Ultralight Ultrafast Enzymes.

Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physicochemical properties from their isotopically natural counterparts. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with most proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in depleted media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase and Pfu DNA polymerase showed up to a 250 % increase in activity compared to the native counterparts, with an additional ∼50 % increase at 10 °C. Diminished conformational and vibrational entropy is hypothesized to be the cause of the accelerated kinetics. Ultralight enzymes may find an application where extreme reaction rates are required.

Proteome Integral Solubility Alteration: A High-Throughput Proteomics Assay for Target Deconvolution.

Various agents, including drugs as well as nonmolecular stimuli, induce alterations in the physicochemical properties of proteins in cell lysates, living cells, and organisms. These alterations can be probed by applying a stability- and solubility-modifying factor, such as elevated temperature, to a varying degree. As a second dimension of variation, drug concentration or agent intensity/concentration can be used. Compared to standard approaches where curves are fitted to protein solubility data acquired at different temperatures and drug concentrations, Proteome Integral Solubility Alteration (PISA) assay increases the analysis throughput by 1 to 2 orders of magnitude for an unlimited number of factor variation points in such a scheme. The consumption of the compound and biological material decreases in PISA by the same factor. We envision widespread use of the PISA approach in chemical biology and drug development.

Multifaceted Proteome Analysis at Solubility, Redox, and Expression Dimensions for Target Identification.

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, the first high-throughput redox proteomics approach integrated with expression analysis (REX) is devised and combined with the Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, HCT116 cells treated with auranofin are analyzed, showing great improvement compared with previous studies. PISA-REX is then applied to study proteome remodeling upon stimulation of human monocytes by interferon α (IFN-α). Applying this tool to study the proteome changes in plasmacytoid dendritic cells (pDCs) isolated from wild-type versus Ncf1-mutant mice treated with interferon α, shows that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility, and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

Redox regulation of PTPN22 affects the severity of T-cell-dependent autoimmune inflammation.

Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor (TCR) signaling. As with all protein tyrosine phosphatases, the activity of PTPN22 is redox regulated, but if or how such regulation can modulate inflammatory pathways in vivo is not known. To determine this, we created a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 (C129S), a residue proposed to alter the redox regulatory properties of PTPN22 by forming a disulfide with the catalytic C227 residue. The C129S mutant mouse showed a stronger T-cell-dependent inflammatory response and development of T-cell-dependent autoimmune arthritis due to enhanced TCR signaling and activation of T cells, an effect neutralized by a mutation in Ncf1, a component of the NOX2 complex. Activity assays with purified proteins suggest that the functional results can be explained by an increased sensitivity to oxidation of the C129S mutated PTPN22 protein. We also observed that the disulfide of native PTPN22 can be directly reduced by the thioredoxin system, while the C129S mutant lacking this disulfide was less amenable to reductive reactivation. In conclusion, we show that PTPN22 functionally interacts with Ncf1 and is regulated by oxidation via the noncatalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T-cell-dependent autoimmunity.

An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation.

Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.

Vibration and glycerol-mediated plasmid DNA transformation for Escherichia coli.

Escherichia coli transformation is an essential step in many molecular biology experiments. Despite earlier advances in the field, many studies including shotgun cloning still require more efficient transformation protocols. Chemical transformation has been the most popular method, in which competent cells are transformed following a brief period of heat shock. Here, we report a novel protocol with higher efficiency, in which competent E. coli cells (treated with CaCl2 ) grown in media containing glycerol experience a gentle vibration. Three E. coli strains DH5α, Jm107 and BL21 (DE3) and three plasmids pGEM-T, pET-28a and pCAMBIA with different sizes (3000, 5369 and 8428 bp, respectively) were used to test the protocol. The results indicated a significant increase in number of transformed colonies compared with heat-shock method. Our findings also demonstrated the favourable impacts of glycerol on transformation of E. coli.