Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation.

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

TRAPP webserver: predicting protein binding site flexibility and detecting transient binding pockets.

The TRAnsient Pockets in Proteins (TRAPP) webserver provides an automated workflow that allows users to explore the dynamics of a protein binding site and to detect pockets or sub-pockets that may transiently open due to protein internal motion. These transient or cryptic sub-pockets may be of interest in the design and optimization of small molecular inhibitors for a protein target of interest. The TRAPP workflow consists of the following three modules: (i) TRAPP structure- generation of an ensemble of structures using one or more of four possible molecular simulation methods; (ii) TRAPP analysis-superposition and clustering of the binding site conformations either in an ensemble of structures generated in step (i) or in PDB structures or trajectories uploaded by the user; and (iii) TRAPP pocket-detection, analysis, and visualization of the binding pocket dynamics and characteristics, such as volume, solvent-exposed area or properties of surrounding residues. A standard sequence conservation score per residue or a differential score per residue, for comparing on- and off-targets, can be calculated and displayed on the binding pocket for an uploaded multiple sequence alignment file, and known protein sequence annotations can be displayed simultaneously. The TRAPP webserver is freely available at http://trapp.h-its.org.

Protein Binding Pocket Dynamics.

The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five different classes of protein pocket dynamics: (1) appearance/disappearance of a subpocket in an existing pocket; (2) appearance/disappearance of an adjacent pocket on the protein surface in the direct vicinity of an already existing pocket; (3) pocket breathing, which may be caused by side-chain fluctuations or backbone or interdomain vibrational motion; (4) opening/closing of a channel or tunnel, connecting a pocket inside the protein with solvent, including lid motion; and (5) the appearance/disappearance of an allosteric pocket at a site on a protein distinct from an already existing pocket with binding of a ligand to the allosteric binding site affecting the original pocket. We suggest that the class of pocket dynamics, as well as the type and extent of protein motion affecting the binding pocket, should be factors considered in choosing the most appropriate computational approach to study a given binding pocket. Furthermore, we examine the relationship between pocket dynamics classes and induced fit, conformational selection, and gating models of ligand binding on binding kinetics and thermodynamics. We discuss the implications of protein binding pocket dynamics for drug design and conclude with potential future directions for computational analysis of protein binding pocket dynamics.

LigDig: a web server for querying ligand-protein interactions.

UNLABELLED: LigDig is a web server designed to answer questions that previously required several independent queries to diverse data sources. It also performs basic manipulations and analyses of the structures of protein-ligand complexes. The LigDig webserver is modular in design and consists of seven tools, which can be used separately, or via linking the output from one tool to the next, in order to answer more complex questions. Currently, the tools allow a user to: (i) perform a free-text compound search, (ii) search for suitable ligands, particularly inhibitors, of a protein and query their interaction network, (iii) search for the likely function of a ligand, (iv) perform a batch search for compound identifiers, (v) find structures of protein-ligand complexes, (vi) compare three-dimensional structures of ligand binding sites and (vii) prepare coordinate files of protein-ligand complexes for further calculations. AVAILABILITY AND IMPLEMENTATION: LigDig makes use of freely available databases, including ChEMBL, PubChem and SABIO-RK, and software programs, including cytoscape.js, PDB2PQR, ProBiS and Fconv. LigDig can be used by non-experts in bio- and chemoinformatics. LigDig is available at: http://mcm.h-its.org/ligdig. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Metabolomics of Ramadan fasting: an opportunity for the controlled study of physiological responses to food intake.

High-throughput screening techniques that analyze the metabolic endpoints of biological processes can identify the contributions of genetic predisposition and environmental factors to the development of common diseases. Studies applying controlled physiological challenges can reveal dysregulation in metabolic responses that may be predictive for or associated with these diseases. However, large-scale epidemiological studies with well controlled physiological challenge conditions, such as extended fasting periods and defined food intake, pose logistic challenges. Culturally and religiously motivated behavioral patterns of life style changes provide a natural setting that can be used to enroll a large number of study volunteers. Here we report a proof of principle study conducted within a Muslim community, showing that a metabolomics study during the Holy Month of Ramadan can provide a unique opportunity to explore the pre-prandial and postprandial response of human metabolism to nutritional challenges. Up to five blood samples were obtained from eleven healthy male volunteers, taken directly before and two hours after consumption of a controlled meal in the evening on days 7 and 26 of Ramadan, and after an over-night fast several weeks after Ramadan. The observed increases in glucose, insulin and lactate levels at the postprandial time point confirm the expected physiological response to food intake. Targeted metabolomics further revealed significant and physiologically plausible responses to food intake by an increase in bile acid and amino acid levels and a decrease in long-chain acyl-carnitine and polyamine levels. A decrease in the concentrations of a number of phospholipids between samples taken on days 7 and 26 of Ramadan shows that the long-term response to extended fasting may differ from the response to short-term fasting. The present study design is scalable to larger populations and may be extended to the study of the metabolic response in defined patient groups such as individuals with type 2 diabetes.

Impact of carbonylation on glutathione peroxidase-1 activity in human hyperglycemic endothelial cells.

AIMS: High levels of glucose and reactive carbonyl intermediates of its degradation pathway such as methylglyoxal (MG) may contribute to diabetic complications partly via increased generation of reactive oxygen species (ROS). This study focused on glutathione peroxidase-1 (GPx1) expression and the impact of carbonylation as an oxidative protein modification on GPx1 abundance and activity in human umbilical vein endothelial cells (HUVEC) under conditions of mild to moderate oxidative stress. RESULTS: High extracellular glucose and MG enhanced intracellular ROS formation in HUVECs. Protein carbonylation was only transiently augmented pointing to an effective antioxidant defense in these cells. Nitric oxide synthase expression was decreased under hyperglycemic conditions but increased upon exposure to MG, whereas superoxide dismutase expression was not significantly affected. Increased glutathione peroxidase (GPx) activity seemed to compensate for a decrease in GPx1 protein due to enhanced degradation via the proteasome. Mass spectrometry analysis identified Lys-114 as a possible carbonylation target which provides a vestibule for the substrate H2O2 and thus enhances the enzymatic reaction. INNOVATION: Oxidative protein carbonylation has so far been associated with functional inactivation of modified target proteins mainly contributing to aging and age-related diseases. Here, we demonstrate that mild oxidative stress and subsequent carbonylation seem to activate protective cellular redox signaling pathways whereas severe oxidative stress overwhelms the cellular antioxidant defense leading to cell damage. CONCLUSIONS: This study may contribute to a better understanding of redox homeostasis and its role in the development of diabetes and related vascular complications.

Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.

ProSAT+: visualizing sequence annotations on 3D structure.

PRO: tein S: tructure A: nnotation T: ool-plus (ProSAT(+)) is a new web server for mapping protein sequence annotations onto a protein structure and visualizing them simultaneously with the structure. ProSAT(+) incorporates many of the features of the preceding ProSAT and ProSAT2 tools but also provides new options for the visualization and sharing of protein annotations. Data are extracted from the UniProt KnowledgeBase, the RCSB PDB and the PDBe SIFTS resource, and visualization is performed using JSmol. User-defined sequence annotations can be added directly to the URL, thus enabling visualization and easy data sharing. ProSAT(+) is available at http://prosat.h-its.org.