Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57%+/-4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.     

HMGB1 is a cofactor in mammalian base excision repair

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.     

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.     

Protein-Induced Membrane Curvature Alters Local Membrane Tension

Adsorption of proteins onto membranes can alter the local membrane curvature. This phenomenon has been observed in biological processes such as endocytosis, tubulation, and vesiculation. However, it is not clear how the local surface properties of the membrane, such as membrane tension, change in response to protein adsorption. In this article, we show that the partial differential equations arising from classical elastic model of lipid membranes, which account for simultaneous changes in shape and membrane tension due to protein adsorption in a local region, cannot be solved for nonaxisymmetric geometries using straightforward numerical techniques; instead, a viscous-elastic formulation is necessary to fully describe the system. Therefore, we develop a viscous-elastic model for inhomogeneous membranes of the Helfrich type. Using the newly available viscous-elastic model, we find that the lipids flow to accommodate changes in membrane curvature during protein adsorption. We show that, at the end of protein adsorption process, the system sustains a residual local tension to balance the difference between the actual mean curvature and the imposed spontaneous curvature. We also show that this change in membrane tension can have a functional impact such as altered response to pulling forces in the presence of proteins.     

Life Satisfaction and the Pursuit of Happiness on Twitter

Life satisfaction refers to a somewhat stable cognitive assessment of one’s own life. Life satisfaction is an important component of subjective well being, the scientific term for happiness. The other component is affect: the balance between the presence of positive and negative emotions in daily life. While affect has been studied using social media datasets (particularly from Twitter), life satisfaction has received little to no attention. Here, we examine trends in posts about life satisfaction from a two-year sample of Twitter data. We apply a surveillance methodology to extract expressions of both satisfaction and dissatisfaction with life. A noteworthy result is that consistent with their definitions trends in life satisfaction posts are immune to external events (political, seasonal etc.) unlike affect trends reported by previous researchers. Comparing users we find differences between satisfied and dissatisfied users in several linguistic, psychosocial and other features. For example the latter post more tweets expressing anger, anxiety, depression, sadness and on death. We also study users who change their status over time from satisfied with life to dissatisfied or vice versa. Noteworthy is that the psychosocial tweet features of users who change from satisfied to dissatisfied are quite different from those who stay satisfied over time. Overall, the observations we make are consistent with intuition and consistent with observations in the social science research. This research contributes to the study of the subjective well being of individuals through social media.     

Interleukin-3 inhibits cycloheximide of C-Jun mRNA in human monocytes: Possible role for a serine/threonine phosphatase

Cycloheximide is a strong inducer of the c-jun protooncogene mRNA at concentrations (≤50 ng/ml) that do not inhibit protein synthesis in human monocytes. This induction is transient lasting 30–60 min in contrast to the sustained induction obtained with concentrations that inhibit protein synthesis. The pluripotent colony stimulating factor interleukin-3 (IL-3) (10 ng/ml) is also a modest inducer of the c-jun gene in these cells; however, in combination with cycloheximide, IL-3 dramatically reduces the c-jun induction below levels induced by cycloheximide alone. This is a true inhibition and is not due to a change in temporal kinetics of induction because the suppression in the presence of IL-3 is observed at both 30 and 60 min after simultaneous addition of both IL-3 and cycloheximide. Preincubation of monocytes with 12.5 nM okadaic acid (a potent inhibitor of protein phosphatases 1 and 2A) and cycloheximide prior to addition of IL-3 restored the level of c-jun induction to that mediated by cycloheximide alone. This concentration of okadaic acid inhibited almost 70% of the phosphorylase phosphatase activity in monocyte lysates. These observations suggest that activation of protein serine/threonine phosphatase(s) underlies the ability of IL-3 to inhibit cycloheximide induction of c-jun in monocytes. © 1993 Wiley-Liss, Inc.     

Retrieval with gene queries

Background  

Accuracy of document retrieval from MEDLINE for gene queries is crucially important for many applications in bioinformatics. We explore five information retrieval-based methods to rank documents retrieved by PubMed gene queries for the human genome. The aim is to rank relevant documents higher in the retrieved list. We address the special challenges faced due to ambiguity in gene nomenclature: gene terms that refer to multiple genes, gene terms that are also English words, and gene terms that have other biological meanings.