A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B.

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

Therapeutic targeting of tetraspanin8 in epithelial ovarian cancer invasion and metastasis.

Epithelial ovarian cancer (EOC) invasion and metastasis are complex phenomena that result from the coordinated action of many metastatic regulators and must be overcome to improve clinical outcomes for patients with these cancers. The identification of novel therapeutic targets is critical because of the limited success of current treatment regimens, particularly in advanced-stage ovarian cancers. In this study, we found that tetraspanin 8 (TSPAN8) is overexpressed in about 52% (14/27) of EOC tissues and correlates with poor survival. Using small interfering RNA-mediated TSPAN8 knockdown and a competition assay with purified TSPAN8 large extracellular loop (TSPAN8-LEL) protein, we identified TSPAN8-LEL as a key regulator of EOC cell invasion. Furthermore, monotherapy with TSPAN8-blocking antibody we developed shows that antibody-based modulation of TSPAN8-LEL can significantly reduce the incidence of EOC metastasis without severe toxicity in vivo. Finally, we demonstrated that the TSPAN8-blocking antibody promotes the internalization and concomitant downregulation of cell surface TSPAN8. Collectively, our data suggest TSPAN8 as a potential novel therapeutic target in EOCs and antibody targeting of TSPAN8 as an effective strategy for inhibiting invasion and metastasis of TSPAN8-expressing EOCs.

Technical assessment of processing plants as exemplified by the sorting of beverage cartons from lightweight packaging wastes.

The recovery of beverage cartons (BC) in three lightweight packaging waste processing plants (LP) was analyzed with different input materials and input masses in the area of 21-50Mg. The data was generated by gravimetric determination of the sorting products, sampling and sorting analysis. Since the particle size of beverage cartons is larger than 120mm, a modified sampling plan was implemented and targeted multiple sampling (3-11 individual samplings) and a total sample size of respectively 1200l (ca. 60kg) for the BC-products and of about 2400l (ca. 120kg) for material-heterogeneous mixed plastics (MP) and sorting residue products. The results infer that the quantification of the beverage carton yield in the process, i.e., by including all product-containing material streams, can be specified only with considerable fluctuation ranges. Consequently, the total assessment, regarding all product streams, is rather qualitative than quantitative. Irregular operation conditions as well as unfavorable sampling conditions and capacity overloads are likely causes for high confidence intervals. From the results of the current study, recommendations can basically be derived for a better sampling in LP-processing plants. Despite of the suboptimal statistical results, the results indicate very clear that the plants show definite optimisation potentials with regard to the yield of beverage cartons as well as the required product purity. Due to the test character of the sorting trials the plant parameterization was not ideal for this sorting task and consequently the results should be interpreted with care.

Extracting the redox orbitals in Li battery materials with high-resolution x-ray compton scattering spectroscopy.

We present an incisive spectroscopic technique for directly probing redox orbitals based on bulk electron momentum density measurements via high-resolution x-ray Compton scattering. Application of our method to spinel Li_{x}Mn_{2}O_{4}, a lithium ion battery cathode material, is discussed. The orbital involved in the lithium insertion and extraction process is shown to mainly be the oxygen 2p orbital. Moreover, the manganese 3d states are shown to experience spatial delocalization involving 0.16±0.05 electrons per Mn site during the battery operation. Our analysis provides a clear understanding of the fundamental redox process involved in the working of a lithium ion battery.

Function and molecular evolution of multicopper blue proteins.

Multicopper blue proteins (MCBPs) are multidomain proteins that utilize the distinctive redox ability of copper ions. There are a variety of MCBPs that have been roughly classified into three different groups, based on their domain organization and functions: (i) nitrite reductase-type with two domains, (ii) laccase-type with three domains, and (iii) ceruloplasmin-type with six domains. Together, the second and third group are often commonly called multicopper oxidases (MCOs). The rapid accumulation of genome sequence information in recent years has revealed several new types of proteins containing MCBP domains, mainly from bacteria. In this review, the recent research on the functions and structures of MCBPs is summarized, mainly focusing on the new types. The latter half of this review focusses on the two domain MCBPs, which we propose as the evolutionary intermediate of the MCBP family.

Dynamical transition of myoglobin in a crystal: comparative studies of X-ray crystallography and Mössbauer spectroscopy.

The crystallographic normal mode refinements of myoglobin at a wide range of temperature from 40 K to 300 K were carried out to study the temperature dependence of the internal atomic fluctuations. The refinement method decomposes the mean square displacement from the average position, (deltar2), into the contributions from the internal degrees of freedom and those from the external degrees of freedom. The internal displacements show linear temperature dependence as (deltar2)=alphaT+beta, throughout the temperature range measured here, and exhibit no obvious change in the slope alpha at the dynamical transition temperature (Tc=ca. 180 K). The slope alpha is practically the same as the value predicted theoretically by normal mode analysis. Such linear dependence is considered to be due to the following reason. The crystallographic Debye-Waller factor represents the static distribution caused by convolution of temperature-dependent normal mode motions and a temperature-independent set of the conformational substates. In contrast, Mössbauer absorption spectroscopy shows a clear increase in the gradient alpha at Tc. This difference from X-ray diffraction originates from the incoherent nature of the Mössbauer effect together with its high-energy resolution, which yields the self-correlation, and the temporal behavior of individual Fe atoms in the myoglobin crystal.

Selection of estrogen receptor beta- and thyroid hormone receptor beta-specific coactivator-mimetic peptides using recombinant peptide libraries.

Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.

Molecular dynamics simulation shows large volume fluctuations of proteins.

In this paper we present a new approach to study the volume fluctuations of proteins. From a 1 ns molecular dynamics simulation, the volume fluctuation of human lysozyme has been calculated. We used two different ways for the calculation. In the first one, the volume fluctuation is extracted directly from the trajectory. For the second one, a newly developed formalism based on principal component analysis is used. The r.m.s. volume fluctuations obtained from the two analyses agree well with each other. The isothermal intrinsic compressibility was found to be larger than the one reported by experiment. The difference is discussed and suggested to exist in the assumed uncertainty of the compressibility of hydrated water to deduce the isothermal intrinsic compressibility from the experimental value. Spectral analysis shows that low-frequency dynamics dominate the total volume fluctuation. The same aspect is found in the study using principal component analysis. This low-frequency region is related to large and slow motions of proteins. Therefore a long time dynamics simulation is necessary to describe the volume fluctuations of proteins.

Dynamical structure of transfer RNA studied by normal mode analysis.

The internal motion of yeast phenylalanine transfer RNA is studied by normal mode analysis in extended dihedral angle space in which the flexibility of five-membered ribose rings is treated faithfully by introducing a variable for its pseudo-rotational motion. Analysis of global molecular motion reveals that the molecule is very soft. We show that this softness comes not from the property of the "material" comprising the molecule but from its slender shape. Analysis of thermal distance fluctuations reveals that this molecule can be regarded as consisting dynamically of three blocks. Thermal fluctuations of the mainchain dihedral angles show rigidity of the anticodon region. They also show flexibility of regions around non-stacking bases. Base-stacking interactions cause suppression of the correlated functions of mainchain dihedral angles beyond a ribose ring. We analyze the thermal fluctuation of parameters describing the positions of two adjacent bases. Fluctuations of relative translational parameters in the anticodon and acceptor stem regions are found to be larger that those in other stem regions. The relative translational motions cause the two stem regions to undergo global twisting and bending motions. We show that the role of pseudo-rotational motion of sugars is important in regions around bases which are involved in nonregular interactions.

Diversity of functions of proteins with internal symmetry in spatial arrangement of secondary structural elements.

We carry out a systematic analysis of the correlation between similarity of protein three-dimensional structures and their evolutionary relationships. The structural similarity is quantitatively identified by an all-against-all comparison of the spatial arrangement of secondary structural elements in nonredundant 967 representative proteins, and the evolutionary relationship is judged according to the definition of superfamily in the SCOP database. We find the following symmetry rule: a protein pair that has similar folds but belong to different superfamilies has (with a very rare exception) certain internal symmetry in its common similar folds. Possible reasons behind the symmetry rule are discussed.

Investigating protein dynamics in collective coordinate space.

Currently, collective coordinates are commonly employed in order to examine protein dynamics. In recent studies, they have been successfully applied to finding functionally relevant motions, to investigating the physical nature of protein dynamics, to sampling of the conformational space and to the analysis of experimental data. Collective coordinates also have other possible applications.

Structural motif of phosphate-binding site common to various protein superfamilies: all-against-all structural comparison of protein-mononucleotide complexes.

In order to search for a common structural motif in the phosphate-binding sites of protein-mononucleotide complexes, we investigated the structural variety of phosphate-binding schemes by an all-against-all comparison of 491 binding sites found in the Protein Data Bank. We found four frequently occurring structural motifs composed of protein atoms interacting with phosphate groups, each of which appears in different protein superfamilies with different folds. The most frequently occurring motif, which we call the structural P-loop, is shared by 13 superfamilies and is characterized by a four-residue fragment, GXXX, interacting with a phosphate group through the backbone atoms. Various sequence motifs, including Walker's A motif or the P-loop, turn out to be a structural P-loop found in a few specific superfamilies. The other three motifs are found in pairs of superfamilies: protein kinase and glutathione synthetase ATPase domain like, actin-like ATPase domain and nucleotidyltransferase, and FMN-linked oxidoreductase and PRTase.

CCR2B-64I chemokine receptor allele and mother-to-child HIV-1 transmission or disease progression in children. French pediatric HIV infection study group.

The beneficial role of a variant of the chemokine receptor CCR2B (CCR2B-641) in the evolution of HIV-1 infection in adults is still controversial. Furthermore, no studies have been performed in HIV-1-infected children. A multicenter and prospective study of 745 infants born to HIV-1-seropositive mothers was performed. The CCR2B-641 allele was studied in 525 non-African children among whom 523 had been previously genotyped for the CCR5delta32 allele and 220 African children. Of the 745 total, 376 children were infected and 369 were uninfected. In the complete population studied, the children homozygous for the CCR2B-64I allele and the heterozygous children were found distributed equally in the infected (respectively, 1.6% and 21%) and uninfected (respectively, 1.9% and 26.3%) groups (p < .22). Among 376 infected children, the incidence of stage C symptoms (U.S. Centers for Disease Control and Prevention [CDC] classification) or the progression of severe immune deficiency (CD4 <15%, CDC stage 3) was not significantly different in heterozygous infected children or children homozygous for the normal allele (p < .17 and p < .75, respectively). The same lack of protective effect was obtained when a separate analysis was performed in the non-African and African HIV-1-infected children.

Energy landscape of a native protein: jumping-among-minima model.

We have investigated energy landscape of human lysozyme in its native state by using principal component analysis and a model, jumping-among-minima (JAM) model. These analyses are applied to 1 nsec molecular dynamics trajectory of the protein in water. An assumption embodied in the JAM model allows us to divide protein motions into intra-substate and inter-substate motions. By examining intra-substate motions, it is shown that energy surfaces of individual conformational substates are nearly harmonic and mutually similar. As a result of principal component analysis and JAM model analysis, protein motions are shown to consist of three types of collective modes, multiply hierarchical modes, singly hierarchical modes, and harmonic modes. Multiply hierarchical modes, the number of which accounts only for 0.5% of all modes, dominate contributions to total mean-square atomic fluctuation. Inter-substate motions are observed only in a small-dimensional subspace spanned by the axes of multiplyhierarchical and singly hierarchical modes. Inter-substate motions have two notable time components: faster component seen within 200 psec and slower component. The former involves transitions among the conformational substates of the low-level hierarchy, whereas the latter involves transitions of the higher level substates observed along the first four multiply hierarchical modes. We also discuss dependence of the subspace, which contains conformational substates, on time duration of simulation.

Molecular dynamics study of femtosecond events in photoactive yellow protein after photoexcitation of the chromophore.

Molecular dynamics simulations were carried out to study what happens in a photoreceptor protein, photoactive yellow protein (PYP), immediately after the vertical transition of the chromophore from the ground to the excited state. A photon absorption simulation was performed to investigate the movement of amino acid residues upon photoexcitation. To calculate the excited state of the chromophore, SCF-CI calculation was carried out with INDO/S Hamiltonian. We observed that some amino acid residues have strong interactions with the chromophore. Most of these amino acid residues are conserved in PYPs from three different species of bacteria. This observation indicates the biological importance of these residues.

Computational analysis of thermal stability: effect of Ile-->Val mutations in human lysozyme.

BACKGROUND: Free energy calculations are carried out to study the change of thermal stability caused by Ile23-->Val, Ile56-->Val, Ile89-->Val and Ile106-->Val mutations in human lysozyme. In order to examine the dependence of the free energy difference, DeltaDeltaG, on the denatured-state structure, extended and native-like conformations are employed as initial conformations in the denatured-state simulations. RESULTS: Calculated values of DeltaDeltaG for the mutations, Ile56-->Val, Ile89-->Val and Ile106-->Val, were in good agreement with experimental values when the native-like structure was employed in the respective denatured-state simulations. In the case of Ile23-->Val, a considerable difference between the calculated and experimental values of DeltaDeltaG was observed. CONCLUSIONS: The physical nature of Ile56-->Val, Ile89-->Val and Ile106-->Val mutations was rationally characterized by a free energy component analysis. It is suggested that the alpha domain in which Ile23 is included is considerably structured even in the denatured state.

Pulmonary mechanics of normal very-low-birth-weight infants at 40 weeks postconception: a comparison with normal term infants.

We evaluated pulmonary function status in healthy, nonventilated very-low-birth-weight (VLBW) infants at 40 weeks postconception because little is known about the pulmonary function status of these infants at the time of discharge. Seven normal VLBW infants were evaluated at 40 weeks postconception, and five normal term infants were examined as a control. The neonates were placed supine with their heads in the neutral position. Data were obtained while the infant was resting quietly and breathing spontaneously prior to feeding. Pulmonary mechanics and energetics were measured with a CP-100 pulmonary monitor (Bicore Co. Ltd.). Pulmonary mechanics and energetics were determined by the least mean square technique. Dynamic compliance (Cdyn), resistance (Rtot, Re), and work of breathing (WOB) were calculated for the total breath, and tidal volume, minute ventilation, ratio of inspiratory time to respiratory period (Ti/Ttot), and respiratory rate were measured. There were no significant differences between normal VLBW infants and term infants in regard to ventilation, mechanics, or energetics. Although the respiratory system of normal VLBW infants is immature at birth, its development caught up to term infants by the time of discharge.

Genetic recombination as a reporter for screening steroid receptor agonists and antagonists.

Reporter cell lines are often used for high throughput screening of chemical libraries to identify new receptor ligands. Here we show how Cre recombinase can be used in mammalian cells to screen for steroid receptor ligands. A translational fusion of Cre recombinase and the ligand binding domain of the human glucocorticoid receptor was transfected into mammalian cells with a loxP/luciferase reporter gene. The recombinase function of the fusion is dependent on ligand binding to the receptor, and Cre-mediated recombination results in constitutive expression of luciferase from the reporter gene. A stable transfected clone was isolated and used to characterize the kinetics, ligand specificity, and dose response to various receptor ligands. The Cre fusion system, unlike a transcriptional reporter using the mouse mammary tumor virus promoter, can detect binding of the receptor antagonist RU486. We also studied the Cre reporter in a sensitive, miniaturized, assay format using an 864-well plate and show that as few as 560 cells per assay well was sufficient to measure a dose response to ligand.

CCR5 chemokine receptor variant in HIV-1 mother-to-child transmission and disease progression in children. French Pediatric HIV Infection Study Group.

CONTEXT: Studies suggest that adults with the CCR5delta32 deletion are less likely to become infected with the human immunodeficiency virus (HIV) and to develop HIV-related disease progression, but the effect of the mutation in children is not known. OBJECTIVE: To study the effect of the CCR5 chemokine receptor mutant allele on mother-to-child transmission of HIV type 1 (HIV-1) and subsequent disease progression in infected children. DESIGN: Multicenter, prospective study of infants born to mothers seropositive for HIV-1. SETTING: A total of 52 medical centers participating in the French Pediatric HIV Cohort studies. PARTICIPANTS: The CCR5delta32 deletion was studied in 512 non-African children, born between 1983 and 1996 to HIV-1-infected mothers. Among them, 276 children were infected and 236 were not. MAIN OUTCOME MEASURES: HIV-1 infection status and, in infected children followed up since birth, incidence of category B and C disease events and severe immunosuppression as defined in the new pediatric Centers for Disease Control and Prevention (CDC) classification, according to CCR5 genotype. RESULTS: The 32-base pair deleted allele was detected at a frequency of 0.05. Only 1 infant, not infected by HIV-1, was homozygous for the delta32 deletion. The 49 heterozygous children (9.6% of the total; 95% confidence interval [CI], 7.1-12.2) were equally distributed into the infected (9.8%) and uninfected (9.3%) groups. The incidence of stage C symptoms in heterozygous infected children was 9% at 36 months vs 28% in children homozygous for the normal allele (P<.004). The proportion of children at 8 years old with no stage B or C symptoms was 49% for heterozygous children and 11% for children homozygous for the normal allele (P<.003). The progression of severe immune deficiency (CD4 <15%, CDC stage 3) was also significantly different between the 2 groups (P<.001). CONCLUSIONS: Heterozygosity for the CCR5delta32 deletion does not protect children from infection by the maternal virus but substantially reduces the progression of the disease in HIV-1-infected children.

Mechanical property of a TIM-barrel protein.

The mechanical response of a TIM-barrel protein to an applied pressure has been studied. We generated structures under an applied pressure by assuming the volume change to be a linear function of normal mode variables. By Delaunay tessellation, the space occupied by protein atoms is divided uniquely into tetrahedra, whose four vertices correspond to atomic positions. Based on the atoms that define them, the resulting Delaunay tetrahedra are classified as belonging to various secondary structures in the protein. The compressibility of various regions identified with respect to secondary structural elements in this protein is obtained from volume changes of respective regions in two structures with and without an applied pressure. We found that the beta barrel region located at the core of the protein is quite soft. The interior of the beta barrel, occupied by side chains of beta strands, is the softest. The helix, strand, and loop segments themselves are extremely rigid, while the regions existing between these secondary structural elements are soft. These results suggest that the regions between secondary structural elements play an important role in protein dynamics. Another aspect of tetrahedra, referred to as bond distance, is introduced to account for rigidities of the tetrahedra. Bond distance is a measure of separation of the atoms of a tetrahedron in terms of number of bonds along the polypeptide chain or side chains. Tetrahedra with longer bond distances are found to be softer on average. From this behavior, we derive a simple empirical equation, which well describes the compressibilities of various regions.