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The 90-kDa heat shock protein (Hsp90) is a homodimeric molecular chaperone with ATPase activity, which has become an intensely studied target for the development of drugs for the treatment of cancer, neurodegenerative and infectious diseases. The equilibrium between Hsp90 dimers and oligomers is important for modulating its function. In the absence of ATP, the passive chaperone activity of Hsp90 dimers and oligomers has been shown to stabilize client proteins as a holdase, which enhances substrate binding and prevents irreversible aggregation and precipitation of the substrate proteins. In the presence of ATP and its associated cochaperones, Hsp90 homodimers act as foldases with the binding and hydrolysis of ATP driving conformational changes that mediate client folding. Crystal structures of both wild type and W320A mutant Hsp90αMC (middle/C-terminal domain) have been determined, which displayed a preference for hexameric and dimeric states, respectively. Structural analysis showed that W320 is a key residue for Hsp90 oligomerization by forming intermolecular interactions at the Hsp90 hexameric interface through cation-π interactions with R367. W320A substitution results in the formation of a more open conformation of Hsp90, which has not previously been reported, and the induction of a conformational change in the catalytic loop. The structures provide new insights into the mechanism by which W320 functions as a key switch for conformational changes in Hsp90 self-oligomerization, and binding cochaperones and client proteins.Communicated by Ramaswamy H. Sarma.

The heat shock protein 90 (Hsp90) family of molecular chaperones mediates the folding and activation of client proteins associated with all 10 hallmarks of cancer. Herein, the design, synthesis, and biological validation of Hsp90α-selective inhibitors that contain a tertiary alcohol are reported. Forty-one analogues were synthesized to modulate hydrogen-bonding interactions and to probe for steric and hydrophobic interactions within the Hsp90α binding site. Cocrystal structures of lead compound

The 90 kDa heat-shock protein (Hsp90) is an abundant molecular chaperone that is essential to activate, stabilize and regulate the function of a plethora of client proteins. As drug targets for the treatment of cancer and neurodegenerative diseases, Hsp90 inhibitors that bind to the N-terminal ATP-binding site of Hsp90 have shown disappointing efficacy in clinical trials. Thus, allosteric regulation of the function of Hsp90 by compounds that interact with its middle and C-terminal (MC) domains is now being pursued as a mechanism to inhibit the ATPase activity and client protein-binding activity of Hsp90 without concomitant induction of the heat-shock response. Here, the crystal structure of the Hsp90αMC protein covalently linked to a coumarin derivative, MDCC {7-diethylamino-3-[N-(2-maleimidoethyl)carbamoyl]coumarin}, which is located in a hydrophobic pocket that is formed at the Hsp90αMC hexamer interface, is reported. MDCC binding leads to the hexamerization of Hsp90, and the stabilization and conformational changes of three loops that are critical for its function. A fluorescence competition assay demonstrated that other characterized coumarin and isoflavone-containing Hsp90 inhibitors compete with MDCC binding, suggesting that they could bind at a common site or that they might allosterically alter the structure of the MDCC binding site. This study provides insights into the mechanism by which the coumarin class of allosteric inhibitors potentially disrupt the function of Hsp90 by regulating its oligomerization and the burial of interaction sites involved in the ATP-dependent folding of Hsp90 clients. The hydrophobic binding pocket characterized here will provide new structural information for future drug design.

Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+signaling, little is known about the mechanisms of bacterial Ca2+signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+regulation of virulence factors production and infectivity inPseudomonas aeruginosa,a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+with 13.7 µM affinity, and that mutations at the +X and −Z positions within each or both EF-hand motifs abolished Ca2+binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins.15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion ofefhPimpairedP. aeruginosasurvival in macrophages and virulence in vivo.Disabling EfhP Ca2+binding abolished Ca2+induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+regulation ofP. aeruginosavirulence, thus establishing the role of EfhP as a Ca2+sensor.

We study a cost effective and easy way to fabricate stable, uniform and large area surface-enhanced Raman scattering (SERS) substrates. The AgNPs with varying diameters have been fabricated through a thermally treating method on SiO2/Si substrate which present a high sensitivity, good uniformity and good stability. By using rhodamine 6G (R6G) molecule as analyte, the maximized detection limit is as low as 10−8 M. The deviation of SERS intensity by using proposed AgNPs SERS substrate is less than 15% which indicates its good uniformity. This work provides an efficiently way of fabricating industry SERS substrates which are good candidate in practical application in medicine, food safety and biotechnology.

Significance SAMD9 and SAMD9L (SAMD9/9L) are important innate immune defenders against viruses and the development of myeloid tumors. They form a crucial host barrier that poxviruses must overcome for successful infection. A myriad of human diseases including many pediatric myelodysplastic syndromes are caused by mutations in SAMD9/9L. However, the molecular functions of SAMD9/9L and how their functions are executed are unknown, hindering progress in developing effective therapies for SAMD9/9L-associated human diseases. Here, we identified the structure and function of a SAMD9/9L effector domain that is essential for their physiological functions as well as the pathogenic effects exerted by patient-derived mutations. Our study revealed a potential therapeutic target for SAMD9/9L-associated human diseases., SAMD9 and SAMD9L (SAMD9/9L) are antiviral factors and tumor suppressors, playing a critical role in innate immune defense against poxviruses and the development of myeloid tumors. SAMD9/9L mutations with a gain-of-function (GoF) in inhibiting cell growth cause multisystem developmental disorders including many pediatric myelodysplastic syndromes. Predicted to be multidomain proteins with an architecture like that of the NOD-like receptors, SAMD9/9L molecular functions and domain structures are largely unknown. Here, we identified a SAMD9/9L effector domain that functions by binding to double-stranded nucleic acids (dsNA) and determined the crystal structure of the domain in complex with DNA. Aided with precise mutations that differentially perturb dsNA binding, we demonstrated that the antiviral and antiproliferative functions of the wild-type and GoF SAMD9/9L variants rely on dsNA binding by the effector domain. Furthermore, we showed that GoF variants inhibit global protein synthesis, reduce translation elongation, and induce proteotoxic stress response, which all require dsNA binding by the effector domain. The identification of the structure and function of a SAMD9/9L effector domain provides a therapeutic target for SAMD9/9L-associated human diseases.

Calcium (Ca

The optimal energy management of a hybrid powertrain has the task to provide the required traction power combining both power sources in the best way. This can be achieved well if the future drive cycle is known/precomputed. However, both speed and traction power requirement may deviate from the expected ones due to many factors, like traffic, weather etc. Against this background, it might be sensible to recompute them whenever needed to keep using the latest future information. Unfortunately, this computation is typically too slow for real time use. In this paper we propose a control structure in which the real time task is solved by a predictive controller which tracks the optimal reference from the cloud, and requests an update of the reference regularly. The update can integrate new information from V2X. This asynchronous operation allows recovering most of the performance of the perfect prediction, while removing tight constraints on the offline computation and copes better with interruptions in communications to the cloud.

Surface-enhanced Raman scattering (SERS) as a label-free, non-contact, highly sensitive, and powerful technique has been widely applied in determining bio- and chemical molecules with fingerprint recognitions. 2-dimensional (2D) materials with layered structures, tunable optical properties, good chemical/physical stabilities, and strong charge–transfer interaction with molecules have attracted researchers’ interests. Two-D materials with a large and flat surface area, as well as good biocompatibility have been considered promising candidates in SERS and widely applied in chemical and bio-applications. It is well known that the noble metallic nanostructures with localized surface plasmon effects dominate the SERS performance. The combination of noble metallic nanostructure with 2D materials is becoming a new and attractive research domain. Until now, the SERS substrates combined with 2D materials, such as 2D graphene/metallic NPs, 2D materials@metallic core-shell structures, and metallic structure/2D materials/metallic structure are intensely studied. In this review, we introduce different kinds of fabrication strategies of 2D and 3D SERS substrates combing with 2D materials as well as their applications. We hope this review will help readers to figure out new ideas in designing and fabricating SERS substrates with high SERS performance that could enlarge the applicable domains of SERS.

Knowledge of the upcoming traffic velocity along a route can help in many respects, among them optimizing energy management for hybrid vehicles, which, for instance, could reduce instantaneous battery usage if a traffic jam is upcoming in the next future. While such kind of knowledge can hardly be precise on a single-vehicle level, we show in this paper that a prediction method which combines present and past Vehicle-to-Everything (V2X) information can strongly improve the energy efficiency. Our approach is first compared with other prevailing prediction methods and its advantages in terms of stability and accuracy are shown. Then the prediction results are applied in a hybrid powertrain control example, in which its potential in fuel savings are illustrated.

A priori information about the future traffic conditions along the planned route can be essential for optimal hybrid powertrain energy management. However, due to the limited sensor range of a single vehicle, it cannot be acquired locally. In recent time, V2X (decentralized wireless vehicle to everything) has been receiving much attention as a way to obtain and share information from different distributed sources. V2X data can provide updated information on traffic at different locations. Still, this information will be obsolete when the corresponding positions are reached due to changing traffic, and an optimal strategy based on outdated information may not bring the full benefit. Against this background, we propose a method based on a velocity prediction approach which utilizes V2X data currently available in the market in combination with historical data, to obtain a prediction of the expected traffic conditions at in the close future. Actual measurements on a city highway in Linz, Austria, are used to estimate the potential of the approach. Even for rather mild changes in traffic conditions, a reduction of up to 4% in terms fuel consumption over this track was found, confirming the potential benefit of this method.

Existing mechanical HVDC circuit breakers (CB) are capable of interrupting HVDC currents within several tens of milliseconds, but this is too slow to satisfy the requirements of a reliable HVDC grid. HVDC CB based on semiconductors can easily overcome the limitations of operating speed, but need large number of the series (parallel) connection of electronic switching devices. To overcome these shortcomings, this paper introduces a topology of hybrid CB; this paper first describes the principle of hybrid CB; then a control strategy of fault pre-treatment is also proposed, that is, the ‘pre-action and action (or recovery)’ of the switches, in order to improve the breaking speed of the hybrid CB and the equalising circuit of its solid-state switch has been designed. The proposed topology and control strategy do not produce loss in the normal operation of CB, and instantaneous trigger solid-state switch with quickly cutting short-circuit current during short-circuit faults; at last the simulation and experiment of the hybrid HVDC CB have also been done. The results turn out when a short circuit occurs, the hybrid HVDC CB can quickly break the short-circuit current; the buffer circuit can be provided for the release of the stored energy circuit.

The STENOFOLIA homeodomain binds promoter DNA as a tetramer and the α3 helix binds DNA in both the major and minor grooves., Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants. In plants, the WUSCHEL-related homeobox (WOX) family of transcription factors function as central organizers of several developmental programs ranging from embryo patterning to meristematic stem-cell maintenance through transcriptional activation and repression mechanisms. The Medicago truncatula STENOFOLIA (STF) gene is a master regulator of leaf-blade lateral development. Here, the crystal structure of the homeodomain (HD) of STF (STF-HD) in complex with its promoter DNA is reported at 2.1 Å resolution. STF-HD binds DNA as a tetramer, enclosing nearly the entire bound DNA surface. The STF-HD tetramer is partially stabilized by docking of the C-terminal tail of one protomer onto a conserved hydrophobic surface on the head of another protomer in a head-to-tail manner. STF-HD specifically binds TGA motifs, although the promoter sequence also contains TAAT motifs. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides DNA binding in the minor groove through basic residues located at its C-terminus. The structural and functional data in planta reported here provide new insights into the DNA-binding mechanisms of plant-specific HDs from the WOX family of transcription factors.

Using knowledge of the future route and its topology is known to offer substantial fuel savings, and this is even more true for hybrid electric vehicles, as the battery use can be planned in advance, for instance to take into account coming slopes. However, traffic or other environmental conditions can force to deviate from the initial planning making it no longer optimal.In this paper, we propose a flexible double layer approach for energy management of hybrid vehicles able to cope with traffic changes. First, before departure, an expected optimal speed and powertrain state reference is computed on a cloud and sent to an on-board controller. Simple, route-specific engine on/off rules are extracted by the controller and used for an on-board fast convex optimization, which can be conducted frequently along the drive, adapting the references to take into account changes of traffic conditions over longer sections of the route as communicated by V2X. Abrupt disturbances are handled by a lower level Model Predictive Control (MPC). If the condition changes are very substantial, so that the empirical on/off rule seems questionable, the cloud can be asked to perform a full optimization again.

In this project we design and study a differential phase surface plasmon resonance sensor combining with microfluidic technology. The differential phase Surface Plasmon Resonance (SPR) sensor is constructed by applying the Kretschman plasmon excitation structure and combing with interference spectroscopy. Through making use of simulation and experimental method, the principle of SPR sensor is theoretically and experimentally studied. In this project, we apply the SPR sensor in testing urea solution with varying weight concentration. Through the results, it can be observed that the plasma resonance absorption ranges from 640 nm to 840 nm and its detection limits can reach 0.0004 RIU. Due to its tunable plasma absorption and lower detection limits, the microfluidic differential phase surface plasmon resonance sensor illustrates a promising future in chemistry, physics and biology.

Programmed cell death (PCD) and apoptosis have key functions in development and disease resistance in diverse organisms; however, the induction of necrosis remains poorly understood. Here, we identified a semi-dominant mutant allele that causes the necrotic death of the entire seedling (DES) of wheat (Triticum aestivum L.) in the absence of any pathogen or external stimulus. Positional cloning of the lethal allele mDES1 revealed that this premature death via necrosis was caused by a point mutation from Asp to Asn at amino acid 441 in a nucleotide-binding leucine-rich repeat protein containing nucleotide-binding domain and leucine-rich repeats. The overexpression of mDES1 triggered necrosis and PCD in transgenic plants. However, transgenic wheat harboring truncated wild-type DES1 proteins produced through gene editing that exhibited no significant developmental defects. The point mutation in mDES1 did not cause changes in this protein in the oligomeric state, but mDES1 failed to interact with replication protein A leading to abnormal mitotic cell division. DES1 is an ortholog of Sr35, which recognizes a Puccinia graminis f. sp. tritici stem rust disease effector in wheat, but mDES1 gained function as a direct inducer of plant death. These findings shed light on the intersection of necrosis, apoptosis, and autoimmunity in plants.

The task of energy management for hybrid electric vehicles (HEV) consists in providing the requested traction power by splitting the demand between electrical and mechanical energy sources. The best performance can be achieved if the power split can be computed over the whole trip at once, e.g. using Dynamic Programming (DP). However, actual travel conditions are prone to change with respect to the assumed ones, so that updates are needed. Recomputing the optimal power split for the whole remaining route via DP is hardly feasible due to the actual travel conditions, so approximate solutions are needed. In this paper, we propose an online-capable strategy for a parallel HEV based on the minimization of total losses and heuristics for engine on/off and gear actions. It yields a very good approximation of the DP solution with little computational burden.

Eco-driving is a way to improve performance — mainly energy consumption — of road vehicles by computing an optimal speed and gear shifting profile based on vehicle data and road profile, e.g. slopes or speed limits. It mainly focuses on long haul scenarios such as highways, considering longitudinal movement only. Lateral acceleration of a vehicle is a critical quantity both in terms of comfort and safety, but its impact on fuel consumption or emissions is rarely considered or believed to be limited [1], as it does not affect directly the operating point of the engine. However, on country roads which usually present much stronger curvatures, lateral acceleration may be a critical constraint. In this paper, the impact of lateral acceleration limits on optimal solutions to multi-objective eco-driving is investigated. It is found that it may play an even more critical role than longitudinal acceleration with respect to fuel consumption and NOx emission. As a consequence, the choice of limits to lateral acceleration on curvy roads should be set very carefully in order to achieve a balance between energy saving, drivers comfort and travel time. The results of this work are validated on a high-feasibility Hardware-in-the-loop (HIL) system calibrated with data from Real Driving Emissions tests.

Entrances into highway represent critical sections, as entering vehicles are bound to merge with the existing traffic at a fixed junction point almost independently from the traffic conditions-in some sense a “constrained” cut-in manoeuver. Advanced driver-assistance systems (ADAS) need to perform correctly under these conditions as well, but could also be used to facilitate merging and reducing risks. In particular, vehicles on the main road could adapt their speeds-or even change lane-provided an estimate of the entering vehicle's time to merge is available, exactly as a human driver would do. This paper is concerned with providing such an estimate. To this end, we observe that the speed profile on the entering ramps is rather well predictable if the acceleration of the entering vehicles is described as a function of their actual distance from the junction point. Still, uncertainties remain and to cope with them, we use a stochastic prediction model based on Dynamic Bayesian Network. The result are probability distribution functions of the time to merge based on the observation of speed and distance of the entering vehicle once they become visible to the traffic on the main road. Experimental data are used to illustrate the model structure and the parameter determination. The model quality is then assessed by comparing statistics from simulations with the one recorded on road. The possible use of the model as a tool for traffic prediction algorithm embedded in ADAS and an extension to existing highway stochastic traffic models are shortly discussed at the end of the paper.

A rapid optical microarray imaging approach for anticancer drug screening at specific cancer protein-protein interface targets with binding kinetics and validation by a mass sensor is reported for the first time. Surface plasmon resonance imager (SPRi) demonstrated a 3.5-fold greater specificity for interactions between murine double minute 2 protein (MDM2) and wild-type p53 over a nonspecific p53 mutant in a real-time microfluidic analysis. Significant percentage reflectivity changes (Δ%R) in the SPRi signals and molecular-level mass changes were detected for both the MDM2-p53 interaction and its inhibition by a small-molecule Nutlin-3 drug analogue known for its anticancer property. We additionally demonstrate that synthetic, inexpensive binding domains of interacting cancer proteins are sufficient to screen anticancer drugs by an array-based SPRi technique with excellent specificity and sensitivity. This imaging array, combined with a mass sensor, can be used to study quantitatively any protein-protein interaction and screen for small molecules with binding and potency evaluations.

Human sterile alpha motif domain-containing 9 (SAMD9) protein is a host restriction factor for poxviruses, but it can be overcome by some poxvirus host-range proteins that share homology with vaccinia virus C7 protein. To understand the mechanism of action for this important family of host-range factors, we determined the crystal structures of C7 and myxoma virus M64, a C7 family member that is unable to antagonize SAMD9. Despite their different functions and only 23% sequence identity, the two proteins have very similar overall structures, displaying a previously unidentified fold comprised of a compact 12-stranded antiparallel β-sandwich wrapped in two short α helices. Extensive structure-guided mutagenesis of C7 identified three loops clustered on one edge of the β sandwich as critical for viral replication and binding with SAMD9. The loops are characterized with functionally important negatively charged, positively charged, and hydrophobic residues, respectively, together forming a unique "three-fingered molecular claw." The key residues of the claw are not conserved in two C7 family members that do not antagonize SAMD9 but are conserved in distantly related C7 family members from four poxvirus genera that infect diverse mammalian species. Indeed, we found that all in the latter group of proteins bind SAMD9. Taken together, our data indicate that diverse mammalian poxviruses use a conserved molecular claw in a C7-like protein to target SAMD9 and overcome host restriction.

Cellular membranes are maintained as closed compartments, broken up only transiently during membrane reorganization or lipid transportation. However, open-ended membranes, likely derived from scissions of the endoplasmic reticulum, persist in vaccinia virus-infected cells during the assembly of the viral envelope. A group of viral membrane assembly proteins (VMAPs) were identified as essential for this process. To understand the mechanism of VMAPs, we determined the 2.2-A crystal structure of the largest member, named A6, which is a soluble protein with two distinct domains. The structure of A6 displays a novel protein fold composed mainly of alpha helices. The larger C-terminal domain forms a unique cage that encloses multiple glycerophospholipids with a lipid bilayer-like configuration. The smaller N-terminal domain does not bind lipid but negatively affects lipid binding by A6. Mutations of key hydrophobic residues lining the lipid-binding cage disrupt lipid binding and abolish viral replication. Our results reveal a protein modality for enclosing the lipid bilayer and provide molecular insight into a viral machinery involved in generating and/or stabilizing open-ended membranes.

DC distribution network topology includes: chain structure, two-terminal structure and ring structure. This paper firstly introduces a device of Flexible Alternative Current Transmission System: static synchronous series compensator with fault current limiting. DC distribution network with SSSC-FCL can develop the power quality of distribution network and improve the stability. When the fault occurs, the SSSC-FCL can effectively limit the short circuit current to ensure the safety of system. And then this paper also introduces the main advantages of the DC distribution network and the topology, basic control methods of SSSC-FCL. Finally put forward a DC distribution network topology structure with SSSC-FCL. With the use of PSCAD/EMTDC to model and simulate the DC distribution network, the result of the simulation verified the feasibility and effectiveness of the DC network topology structure with SSSC-FCL.

In the new economic situation, more and more researchers began to pay attention to the electricity market. Government departments attach great importance to the growth of electricity consumption. This paper tries to investigate the relationship between electricity consumption and economic growth in Fujian Province China, using monthly data during January 2014 (2014m01) to May 2017 (2017m05). Investment, consumption and export data instead of only Gross Domestic Product (GDP) data is used to represent the economic growth, which indicates their individual effect on electricity consumption. Dickey-Fuller (DF) tests reveal that all the time series are non-stationary and individually integrated of order one. Long-run cointegration relationships between investment, consumption, export and electricity consumption individually are revealed by Engel-Granger (EG) tests. Furthermore, this paper shows that there exist unidirectional Granger causalities running from investment, consumption and export to electricity consumption based on Vector Auto-Regression (VAR) models, which are verified robust and can be used for policy analysis.

Compared with the AC distribution network, the DC distribution network has the advantages of lower line losses, better power quality, higher reliability and being suitable for all kinds distributed generation and DC load access, etc. This paper firstly studies the models of PV and energy storage connected to the DC distribution network. Then, a new type of DC distribution network topology with hybrid DC circuit breaker is proposed. A coordinated control strategy is also proposed, which can control energy storage to improve the stability of the system. Finally, using PSCAD/EMTDC to model and simulate. The simulation results show that the proposed topology of DC distribution network and control strategy can quickly remove the fault, shorten the fault power failure time and ensure the safe and stable operation of the system, which greatly improves the operation efficiency and safety level of the power grid.

Interleukin-18 (IL-18) is a pleiotropic pro-inflammatory cytokine belonging to the IL-1 superfamily. IL-18 plays an important role in host innate and acquired immune defense, with its activity being modulatedin vivoby its naturally occurring antagonist IL-18 binding protein (IL-18BP). Recent crystal structures of human IL-18 (hIL-18) in complex with its antagonist or cognate receptor(s) have revealed a conserved binding interface on hIL-18 representing a promising drug target. An important step in this process is obtaining crystals of apo hIL-18 or hIL-18 in complex with small-molecule inhibitors, preferably under low ionic strength conditions. In this study, surface-entropy reduction (SER) and rational protein design were employed to facilitate the crystallization of hIL-18. The results provide an excellent platform for structure-based drug design.

Phosphoinositides and phosphoinositide binding proteins play a critical role in membrane and protein trafficking in eukaryotes. Their critical role in replication of cytoplasmic viruses has just begun to be understood. Poxviruses, a family of large cytoplasmic DNA viruses, rely on the intracellular membranes to develop their envelope, and poxvirus morphogenesis requires enzymes from the cellular phosphoinositide metabolic pathway. However, the role of phosphoinositides in poxvirus replication remains unclear, and no poxvirus proteins show any homology to eukaryotic phosphoinositide binding domains. Recently, a group of poxvirus proteins, termed viral membrane assembly proteins (VMAPs), were identified as essential for poxvirus membrane biogenesis. A key component of VMAPs is the H7 protein. Here we report the crystal structure of the H7 protein from vaccinia virus. The H7 structure displays a novel fold comprised of seven α-helices and a highly curved three-stranded antiparallel β-sheet. We identified a phosphoinositide binding site in H7, comprised of basic residues on a surface patch and the flexible C-terminal tail. These residues were found to be essential for viral replication and for binding of H7 to phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Our studies suggest that phosphoinositide binding by H7 plays an essential role in poxvirus membrane biogenesis. IMPORTANCE Poxvirus viral membrane assembly proteins (VMAPs) were recently shown to be essential for poxvirus membrane biogenesis. One of the key components of VMAPs is the H7 protein. However, no known structural motifs could be identified from its sequence, and there are no homologs of H7 outside the poxvirus family to suggest a biochemical function. We have determined the crystal structure of the vaccinia virus (VACV) H7 protein. The structure displays a novel fold with a distinct and positively charged surface. Our data demonstrate that H7 binds phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate and that the basic surface patch is indeed required for phosphoinositide binding. In addition, mutation of positively charged residues required for lipid binding disrupted VACV replication. Phosphoinositides and phosphoinositide binding proteins play critical roles in membrane and protein trafficking in eukaryotes. Our study demonstrates that VACV H7 displays a novel fold for phosphoinositide binding, which is essential for poxvirus replication.

Sperm-associated antigen 9 (SPAG9) is a recently characterized oncoprotein involved in the progression of several human malignancies. To elucidate the role of SPAG9 in the development of human prostate cancer (PCa), tissue microarray (TMA) and immunohistochemistry were used to detect the clinical relevance of SPAG9 in PCa tissues. We found that SPAG9 expression was increased in the PCa tissues when compared with the level in the tumor adjacent normal prostate tissues, and increased SPAG9 staining was significantly correlated with TNM stage and tumor grade. We also examined prostate cancer cell motility, invasion and angiogenesis ability following reduced SPAG9 expression by siRNA. Our data showed that knockdown of SPAG9 in prostate cancer cell lines inhibited cell motility and invasion due to the inactivation of metalloproteinase-2 (MMP‑2)/MMP-9 by upregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1)/TIMP-2. Furthermore, downregulation of vascular endothelial growth factor (VEGF) secretion greatly contributed to the reduced ability of angiogenesis. Our data indicate that SPAG9 expression is significantly increased in PCa and it may be involved in the process of prostate cancer cell motility, migration and angiogenesis.

Poxvirus virion biogenesis is a complex, multistep process, starting with the formation of crescent-shaped viral membranes, followed by their enclosure of the viral core to form spherical immature virions. Crescent formation requires a group of proteins that are highly conserved among poxviruses, including A6 and A11 of vaccinia virus (VACV). To gain a better understanding of the molecular function of A6, we established a HeLa cell line that inducibly expressed VACV-A6, which allowed us to construct VACV mutants with an A6 deletion or mutation. As expected, the A6 deletion mutant of VACV failed to replicate in noncomplementing cell lines with defects in crescent formation and A11 localization. Surprisingly, a VACV mutant that had A6 replaced with a close ortholog from the Yaba-like disease virus YLDV-97 also failed to replicate. This mutant, however, developed crescents and had normal A11 localization despite failing to form immature virions. Limited proteolysis of the recombinant A6 protein identified an N domain and a C domain of approximately 121 and 251 residues, respectively. Various chimeras of VACV-A6 and YLDV-97 were constructed, but only one that precisely combined the N domain of VACV-A6 and the C domain of YLDV-97 supported VACV replication albeit at a reduced efficiency. Our results show that VACV-A6 has a two-domain architecture and functions in both crescent formation and its enclosure to form immature virions. While a cognate N domain is not required for crescent formation, it is required for virion formation, suggesting that interactions of the N domain with cognate viral proteins may be critical for virion assembly. IMPORTANCE Poxviruses are unique among enveloped viruses in that they acquire their primary envelope not through budding from cellular membranes but by forming and extending crescent membranes. The crescents are highly unusual, open-ended membranes, and their origin and biogenesis have perplexed virologists for decades. A group of five viral proteins were recently identified as being essential for crescent formation, including the A6 protein of vaccinia virus. It is thus important to understand the structure and function of A6 in order to solve the long-standing mystery of poxvirus membrane biogenesis. Here, we established an experimental system that allowed the genetic manipulation of the essential A6L gene. By studying A6 mutant viruses, we found that A6 plays an essential role not only in the formation of crescents but also in their subsequent enclosure to form immature virions. We defined the domain architecture of A6 and suggested that one of its two domains cooperates with cognate viral proteins.

Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling., Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′ins) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′ins helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

IL-17 cytokines play a crucial role in a variety of inflammatory and autoimmune diseases. They signal through heterodimeric receptor complexes consisting of members of IL-17R family. A unique intracellular signaling domain was identified within all IL-17Rs, termed similar expression to fibroblast growth factor genes and IL-17R (SEFIR). SEFIR is also found in NF-κB activator 1 (Act1), an E3 ubiquitin ligase, and mediates its recruitment to IL-17Rs. In this study, to our knowledge, we report the structure of the first SEFIR domain from IL-17RB at 1.8Å resolution. SEFIR displays a five-stranded parallel β-sheet that is wrapped by six helices. Site-directed mutagenesis on IL-17RB identified helix αC as being critical for its interaction with Act1 and IL-25 (IL-17E) signaling. Using the current SEFIR structure as a template, the key functional residues in Act1 are also mapped as part of helix αC, which is conserved in IL-17RA and RC, suggesting this helix as a common structural signature for heterotypic SEFIR–SEFIR association. In contrast, helix αB′ is important for homodimerization of Act1, implicating a dual ligand-binding model for SEFIR domain, with distinct structural motifs participating in either homotypic or heterotypic interactions. Furthermore, although the IL-17RB-SEFIR structure resembles closest to the Toll/IL-1R domain of TLR10 with low sequence homology, substantial differences were observed at helices αC, αD, and DD′ loop. To our knowledge, this study provides the first structural view of the IL-17R intracellular signaling, unraveling the mechanism for the specificity of SEFIR versus Toll/IL-1R domain in their respective signaling pathways.

A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

Heat shock protein 90 (Hsp90) is a highly conserved chaperone protein that enables the proper folding of a large number of structurally diverse proteins (a.k.a., clients) in the crowded cytosolic environment and plays a key role in regulating the heat shock response. An outstanding question is how Hsp90 accommodates the structural diversity of a large cohort of client proteins? We report ATR FTIR study on structural properties of Hsp90 C-terminal domain (CTD) and their temperature dependences. Our data reveal that within a narrow temperature window, from 35o to 45 oC around physiological temperatures, Hsp90CTD exhibits significant increases in protein aggregation and increases in unordered structures. Binding of Hsp90 inhibitor Clorobiocin elevates the transition temperature of protein aggregation from 38 °C (a physiological temperature) to 65 °C (far above the physiological temperatures). In striking contrast, the Hsp90 N-terminal domain and middle domain (NTMD) demonstrates no protein aggregation from 20° to 90 °C. Despite the intrinsically disordered nature of Hsp90CTD, it retains a protected hydrophobic core at 40 °C. We introduce a hydrophobic interlock (HPI) model to account for the functional capability of intrinsically disordered Hsp90. Implications of these results and the HPI model will be discussed in the light of the structural dynamics and client diversity of Hsp90.

Previous studies indicated a role of hepatocellular carcinoma-related protein-1(HCRP-1) in human cancers, however, its expression pattern in renal cell carcinoma (RCC) and the molecular mechanism of HCRP-1 on cancer progression have not been characterized. In the present study, HCRP-1 expression was examined in a RCC tissue microarray. The negative expression of HCRP-1 was significantly correlated with tumor grade (P = 0.002), TNM stage (P = 0.001) and pT status (P = 0.003). Furthermore, we showed a strong correlation between negative HCRP-1 expression and worse overall and disease-specific survival (P = 0.0003 and P = 0.0012, respectively). Knockdown of HCRP-1 promoted cell migration and invasion in 786-O and OS-RC-2 cell lines. HCRP-1 depletion increased matrix metalloproteinase (MMP)-2 protein level, with increased extracellular signal-regulatedkinase (ERK) phosphorylation, which could be reversed by ERK siRNA or ERK inhibitor, PD98059. Further analysis showed that HCRP-1 knockdown induced epidermal growth factor receptor (EGFR) phosphorylation. Treatment with EGFR inhibitor or EGFR siRNA blocked HCRP-1-mediated up-regulation of EGFR, ERK phosphorylation and MMP-2 expression. In summary, our results showed that negative HCRP-1 expression is an independent prognostic factor for RCC patients and promotes migration and invasion by EGFR-ERK-mediated up-regulation of MMP-2. HCRP-1 may serve as a therapeutic target for RCC.

Inhibitor of differentiation or DNA binding-1 (ID1) is an important helix-loop-helix (HLH) transcription factor involved in diverse biological functions including cell differentiation, proliferation, apoptosis, and senescence. Recently, it was reported that ID1 can activate the NF-κB signaling pathway in a variety of cancer cells and a T cell line, but the mechanisms involved in ID1-mediated transactivation of NF-κB are not clear. In this study, we demonstrate by both in vitro pull-down assays and a cell-based in vivo two-hybrid system that ID1-mediated NF-κB activation is due to its physical interaction with p65. We have identified that the transcriptional activation domain (TAD) in p65 and the HLH domain in ID1 are vital for their interaction. Interestingly, a single site mutation (Leu76) in the HLH domain of ID1 protein drastically decreased its ability to bind with p65. Using a dual-luciferase assay, we demonstrated that the interaction between ID1 and p65 modulates activation of the NF-κB signaling pathway in vivo. In addition, we demonstrated that, by affecting the nuclear translocation of p65, ID1 is essential in regulating TNF-α-induced p65 recruitment to its downstream target, the cellular inhibitor of apoptosis protein 2 (cIAP2) promoter.

Previous studies indicate that prostate cancer antigen 3 (PCA3) is highly expressed in prostatic tumors. However, its clinical value has not been characterized. The aim of this study was to investigate the clinical value of the urine PCA3 test in the diagnosis of prostate cancer by pooling the published data. Clinical trials utilizing the urine PCA3 test for diagnosing prostate cancer were retrieved from PubMed and Embase. A total of 46 clinical trials including 12,295 subjects were included in this meta-analysis. The pooled sensitivity, specificity, positive likelihood ratio (+LR), negative likelihood ratio (−LR), diagnostic odds ratio (DOR) and area under the curve (AUC) were 0.65 (95% confidence interval [CI]: 0.63–0.66), 0.73 (95% CI: 0.72–0.74), 2.23 (95% CI: 1.91–2.62), 0.48 (95% CI: 0.44–0.52), 5.31 (95% CI: 4.19–6.73) and 0.75 (95% CI: 0.74–0.77), respectively. In conclusion, the urine PCA3 test has acceptable sensitivity and specificity for the diagnosis of prostate cancer and can be used as a non-invasive method for that purpose.

LRRK2 is a 250 kDa multidomain protein, mutations in which cause familial Parkinson's disease. Previously, we have demonstrated that the R1441C mutation in the ROC domain decreases GTPase activity. Here we show that the R1441C alters the folding properties of the ROC domain, lowering its thermodynamic stability. Similar to small GTPases, binding of different guanosine nucleotides alters the stability of the ROC domain, suggesting that there is an alteration in conformation dependent on GDP or GTP occupying the active site. GTP/GDP bound state also alters the self-interaction of the ROC domain, accentuating the impact of the R1441C mutation on this property. These data suggest a mechanism whereby the R1441C mutation can reduce the GTPase activity of LRRK2, and highlights the possibility of targeting the stability of the ROC domain as a therapeutic avenue in LRRK2 disease.

Mutations in leucine-rich repeat kinase 2 ( LRRK2 ) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity. The structure of ROC and the mechanism(s) by which it regulates kinase activity are not known. Here, we report the crystal structure of the LRRK2 ROC domain in complex with GDP-Mg 2+ at 2.0-Å resolution. The structure displays a dimeric fold generated by extensive domain-swapping, resulting in a pair of active sites constructed with essential functional groups contributed from both monomers. Two PD-associated pathogenic residues, R1441 and I1371, are located at the interface of two monomers and provide exquisite interactions to stabilize the ROC dimer. The structure demonstrates that loss of stabilizing forces in the ROC dimer is likely related to decreased GTPase activity resulting from mutations at these sites. Our data suggest that the ROC domain may regulate LRRK2 kinase activity as a dimer, possibly via the C-terminal of ROC (COR) domain as a molecular hinge. The structure of the LRRK2 ROC domain also represents a signature from a previously undescribed class of GTPases from complex proteins and results may provide a unique molecular target for therapeutics in PD.

A series of novel anthracene L-rhamnopyranosides compounds were designed and synthesized and their anti-proliferative activities on cancer cell lines were investigated. We found that one derivative S-8 (EM-d-Rha) strongly inhibited cell proliferation of a panel of different human cancer cell lines including A549, HepG2, OVCAR-3, HeLa and K562 and SGC-790 cell lines, and displayed IC50 values in low micro-molar ranges, which are ten folds more effective than emodin. In addition, we found EM-d-Rha (3-(2",3"-Di-O-acetyl-α-L-rhamnopyranosyl-(1→4)-2',3'-di-O-acetyl-α-L-rhamnopyranosyl)-emodin) substantially induced cellular apoptosis of HepG2 and OVCAR-3 cells in the early growth stage. Furthermore, EM-d-Rha led to the decrease of mitochondrial transmembrane potential, and up-regulated the express of cells apoptosis factors in a concentration- and time-dependent manner. The results indicated the EM-d-Rha may inhibit the growth and proliferation of HepG2 cells through the pathway of apoptosis induction, and the possible molecular mechanism may due to the activation of intrinsic apoptotic signal pathway.

Trypanosomatids are pathogenic protozoa that undergo a unique form of post-transcriptional RNA editing that inserts or deletes uridine nucleotides in many mitochondrial pre-mRNAs. Editing is catalyzed by a large multiprotein complex, the editosome. A key editosome enzyme, RNA editing terminal uridylyl transferase 2 (TUTase 2; RET2) catalyzes the uridylate addition reaction. Here, we report the 1.8 A crystal structure of the Trypanosoma brucei RET2 apoenzyme and its complexes with uridine nucleotides. This structure reveals that the specificity of the TUTase for UTP is determined by a crucial water molecule that is exquisitely positioned by the conserved carboxylates D421 and E424 to sense a hydrogen atom on the N3 position of the uridine base. The three-domain structure also unveils a unique domain arrangement not seen before in the nucleotidyltansferase superfamily, with a large domain insertion between the catalytic aspartates. This insertion is present in all trypanosomatid TUTases. We also show that TbRET2 is essential for survival of the bloodstream form of the parasite and therefore is a potential target for drug therapy.

Trypanosomatids are causative agents of several devastating tropical diseases such as African sleeping sickness, Chagas’ disease and leishmaniasis. There are no effective vaccines available to date for treatment of these protozoan diseases, while current drugs have limited efficacy, significant toxicity and suffer from increasing resistance. Trypanosomatids have several remarkable and unique metabolic and structural features that are of great interest for developing new anti-protozoan therapeutics. One such feature is “RNA editing”, an essential process in these pathogenic protozoa. Transcripts for key trypanosomatid mitochondrial proteins undergo extensive post-transcriptional RNA editing by specifically inserting or deleting uridylates from premature mRNA in order to create mature mRNAs that encode functional proteins. The RNA editing process is carried out in aw1.6 MDa multi-protein complex, the editosome. In Trypanosoma brucei, one of the editosome’s core enzymes, the RNA editing ligase 1 (TbREL1), has been shown to be essential for survival of both insect and bloodstream forms of the parasite. We report here the crystal structure of the catalytic domain of TbREL1 at 1.2 A u resolution, in complex with ATP and magnesium. The magnesium ion interacts with the b and g-phosphate groups and is almost perfectly octahedrally coordinated by six phosphate and water oxygen atoms. ATP makes extensive direct and indirect interactions with the ligase via essentially all its atoms while extending its base into a deep pocket. In addition, the ATP makes numerous interactions with residues that are conserved in the editing ligases only. Further away from the active site, TbREL1 contains a unique loop containing several hydrophobic residues that are highly conserved among trypanosomatid RNA editing ligases which may play a role in protein– protein interactions in the editosome. The distinct characteristics of the adenine-binding pocket, and the absence of any close homolog in the human genome, bode well for the design of selective inhibitors that will block the essential RNA ligase function in a number of major protozoan pathogens. q 2004 Elsevier Ltd. All rights reserved.

Purine-rich regions in DNA and RNA may contain both guanines and adenines, which have various biological functions. Here we report the crystal structure of an RNA purine-rich fragment containing both guanine and adenine at 1.4 A resolution. Adenines form an adenine tetrad in the N6-H em leader N7 conformation. Substitution of an adenine tetrad in the guanine tetraplex does not change the global conformation but introduces irregularity in both the hydrogen bonding interaction pattern in the groove and the metal ion binding pattern in the central cavity of the tetraplex. The irregularity in groove binding may be critical for specific binding in tetraplexes. The formation of G-U octads provides a mechanism for interaction in the groove. Ba(2+) ions prefer to bind guanine tetrads, and adenine tetrads can only be bound by Na(+) ions, illustrating the binding selectivity of metal ions for the tetraplex.

The crystal structure of an RNA dodecamer, r(GCGUCAGGUC(Br)CG)/r(CGGAAGCAG(Br)CGC), containing a fragment from the signal recognition particle (SRP) RNA (domain IV) of Escherichia coli, has been determined at 1.7 A resolution with 21 666 independent reflections and an R(work) and R(free) of 20.1 and 22.5%, respectively. The structure exhibits a novel crystal packing pattern for RNA oligomer duplexes: one end of the duplex adopts the stacking interaction, while the other end adopts the abutting interaction in the minor groove. The symmetric loop of the SRP, r(CAGG)/r(AGCA), in the center of the dodecamer forms two different conformations of the A.C mismatch, a sheared G.G and a symmetrical G.A mismatch. These four mismatches present a unique surface for the abutting interaction. The involvement of the two A.C mismatches in the abutting interaction implies that these mismatches are the important sites for interaction with proteins. The conformation of the symmetric loop is greatly stabilized by hydrated metal ions, which display flexibility in adjusting their geometry and coordination in interaction with nucleic acids. Comparison with other crystal structures of fragments of 4.5S RNA indicates that the conformation of the symmetric loop is independent of the asymmetrical loop in domain IV.