Hitting the Bullseye: Endogenous Electrophiles Show Remarkable Nuance in Signaling Regulation.

Our bodies produce a host of electrophilic species that can label specific endogenous proteins in cells. The signaling roles of these molecules are under active debate. However, in our opinion, it is becoming increasingly likely that electrophiles can rewire cellular signaling processes at endogenous levels.

Function-guided proximity mapping unveils electrophilic-metabolite sensing by proteins not present in their canonical locales.

Enzyme-assisted posttranslational modifications (PTMs) constitute a major means of signaling across different cellular compartments. However, how nonenzymatic PTMs-despite their direct relevance to covalent drug development-impinge on cross-compartment signaling remains inaccessible as current target-identification (target-ID) technologies offer limited spatiotemporal resolution, and proximity mapping tools are also not guided by specific, biologically-relevant, ligand chemotypes. Here we establish a quantitative and direct profiling platform (Localis-rex) that ranks responsivity of compartmentalized subproteomes to nonenzymatic PTMs.

Passive coupling of membrane tension and cell volume during active response of cells to osmosis.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks.

Phosphoproteomic Effects of Acute Depletion of PP2A Regulatory Subunit Cdc55.

Protein phosphatase regulatory subunits are increasingly recognized as promising drug targets. In the absence of an existing drug, inducible degradation provides a means of predicting candidate targets. Here auxin-inducible degradation of Saccharomyces cerevisiae PP2A regulatory subunit Cdc55 in combination with quantitative phosphoproteomics is employed.

Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9.

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif.

Structure-function insights into direct lipid transfer between membranes by Mmm1-Mdm12 of ERMES.

The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from with that of Mdm12 from and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding.

Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro.

Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner.

TORC2 Structure and Function.

The target of rapamycin (TOR) kinase functions in two multiprotein complexes, TORC1 and TORC2. Although both complexes are evolutionarily conserved, only TORC1 is acutely inhibited by rapamycin. Consequently, only TORC1 signaling is relatively well understood; and, at present, only mammalian TORC1 is a validated drug target, pursued in immunosuppression and oncology.

Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2.

Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser characterized TORC2, which is not. TORC2 is a key regulator of lipid biosynthesis and Akt-mediated survival signaling. In spite of its importance, its structure and the molecular basis of its rapamycin insensitivity are unknown.

The SwissLipids knowledgebase for lipid biology.

Motivation: Lipids are a large and diverse group of biological molecules with roles in membrane formation, energy storage and signaling. Cellular lipidomes may contain tens of thousands of structures, a staggering degree of complexity whose significance is not yet fully understood. High-throughput mass spectrometry-based platforms provide a means to study this complexity, but the interpretation of lipidomic data and its integration with prior knowledge of lipid biology suffers from a lack of appropriate tools to manage the data and extract knowledge from it.

Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis.

The regulatory pathways required to maintain eukaryotic lipid homeostasis are largely unknown. We developed a systematic approach to uncover new players in the regulation of lipid homeostasis. Through an unbiased mass spectrometry-based lipidomic screening, we quantified hundreds of lipid species, including glycerophospholipids, sphingolipids, and sterols, from a collection of 129 mutants in protein kinase and phosphatase genes of Saccharomyces cerevisiae.

Fluorescent labeling agents for quorum-sensing receptors (FLAQS) in live cells.

Lighten up: A selective fluorescent-labeling agent for quorum sensing (FLAQS) can be used for the visualization of the communication pathway of bacteria in live cells (see figure). This represents a new, operationally simple, fast, and inexpensive tool for the imaging of quorum-sensing receptors by using fluorescently labeled signaling-molecule analogues.

Telomerase inhibitors from cyanobacteria: isolation and synthesis of sulfoquinovosyl diacylglycerols from Microcystis aeruguinosa PCC 7806.

By using the Telospot assay, 27 different extracts of cyanobacteria were evaluated for telomerase inhibition. All extracts showed varying, but significant activity. We selected Microcystis aeruguinosa PCC 7806 to identify the active compound and a bioassay guided fractionation led us to isolate mixtures of sulfoquinovosyl diacylglycerols (SQDGs), which were identified by 2D NMR and MS/MS experiments.

A semisynthetic fluorescent sensor protein for glutamate.

We report the semisynthesis of a fluorescent glutamate sensor protein on cell surfaces. Sensor excitation at 547 nm yields a glutamate-dependent emission spectrum between 550 and 700 nm that can be exploited for ratiometric sensing. On cells, the sensor displays a ratiometric change of 1.

Semisynthesis of fluorescent metabolite sensors on cell surfaces.

Progress in understanding signal transduction and metabolic pathways is hampered by a shortage of suitable sensors for tracking metabolites, second messengers, and neurotransmitters in living cells. Here we introduce a class of rationally designed semisynthetic fluorescent sensor proteins, called Snifits, for measuring metabolite concentrations on the cell surface of mammalian cells. Functional Snifits are assembled on living cells through two selective chemical labeling reactions of a genetically encoded protein scaffold.