnessed someone seriously injured and felt scared remained as significant predictors for depression. Having witnessed someone seriously injured, witnessed someone being killed, and felt scared remained as significant predictors for anxiety.
Integrin cytoplasmic domain-associated protein-1 (ICAP1) is a suppressor of integrin activation and directly binds to the cytoplasmic tail of ?1 integrins; its binding suppresses integrin activation by competition with talin. Krev/Rap1 interaction trapped-1 (KRIT1) releases ICAP1 suppression of integrin activation by sequestering ICAP1 away from integrin cytoplasmic tails. Here, the cocrystal structure of the PTB domain of ICAP1 in complex with a 29-amino-acid fragment (residues 170-198) of KRIT1 is presented to 1.7?Å resolution [the resolution at which ?I/?(I)? = 2.9 was 1.83?Å]. In previous studies, the structure of ICAP1 with integrin ?1 was determined to 3.0?Å resolution and that of ICAP1 with the N-terminal portion of KRIT1 (residues 1-198) was determined to 2.54?Å resolution; therefore, this study provides the highest resolution structure yet of ICAP1 and allows further detailed analysis of the interaction of ICAP1 with its minimal binding region in KRIT1.
KRIT1 (Krev/Rap1 Interaction Trapped-1) mutations are observed in ?40% of autosomal-dominant cerebral cavernous malformations (CCMs), a disease occurring in up to 0.5% of the population. We show that KRIT1 functions as a switch for ?1 integrin activation by antagonizing ICAP1 (Integrin Cytoplasmic Associated Protein-1)-mediated modulation of "inside-out" activation. We present cocrystal structures of KRIT1 with ICAP1 and ICAP1 with integrin ?1 cytoplasmic tail to 2.54 and 3.0 Å resolution (the resolutions at which I/?I = 2 are 2.75 and 3.0 Å, respectively). We find that KRIT1 binds ICAP1 by a bidentate surface, that KRIT1 directly competes with integrin ?1 to bind ICAP1, and that KRIT1 antagonizes ICAP1-modulated integrin activation using this site. We also find that KRIT1 contains an N-terminal Nudix domain, in a region previously designated as unstructured. We therefore provide insights to integrin regulation and CCM-associated KRIT1 function.
Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²?G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m?G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m?G-cap. Nematode eIF4E binding to the m?G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5-UTRs in mRNA translation and the function of different eIF4E isoforms.
Polybrominated diphenyl ethers (PBDEs) were measured in atmosphere and soil samples taken in winter and summer at a PBDE production area of Laizhou Bay in China. The concentrations of Sigma11PBDE were 0.017-1.17 ng/m3 in gaseous phase, 0.5-161.1 ng/m3 in particulate phase, and 73-2629 ng/g dry weight in soil samples. The PBDE congener pattern in the gaseous phase differed from that in the particulate phase, and the PBDE congener pattern in the particulate phase was similar with that in soil. This demonstrated that there was little difference with atmospheric particle-soil transfer efficiency among PBDE congeners. In addition, there were seasonal variations in percentages on particle for lower brominated congeners. The BDE-28 was mostly in the gaseous phase in summer (88.3%), whereas the average proportion of BDE-28 in gaseous phase in winter was 38.9%. Higher brominated congeners (i.e., BDE-206, BDE-207, BDE-208, and BDE-209) were bound to the atmospheric particulate phase, and their potentials for long-range migration were mainly affected by the environmental behavior of atmospheric particles. Results indicated that PBDE congeners in summer were closer to gas-particle partition equilibrium than in winter. Temperature should be considered the main factor causing nonequilibrium in winter.
The concentrations of polybrominated diphenyl ethers (PBDEs) were detected in air and aquatic products in PBDEs production areas which are located at the south coast area of Laizhou Bay, Shandong province, China in this study. Concentrations of SigmaPBDEs in the air ranged from 0.47 ng/m3 to 161 ng/m3. In aquatic products, concentrations of SigmaPBDEs ranged from 2.7 ng/g wet weight to 42 ng/g wet weight. The mean dietary intake of SigmaPBDEs via aquatic products consumption in this study was 218 ng/day. Daily intake of SigmaPBDEs via inhalation in this study was 612 ng for men and 455 ng for women. With a contribution of 80%, BDE-209 was predominant in the total intake. Dietary intake and breathing inhalation contributed 29 and 71%, respectively, to the total PBDEs intake. The results indicate that breathing inhalation also plays a very significant pathway for the population of the PBDEs production area. Compared with similar studies in other countries, human exposure to PBDEs via diet and inhalation in this study was the highest in the world.
The association between ages and psychological impact of natural disasters has not been well characterized. A population-based study was conducted 15 months after the 2008 Sichuan earthquake to assess whether elderly survivors were more likely to develop posttraumatic stress disorder (PTSD) and general psychiatric morbidity.
The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m(7)G) or a trimethylguanosine (m(2,2,7)G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m(7)G and m(2,2,7)G caps. The eIF4E.m(7)GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m(7)GpppG and m(2,2,7)GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m(7)G versus m(2,2,7)G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m(2,2,7)G cap.
Polybrominated diphenyl ethers (PBDEs) have been produced in the south coast area of Laizhou Bay, Shandong Province in China, but little is known about the PBDE exposure level of residents to these compounds. We set out to assess potential health risks of PBDEs in the south coast area of the Laizhou Bay by determining the concentrations of PBDEs in serum and breast milk. We measured concentrations of eight PBDE congeners in serum and breast milk. The arithmetic means of Sigma(8)PBDE in pooled serum and breast milk were 613 ng/g lipid and 81.5 ng/g lipid, respectively. The highest concentration for Sigma(8)PBDE in all serum pools was 1830 ng/g lipid from the 41-50 year old female group. BDE-209 was the predominant congener, with the mean concentrations of 403 ng/g lipid in serum and 45.6 ng/g lipid in breast milk, respectively. BDE-209 averagely accounted for 65.8% and 54.2% of the total PBDEs, respectively. Our results suggest that high exposures to PBDEs have led to very high PBDE concentrations in serum and breast milk from the residents living in the south coast area of Laizhou Bay. High PBDE concentrations in human serum, particularly in women, pose a potential public health threat to local residents.
Cerebral cavernous malformations (CCMs) affect 0.1-0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with ?40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 ? co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain "modules."
Integration of the retroviral genome into host DNA is a critical step in the life cycle of a retrovirus. Although assays for in vitro integration have been developed, the actual DNA sequences targeted by murine leukemia retrovirus (MLV) during in vitro reproduction are unknown. While previous studies used artificial target sequences, we developed an assay using target DNA sequences from common MLV integration sites in Stat5a and c-myc in the genome of murine lymphomas and successfully integrated MLV into the target DNA in vitro. We calculated the free energy change during folding of the target sequence DNA and found a close correlation between the calculated free energy change and the number of integrations. Indeed, the integrations closely correlated with fluctuation of the structure of the target DNA segment. These data suggest that the fluctuation may generate a DNA structure favorable for in vitro integration into the target DNA. The approach described here can provide data on the biochemical properties of the integration reaction to which the target DNA structure may contribute.
Crystallization of contaminating proteins is a frequently encountered problem for macromolecular crystallographers. In this study, an attempt was made to obtain a binary cocrystal structure of the SH3 domain of cortactin and a 17-residue peptide from the Arg nonreceptor tyrosine kinase encompassing a PxxPxxPxxP (PxxP1) motif. However, cocrystals could only be obtained in the presence of trace amounts of a contaminating protein. A structure solution obtained by molecular replacement followed by ARP/wARP automatic model building allowed a sequence-by-crystallography approach to discover that the contaminating protein was lysozyme. This 1.65 Å resolution crystal structure determination of a 1:1:1 heterotrimeric complex of Arg, cortactin and lysozyme thus provides an unusual `caveat emptor warning of the dangers that underpurified proteins harbor for macromolecular crystallographers.
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