Molecular architecture of the 40S⋅eIF1⋅eIF3 translation initiation complex.

Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits.

Assembly and dynamics of the autophagy-initiating Atg1 complex.

The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17-Atg31-Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold.

E pluribus unum, no more: from one crystal, many conformations.

Several distinct computational approaches have recently been implemented to represent conformational heterogeneity from X-ray crystallography datasets that are averaged in time and space. As these modeling methods mature, newly discovered alternative conformations are being used to derive functional protein mechanisms. Room temperature X-ray data collection is emerging as a key variable for sampling functionally relevant conformations also observed in solution studies.

Mechanisms of action of hESC-secreted proteins that enhance human and mouse myogenesis.

Adult stem cells grow poorly in vitro compared to embryonic stem cells, and in vivo stem cell maintenance and proliferation by tissue niches progressively deteriorates with age. We previously reported that factors produced by human embryonic stem cells (hESCs) support a robust regenerative capacity for adult and old mouse muscle stem/progenitor cells. Here we extend these findings to human muscle progenitors and investigate underlying molecular mechanisms.

Molecular architecture and function of the SEA complex, a modulator of the TORC1 pathway.

The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation.

Optically detected cross-relaxation spectroscopy of electron spins in diamond.

The application of magnetic resonance spectroscopy at progressively smaller length scales may eventually permit 'chemical imaging' of spins at the surfaces of materials and biological complexes. In particular, the negatively charged nitrogen-vacancy (NV(-)) centre in diamond has been exploited as an optical transducer for nanoscale nuclear magnetic resonance. However, the spectra of detected spins are generally broadened by their interaction with proximate paramagnetic NV(-) centres through coherent and incoherent mechanisms.

Prediction of substrates for glutathione transferases by covalent docking.

Enzymes in the glutathione transferase (GST) superfamily catalyze the conjugation of glutathione (GSH) to electrophilic substrates. As a consequence they are involved in a number of key biological processes, including protection of cells against chemical damage, steroid and prostaglandin biosynthesis, tyrosine catabolism, and cell apoptosis. Although virtual screening has been used widely to discover substrates by docking potential noncovalent ligands into active site clefts of enzymes, docking has been rarely constrained by a covalent bond between the enzyme and ligand.

The protein-vesicle network of autophagy.

The biogenesis of autophagosomes entails the nucleation and growth of a double-membrane sheet, the phagophore, which engulfs cytosol for delivery to the lysosome. Genetic studies have identified a class of Atg proteins that are essential for the process, yet the molecular mechanism of autophagosome biogenesis has been elusive. Proteomic, structural, super-resolution imaging, and biochemical reconstitution experiments have begun to fill in some of the gaps.

Hydrogen tunneling in a prokaryotic lipoxygenase.

A bacterial lipoxygenase (LOX) shows a deuterium kinetic isotope effect (KIE) that is similar in magnitude and temperature dependence to the very large KIE of eukaryotic LOXs. This occurs despite the evolutionary distance, an ~25% smaller catalytic domain, and an increase in Ea of ~11 kcal/mol. Site-specific mutagenesis leads to a protein variant with an Ea similar to that of the prototypic plant LOX, providing possible insight into the origin of evolutionary differences.

A future of the model organism model.

Changes in technology are fundamentally reframing our concept of what constitutes a model organism. Nevertheless, research advances in the more traditional model organisms have enabled fresh and exciting opportunities for young scientists to establish new careers and offer the hope of comprehensive understanding of fundamental processes in life. New advances in translational research can be expected to heighten the importance of basic research in model organisms and expand opportunities.

Nitric oxide-induced conformational changes in soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is the primary mediator of nitric oxide (NO) signaling. NO binds the sGC heme cofactor stimulating synthesis of the second messenger cyclic-GMP (cGMP). As the central hub of NO/cGMP signaling pathways, sGC is important in diverse physiological processes such as vasodilation and neurotransmission.

Joint effects of known type 2 diabetes susceptibility loci in genome-wide association study of Singapore Chinese: the Singapore Chinese health study.

Background: Genome-wide association studies (GWAS) have identified genetic factors in type 2 diabetes (T2D), mostly among individuals of European ancestry. We tested whether previously identified T2D-associated single nucleotide polymorphisms (SNPs) replicate and whether SNPs in regions near known T2D SNPs were associated with T2D within the Singapore Chinese Health Study.

Methods: 2338 cases and 2339 T2D controls from the Singapore Chinese Health Study were genotyped for 507,509 SNPs.

Mycobacterium tuberculosis RpfE crystal structure reveals a positively charged catalytic cleft.

Resuscitation promoting factor (Rpf) proteins, which hydrolyze the sugar chains in cell-wall peptidoglycan (PG), play key roles in prokaryotic cell elongation, division, and escape from dormancy to vegetative growth. Like other bacteria, Mycobacterium tuberculosis (Mtb) expresses multiple Rpfs, none of which is individually essential. This redundancy has left unclear the distinct functions of the different Rpfs.

Structural and biochemical basis for ubiquitin ligase recruitment by arrestin-related domain-containing protein-3 (ARRDC3).

After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4.

Hyperpolarized xenon-based molecular sensors for label-free detection of analytes.

Nuclear magnetic resonance (NMR) can reveal the chemical constituents of a complex mixture without resorting to chemical modification, separation, or other perturbation. Recently, we and others have developed magnetic resonance agents that report on the presence of dilute analytes by proportionately altering the response of a more abundant or easily detected species, a form of amplification. One example of such a sensing medium is xenon gas, which is chemically inert and can be optically hyperpolarized, a process that enhances its NMR signal by up to 5 orders of magnitude.

The Structure-Function Linkage Database.

The Structure-Function Linkage Database (SFLD, http://sfld.rbvi.ucsf.

Optimized atomic statistical potentials: assessment of protein interfaces and loops.

Motivation: Statistical potentials have been widely used for modeling whole proteins and their parts (e.g. sidechains and loops) as well as interactions between proteins, nucleic acids and small molecules.

Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.

Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans.

Mapping polymerization and allostery of hemoglobin S using point mutations.

Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J.

De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes.

hESC-secreted proteins can be enriched for multiple regenerative therapies by heparin-binding.

This work builds upon our findings that proteins secreted by hESCs exhibit pro-regenerative activity, and determines that hESC-conditioned medium robustly enhances the proliferation of both muscle and neural progenitor cells. Importantly, this work establishes that it is the proteins that bind heparin which are responsible for the pro-myogenic effects of hESC-conditioned medium, and indicates that this strategy is suitable for enriching the potentially therapeutic factors. Additionally, this work shows that hESC-secreted proteins act independently of the mitogen FGF-2, and suggests that FGF-2 is unlikely to be a pro-aging molecule in the physiological decline of old muscle repair.

Multistep, eight-electron oxidation catalyzed by the cofactorless oxidase, PqqC: identification of chemical intermediates and their dependence on molecular oxygen.

The final step of the biosynthesis of prokaryotic cofactor PQQ is catalyzed by PqqC, a cofactorless oxidase that brings about a ring closure and overall eight-electron oxidation of its substrate. Time-dependent acid quenching and subsequent high-performance liquid chromatography separation and mass spectrometric analyses of reaction mixtures were performed to correlate the structures of intermediates with previously observed UV-visible signatures. The reaction is composed of four stepwise oxidations: three steps use O2 as the two-electron acceptor, and the fourth uses hydrogen peroxide (H2O2).

Importance of protein dynamics during enzymatic C-H bond cleavage catalysis.

Quantum tunneling and protein dynamics have emerged as important components of enzyme function. This review focuses on soybean lipoxygenase-1, to illustrate how the properties of enzymatic C-H bond activation link protein motions to the fundamental bond making-breaking processes.

The enigmatic conservation of a Rap1 binding site in the Saccharomyces cerevisiae HMR-E silencer.

Silencing at the HMR and HML loci in Saccharomyces cerevisiae requires recruitment of Sir proteins to the HML and HMR silencers. The silencers are regulatory sites flanking both loci and consisting of binding sites for the Rap1, Abf1, and ORC proteins, each of which also functions at hundreds of sites throughout the genome in processes unrelated to silencing. Interestingly, the sequence of the binding site for Rap1 at the silencers is distinct from the genome-wide binding profile of Rap1, being a weaker match to the consensus, and indeed is bound with low affinity relative to the consensus sequence.

Discovery of potent, selective multidrug and toxin extrusion transporter 1 (MATE1, SLC47A1) inhibitors through prescription drug profiling and computational modeling.

The human multidrug and toxin extrusion (MATE) transporter 1 contributes to the tissue distribution and excretion of many drugs. Inhibition of MATE1 may result in potential drug-drug interactions (DDIs) and alterations in drug exposure and accumulation in various tissues. The primary goals of this project were to identify MATE1 inhibitors with clinical importance or in vitro utility and to elucidate the physicochemical properties that differ between MATE1 and OCT2 inhibitors.