Dynamic Redox Regulation of IL-4 Signaling

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation.     

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Condition‐dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State‐of‐the‐art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double‐mutant strains, does not scale readily to multi‐condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG‐GI), by which double‐mutant strains generated via en masse “party” mating can also be monitored en masse for growth to detect genetic interactions. By using site‐specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG‐GI enables multiplexed quantitative tracking of double mutants via next‐generation sequencing. We applied BFG‐GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4‐nitroquinoline 1‐oxide (4NQO), bleomycin, zeocin, and three other DNA‐damaging environments. BFG‐GI recapitulated known genetic interactions and yielded new condition‐dependent genetic interactions. We validated and further explored a subnetwork of condition‐dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.     

CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9

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Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses

Peste des petits ruminants (PPR) is a highly contagious transboundary animal disease with a severe socio-economic impact on the livestock industry, particularly in poor countries where it is endemic. Full understanding of PPR virus (PPRV) pathobiology and molecular biology is critical for effective control and eradication of the disease. To achieve these goals, establishment of stable reverse genetics systems for PPRV would play a key role. Unfortunately, this powerful technology remains less accessible and poorly documented for PPRV. In this review, we discussed the current status of PPRV reverse genetics as well as the recent innovations and advances in the reverse genetics of other non-segmented negative-sense RNA viruses that could be applicable to PPRV. These strategies may contribute to the improvement of existing techniques and/or the development of new reverse genetics systems for PPRV.     

A twinfilin‐like protein coordinates karyokinesis by influencing mitotic spindle elongation and DNA replication in Leishmania

Twinfilin is an evolutionarily conserved actin‐binding protein, which regulates actin‐dynamics in eukaryotic cells. Homologs of this protein have been detected in the genome of various protozoan parasites causing diseases in human. However, very little is known about their core functions in these organisms. We show here that a twinfilin homolog in a human pathogen Leishmania, primarily localizes to the nucleolus and, to some extent, also in the basal body region. In the dividing cells, nucleolar twinfilin redistributes to the mitotic spindle and remains there partly associated with the spindle microtubules. We further show that approximately 50% depletion of this protein significantly retards the cell growth due to sluggish progression of S phase of the cell division cycle, owing to the delayed nuclear DNA synthesis. Interestingly, overexpression of this protein results in significantly increased length of the mitotic spindle in the dividing Leishmania cells, whereas, its depletion adversely affects spindle elongation and architecture. Our results indicate that twinfilin controls on one hand, the DNA synthesis and on the other, the mitotic spindle elongation, thus contributing to karyokinesis in Leishmania.     

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of DeltaCp of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.     

Tensional homeostasis in dermal fibroblasts: Mechanical responses to mechanical loading in three-dimensional substrates

Many soft connective tissues are under endogenous tension, and their resident cells generate considerable contractile forces on the extracellular matrix. The present work was aimed to determine quantitatively how fibroblasts, grown within three-dimensional collagen lattices, respond mechanically to precisely defined tensional loads. Forces generated in response to changes in applied load were measured using a tensional culture force monitor. In a number of variant systems, resident cells consistently reacted to modify the endogenous matrix tension in the opposite direction to externally applied loads. That is, increased external loading was followed immediately by a reduction in cell-mediated contraction whilst decreased external loading elicited increased contraction. Responses were cell-mediated and not a result of material properties of the matrices. This is the first detailed characterisation of a tensional homeostatic response in cells. The maintained force, after 8 h in culture, was typically around 40–60 dynes/million cells. Maintenance of an active tensional homeostasis has widespread implications for cells in culture and forwhole tissue function. J. Cell. Physiol. 175:323–332, 1998. © 1998 Wiley-Liss, Inc.