Among SNARE proteins mediating synaptic vesicle fusion, syntaxin‐1 uniquely includes an N‐terminal peptide (‘N‐peptide’) that binds to Munc18‐1, and a large, conserved Habc‐domain that also binds to Munc18‐1. Previous in vitro studies suggested that the syntaxin‐1 N‐peptide is functionally important, whereas the syntaxin‐1 Habc‐domain is not, but limited information is available about the in vivo functions of these syntaxin‐1 domains. Using rescue experiments in cultured syntaxin‐deficient neurons, we now show that the N‐peptide and the Habc‐domain of syntaxin‐1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N‐peptide is essential for vesicle fusion as such, whereas the Habc‐domain regulates this fusion, in part by forming the closed syntaxin‐1 conformation. Moreover, we observed that deletion of the Habc‐domain but not deletion of the N‐peptide caused a loss of Munc18‐1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the Habc‐domain stabilizes Munc18‐1. Thus, the N‐terminal syntaxin‐1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE‐protein complexes. 相似文献
The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The Kd values for binding of [14C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [3H]Ifenprodil bound to NR1‐R (Kd, 0.18 μM) and NR2B‐R (Kd, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These Kd values were confirmed by circular dichroism measurements. The Kd values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct. 相似文献
The cannabinoid type 2 (CB2) receptor plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease and is therefore a very promising target for therapeutic approaches as well as for imaging. Based on the literature, we identified one 4‐oxoquinoline derivative (designated KD2) as the lead structure. It was synthesized, radiolabeled and evaluated as a potential imaging tracer for CB2. [11C]KD2 was obtained in 99% radiochemical purity. Moderate blood–brain barrier (BBB) passage was predicted for KD2 from an in vitro transport assay with P‐glycoprotein‐transfected Madin Darby canine kidney cells. No efflux of KD2 by P‐glycoprotein was detected. In vitro autoradiography of rat and mouse spleen slices demonstrated that [11C]KD2 exhibits high specific binding towards CB2. High spleen uptake of [11C]KD2 was observed in dynamic positron emission tomography (PET) studies with Wistar rats and its specificity was confirmed by displacement study with a selective CB2 agonist, GW405833. A pilot autoradiography study with post‐mortem spinal cord slices from amyotrophic lateral sclerosis (ALS) patients with [11C]KD2 suggested the presence of CB2 receptors under disease conditions. Specificity of [11C]KD2 binding could also be demonstrated on these human tissues. In conclusion, [11C]KD2 shows good in vitro and in vivo properties as a potential PET tracer for CB2.
Mis18 is a key regulator responsible for the centromere localization of the CENP‐A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP‐A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N‐terminal Yippee‐like domain, is crucial for its centromere localization and function. The crystal structure of the N‐terminal Yippee‐like domain reveals a fold containing a cradle‐shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N‐terminal Yippee‐like domain forms a homodimer in vitro and in vivo, full‐length Mis18, including the C‐terminal α‐helical domain, forms a homotetramer in vitro. We also show that the Yippee‐like domains of human Mis18α/Mis18β interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation. 相似文献
Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin‐dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. Results. In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N‐ and C‐terminal regions of CDC25B and requires CDC25B binding to its CDK—cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. Conclusions. Our results therefore suggest that CDC25B associates with a Ctn2‐containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components. 相似文献
The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (KD) for vitronectin binding to UspA2 was 2.3 × 10?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a KD of 7.9 × 10?8 M. Electron microscopy verified that the active binding domain (UspA230–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA230–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance. 相似文献
PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σT and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ2 and σ4, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ2 and σ4 in the SL domain to open about a flexible connector loop and bind anti‐σ factor. 相似文献
Representative members of a group of linear, N‐acylated polypeptide antibiotics (peptaibols) containing α‐aminoisobutyric acid (Aib) and, in part, isovaline (Iva), as well as proteinogenic amino acids and a C‐terminal‐bonded 2‐amino alcohol, were treated with anhydrous trifluoroacetic acid (TFA) at 37° for 0.5–26 h. The resulting fragments were separated by HPLC and characterized by electrospray ionization collision‐induced dissociation mass spectrometry (ESI‐CID‐MS). The following 16–20‐residue peptaibols were investigated: natural, microheterogeneous mixtures of antiamoebins and alamethicin F50, uniform paracelsin A, and synthetic trichotoxin A50/E. In the natural peptides, bonds formed between Aib (Iva) and Pro (Hyp) were rapidly and selectively cleaved within 0.5 h. Furthermore, TFA esters of the C‐terminal amino alcohols were formed. Depending on time, release of C‐terminal tri‐ and tetrapeptides as well as amino acids from the major fragments was observed. Synthetic homooligopeptides, namely Z‐ and Ac‐(Aib)10‐OtBu and Z‐(Aib)7‐OtBu, were analyzed for comparison. On treatment with TFA, a regular series of Z‐(Aib)10–5‐OH from Z‐(Aib)10‐OtBu were detected within 0.5 h, and, after 3 h, release of a regular series of Z‐(Aib)7–3‐OH from Z‐(Aib)7‐OtBu were observed. Moreover, concomitant release of the series of H‐(Aib)10–3‐OH from the decapeptide occurred. From these data, a repetitive cleavage mechanism via intermediate formation of C‐terminal oxazolones on trifluoroacetolysis is proposed. Furthermore, their formation and stability in native peptaibols are correlated with subtle structural differences in protein amino acids linked to Aib. From the conspicuous concordance of the formation and abundance of regular series of trifluoroacetolytic fragments and of positive ions of the b‐series in CID‐MS, the generation of intermediate oxazolonium ions in both gas and liquid phase is concluded. 相似文献
Viperin is an interferon‐induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S‐adenosyl‐l ‐methionine (SAM) domain which has been shown in vitro to hold a [4Fe‐4S] cluster. We identified tick‐borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of 55Fe, indicative for the presence of an Fe‐S cluster. Mutation of the cysteine residues ligating the Fe‐S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of 55Fe. Mutants lacking the extreme C‐terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe‐4S] cluster assembly, and acts through an enzymatic, Fe‐S cluster‐ and SAM‐dependent mechanism to inhibit viral RNA synthesis. 相似文献
A new type of planar chiral (Rp)‐ and (Sp)‐4,7,12,15‐tetrasubstituted [2.2]paracyclophanes was prepared from racemic 4,7,12,15‐tetrabromo[2.2]paracyclophane as the starting substrate. Regioselective lithiation and transformations afforded racemic bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophane (4,15‐dibromo‐7,12‐dihydroxy[2.2]paracyclophane). Its optical resolution was performed by the diastereomer method using a chiral camphanoyl group as the chiral auxiliary. The diastereoisomers were readily isolated by simple silica gel column chromatography, and the successive hydrolysis afforded (Rp)‐ and (Sp)‐bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophanes ((Rp)‐ and (Sp)‐4,15‐dibromo‐7,12‐dihydroxy[2.2]paracyclophanes). They can be used as pseudo‐meta‐substituted chiral building blocks. 相似文献
Insulin sensitivity (IS) is measured by the euglycemic–hyperinsulinemic clamp under a nonphysiological condition. Daily C‐peptide urinary excretion may be a physiological index of IS, because C‐peptide is co‐secreted with insulin as a function of nutrient intake and IS. The amount of 2H2O released from glycolytic glucose metabolism after [6,6‐2H2]‐glucose ingestion was recently proposed as a physiological measure of IS. We compared these IS surrogates to the gold standard (euglycemic–hyperinsulinemic clamp). Thirty (15 male/15 female) sedentary, nondiabetic participants (27.2 ± 4.0 (s.d.) kg/m2, 35 ± 12 years) were admitted for 3 days to our in‐patient unit. After a 10‐h fast, they received 60 g of glucose and 15 g of [6,6‐2H2]‐glucose. Before glucose ingestion and hourly thereafter for 4 h, plasma glucose and insulin concentrations, and plasma deuterium enrichment were determined. Plasma 2H2O production divided by insulin response was used as the glycolytic index. On day 2, subjects spent 23 h in a metabolic chamber (eucaloric diet, 50% carbohydrate, 30% fat). Urinary C‐peptide excretion was divided by energy intake yielding the C‐peptide to energy intake ratio (CPEP/EI). After leaving the chamber (day 3, 10‐h fast), IS was measured by a 2‐h clamp (120 mU/m2/min). Average IS (clamp) was 7.3 ± 2.6 mg glucose/kg estimated metabolic body size/min (range: 3.6–13.2). These values were inversely correlated with CPEP/EI (r = ?0.62; P < 0.01) and positively with the glycolytic rate (r = 0.60; P < 0.01). In nondiabetic subjects, two novel estimates of IS—daily urinary C‐peptide urinary excretion and glycolytic rate during an oral glucose tolerance test —were related to IS by a clamp. 相似文献
The type VI secretion system (T6SS) of bacteria plays a key role in competing for specific niches by the contact‐dependent killing of competitors. Recently, Rhs proteins with polymorphic C‐terminal toxin‐domains that inhibit or kill neighboring cells were identified. In this report, we identified a novel Rhs with an MPTase4 (Metallopeptidase‐4) domain (designated as Rhs‐CT1) that showed an antibacterial effect via T6SS in Escherichia coli. We managed to develop a specific strategy by matching the diagnostic domain‐architecture of Rhs‐CT1 (Rhs with an N‐terminal PAAR‐motif and a C‐terminal toxin domain) for effector retrieval and discovered a series of Rhs‐CTs in E. coli. Indeed, the screened Rhs‐CT3 with a REase‐3 (Restriction endonuclease‐3) domain also mediated interbacterial antagonism. Further analysis revealed that vgrGO1 and eagR/DUF1795 (upstream of rhs‐ct) were required for the delivery of Rhs‐CTs, suggesting eagR as a potential T6SS chaperone. In addition to chaperoned Rhs‐CTs, neighborless Rhs‐CTs could be classified into a distinct family (Rhs‐Nb) sharing close evolutionary relationship with T6SS2‐Rhs (encoded in the T6SS2 cluster of E. coli). Notably, the Rhs‐Nb‐CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B‐VgrG2 module. In a further retrieval analysis, we discovered various toxin/immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans, suggesting that Rhs‐CTs greatly diversify the antibacterial pathways of T6SS. 相似文献
The synthetic peptide Z‐(Aib)10‐OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection reflected synchrotron radiation to sub‐atomic resolution, where the bonding electron density becomes visible between the non‐hydrogen atoms. Crystals belong to the centrosymmetric space group P . Both molecules in the asymmetric unit form regular 310‐helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure determined to date of longer Aib‐homopeptides. These other peptides are C‐terminal protected by OtBu or OMe. In these cases, because of the missing ability of the C‐terminal protection group to form a hydrogen bond to the residue i‐3, the sense of the helix is reversed in the last residue. Here, the C‐terminal OH‐groups form hydrogen bonds to the residues i‐3, in part mediated by water molecules. This makes Z‐(Aib)10‐OH an Aib‐homopeptide with three complete 310‐helical turns in spite of the shorter length it has compared with Z‐(Aib)11‐OtBu, the only homopeptide to date with three complete turns. 相似文献
Human coagulation factor X is a central component of the blood coagulation cascade that converts, under its activated form, prothrombin into thrombin. Generation of thrombin is the final step of the clotting cascade that leads to the clot by polymerization of fibrinogen molecules into a fibrin network. Today, research of new by‐passing agents of the coagulation may contribute to an increased interest for human factor X, which may, in consequence, lead to the need of a more exhaustive picture of its structural features. Several post‐translational modifications of human factor X such as γ‐carboxylation/β‐hydroxylation of the N‐terminal light chain and N‐/O‐glycosylation of the activation peptide have been described. But, so far as we know, no comprehensive studies of its post‐translational modifications have been reported. In this article we report an exhaustive structural analysis of human factor X by mass spectrometry using successive protein and peptide mapping. Surprisingly, human factor X was found to be mostly O‐glucosylated on its light chain at Ser106 position, Ser9 of its activation peptide is phosphorylated at about 30% and its C‐terminal heavy chain is fully O‐glycosylated at Thr249 by a mucin‐type O‐glycan (HexNAc‐Hex‐NeuAc). The knowledge of these post‐translational modifications is mandatory for the development of recombinant molecules. 相似文献