Folding Circular Permutants of IL-1β: Route Selection Driven by Functional Frustration

Interleukin-1β (IL-1β) is the cytokine crucial to inflammatory and immune response. Two dominant routes are populated in the folding to native structure. These distinct routes are a result of the competition between early packing of the functional loops versus closure of the β-barrel to achieve efficient folding and have been observed both experimentally and computationally. Kinetic experiments on the WT protein established that the dominant route is characterized by early packing of geometrically frustrated functional loops. However, deletion of one of the functional loops, the β-bulge, switches the dominant route to an alternative, yet, as accessible, route, where the termini necessary for barrel closure form first. Here, we explore the effect of circular permutation of the WT sequence on the observed folding landscape with a combination of kinetic and thermodynamic experiments. Our experiments show that while the rate of formation of permutant protein is always slower than that observed for the WT sequence, the region of initial nucleation for all permutants is similar to that observed for the WT protein and occurs within a similar timescale. That is, even permutants with significant sequence rearrangement in which the functional-nucleus is placed at opposing ends of the polypeptide chain, fold by the dominant WT “functional loop-packing route”, despite the entropic cost of having to fold the N- and C- termini early. Taken together, our results indicate that the early packing of the functional loops dominates the folding landscape in active proteins, and, despite the entropic penalty of coalescing the termini early, these proteins will populate an entropically unfavorable route in order to conserve function. More generally, circular permutation can elucidate the influence of local energetic stabilization of functional regions within a protein, where topological complexity creates a mismatch between energetics and topology in active proteins

The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors

Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (?-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain

Diffusion, Crowding & Protein Stability in a Dynamic Molecular Model of the Bacterial Cytoplasm

A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo. A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously. In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations. Brownian dynamics (BD) simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein (GFP) observed in vivo, and “snapshots” of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events. The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited “crowding” effect must be included in attempts to understand macromolecular behavior in vivo

The macrophage at the intersection of immunity and metabolism in obesity

Obesity is a worldwide pandemic representing one of the major challenges that societies face around the globe. Identifying the mechanisms involved in its development and propagation will help the development of preventative and therapeutic strategies that may help control its rising rates

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia

Antimicrobial agents exert a selection pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. The aim of this work was to determine the occurrence of different enterococcal species and to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the genetic diversity in enterococci recovered from faecal samples of food-producing animals (poultry, beef and sheep) in Tunisia. Antimicrobial resistance and the mechanisms implicated were studied in 87 enterococci recovered from 96 faecal samples from animals of Tunisian farms. Enterococcus faecium was the most prevalent species detected (46 %), followed by E. hirae (33.5 %). High percentages of resistance to erythromycin and tetracycline were found among our isolates, and lower percentages to aminoglycosides and ciprofloxacin were identified. Most of the tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in all erythromycin-resistant isolates. The ant(6)-Ia, aph(3)-Ia and aac(6)-aph(2) genes were detected in nine aminoglycoside-resistant isolates. Of our isolates, 11.5 % carried the gelE gene and exhibited gelatinase acitivity. The esp gene was detected in 10 % of our isolates and the hyl gene was not present in any isolate. The predominant species (E. faecium and E. hirae) showed a high genetic diversity by repetitive extragenic palindromic (REP)-PCR. Food animals might play a role in the spread through the food chain of enterococci with virulence and resistance traits to humans. © 2014 Springer-Verlag Berlin Heidelberg and the University of Milan

The Epidermal Growth Factor Receptor Critically Regulates Endometrial Function during Early Pregnancy

<div><p>Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (<i>Egfr</i>) to be critical for endometrial function during early pregnancy. While ablation of <i>Her2</i> or <i>Erbb3</i> led to only a modest reduction in litter size, mice lacking <i>Egfr</i> expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place <i>Egfr</i> in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of <i>Egfr</i>, wingless-related MMTV integration site 4 (<i>Wnt4</i>) or boneless morphogenic protein 2 (<i>Bmp2</i>); revealing that not only are <i>Bmp2</i> and <i>Wnt4</i> key downstream effectors of <i>Egfr</i>, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that <i>EGFR</i> is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries <i>WNK1</i> and <i>AKT1S1</i>, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets.</p></div