Loss of cysteinyl-tRNA synthetase (CARS) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation.

Ferroptosis is a form of regulated non-apoptotic cell death that has been implicated in several disease contexts. A better understanding of the ferroptotic death mechanism could lead to the development of new therapeutics for degenerative diseases, and a better understanding of how to induce ferroptosis in specific tumor contexts. We performed an unbiased genome-wide siRNA screen to find genetic suppressors of ferroptosis. We determined that loss of CARS, the cysteinyl-tRNA synthetase, suppresses ferroptosis induced by erastin, which inhibits the cystine-glutamate antiporter known as system xc(-). Knockdown of CARS inhibited erastin-induced death by preventing the induction of lipid reactive oxygen species, without altering iron homeostasis. Knockdown of CARS led to the accumulation of cystathionine, a metabolite on the transsulfuration pathway, and upregulated genes associated with serine biosynthesis and transsulfuration. In addition, inhibition of the transsulfuration pathway resensitized cells to erastin, even after CARS knockdown. These studies demonstrate a new mechanism of resistance to ferroptosis and may lead to strategies for inducing and suppressing ferroptosis in diverse contexts.

Frontiers in chemical genetics.

New methods enable the identification of compounds that both induce a specific cellular state and lead to identification of proteins that regulate that state. Together, developments in three critical areas: chemical diversity, phenotype-based screening and target identification, enable the systematic application of this chemical genetic approach to almost any biological problem or disease process.

The first regioselective alpha-deprotonation and functionalization of allenamides. An application in intramolecular Pauson-Khand-type cycloadditions.

[rection: see text] The first regioselective alpha-deprotonation and functionalization of electron-deficient allenamines are described here. The acidities of alpha- and gamma-allenic protons of these allenamides are readily differentiated using strong bases, thereby allowing regioselective substitutions at either the alpha- or gamma-allenic position. A specific synthetic application of the novel alpha-substituted allenamides in intramolecular Pauson-Khand-type cycloadditions is also described here.

Chemical genetics: ligand-based discovery of gene function.

Chemical genetics is the study of gene-product function in a cellular or organismal context using exogenous ligands. In this approach, small molecules that bind directly to proteins are used to alter protein function, enabling a kinetic analysis of the in vivo consequences of these changes. Recent advances have strongly enhanced the power of exogenous ligands such that they can resemble genetic mutations in terms of their general applicability and target specificity. The growing sophistication of this approach raises the possibility of its application to any biological process.

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

BACKGROUND: Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. RESULTS: We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. CONCLUSIONS: We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

Probing the role of homomeric and heteromeric receptor interactions in TGF-beta signaling using small molecule dimerizers.

BACKGROUND: Transforming growth factor Beta (TGF-Beta) arrests many cell types in the G1 phase of the cell and upregulates plasminogen activator inhibitor 1 (PAI-1). The type 1 (TGF-Beta RI) an II (TGF-Beta RII) TGF-Beta receptors mediate these and other effects of TGF-Beta on target cells. TGF-Beta initially binds to TGF-Beta RII and subsequently TGF-Beta RI is recruited to form a heteromeric complex. TGF-Beta RI phosphorylates the downstream effectors Smad2 and Smad3, leading to their translocation into the nucleus. Here, we explored the role of receptor oligomerization in TGF-Beta signaling. RESULTS: We constructed fusion proteins containing receptor cytoplasmic tails linked to binding domains for small-molecule dimerizers. In COS-1 cells, recruitment of a soluble TGF-Beta RII tail to a myristoylated TGF-Beta RI tail promoted Smad2 nuclear translocation. In mink lung cells, homo-oligomerization of a myristoylated TGF-Beta Ri tail in presence of a myristoylated TGF-Beta RII tail activated the PAI-1 promoter. Oligomerization of an acidic mutant of the TGF-Beta RI tail in absence of TGF-Beta RII activated the PAI-A promoter and inhibited the growth of mink lung cells. CONCLUSIONS: Non-toxic, small molecules designed to oligomerize cytoplasmic tails of TGF-Beta receptors at the plasma membrane can activate TGF-Beta signaling. Although TGF-Beta normally signals through two receptors that are both necessary for signaling, in one small-molecule system, a dimerizer activates signaling through a single type of receptor that is sufficient to induce TGF-Beta signaling. These methods of activating TGF-Beta signaling could be extended to signaling pathways of other TGF-Beta superfamily members such as activin and the bone morphogenetic proteins.

TGF-beta-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-beta type I receptor fusion proteins.

BACKGROUND: Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-beta), which signals through two TGF-beta receptors (type I, TGF-beta RI, and type II, TGF-beta). In the signaling pathway, TGF-beta binds to the extracellular domain of TGF-betaRII, which can then transphosphorylate TGF-betaRI in its glycine/serine (GS)-rich box. Activated TGF-betaRI phosphorylates two downstream effectors, Smad2 and Smad3, leading to their translocation into the nucleus. Cell growth is arrested and plasminogen activator inhibitor 1 (PAI-1) is upregulated. We investigated the role of the immunophilin FKBP12, which can bind to the GS box of TGF-betaRI, in TGF-beta signaling. RESULTS: Overexpression of myristoylated TGF-betaRI and TGF-betaRII cytoplasmic tails caused constitutive nuclear translocation of a green-fluorescent-protein-Smad2 construct in COS-1 cells, and constitutive activation of a PAI-1 reporter plasmid in mink lung cells. Fusing FKBP12 to TGF-betaRI resulted in repression of autosignaling that could be alleviated by FK506M or rapamycin (two small molecules that can bind to FKBP12). Mutation of the FKBP12-binding site in the FKBP1-TGF-betaRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGF-betaRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-betaRII. Further mutations in the TGF-betaRI FKBP12-binding site resulted in TGF-beta signaling that was independent of both TGF-betaRII and FKBP12 antagonists. CONCLUSIONS: Fusing FKBP12 to TGF-betaRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-betaRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-beta superfamily signals.

An induced proximity model for caspase-8 activation.

The assembly of the CD-95 (Fas/Apo-1) receptor death-inducing signaling complex occurs in a hierarchical manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8 (FLICE/Mach-1) by a homophilic interaction involving the death effector domains. Immediately after recruitment, the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large and small catalytic subunits. Since all caspases cleave their substrates after Asp residues and are themselves processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it follows that an upstream caspase can process a downstream zymogen. However, since FLICE represents the most apical caspase in the Fas pathway, its mode of activation has been enigmatic. We hypothesized that the FLICE zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active protease. Support for this was provided by (i) the synthesis of chimeric Fpk3FLICE molecules that can be oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2. Cells transfected with Fpk3FLICE underwent apoptosis after exposure to FK1012H2; (ii) the creation of a nonprocessable zymogen form of FLICE that retained low but detectable protease activity.

Liquid chromatography combined with tandem mass spectrometry for the confirmation of sarafloxacin in catfish tissue.

A specific chromatographic LC/MS/MS assay is described for the confirmatory identification of residues of sarafloxacin, an arylfluoroquinolone antibacterial agent, in catfish tissue. This confirmatory method takes advantage of the specificity provided by sample preparation, liquid chromatography, and tandem mass spectrometry. This kind of multidimensional analysis is commonly used in environmental, pharmacokinetic, residue, and other studies. However, we demonstrate the addition of a previously unreported criterion, the use of ion ratio ranges from the tandem mass spectrometry (MS/MS) experiment as an aid in confirmation. Using the described method, we were able to achieve MS/MS product ion ratios with <7% variation during 1 day of analysis for over 25 injections. We believe the addition of this criterion will increase the scientific certainty of the confirmatory method.

Hemoglobin S antigelation agents based on 5-bromotryptophan with potential for sickle cell anemia.

5-Bromotryptophan (5-BrTrp) is the most potent amino acid derivative reported in the literature to inhibit the gelation of hemoglobin S (from sickle cell anemia patients). Trp-Trp is also more potent than Trp as an antigelation agent. Therefore, we have prepared a series of dipeptides containing 5-BrTrp and evaluated the antigelation activity. 5-BrTrp-5-BrTrp is the most potent, i.e., 5.9 times the activity of Trp, followed by 5-BrTrp-Trp and then Trp-5-BrTrp. This improved antigelation potency for 5-BrTrp-5-BrTrp and 5-BrTrp-Trp is very significant and will be pursued further as lead compounds with potential for sickle cell anemia.

The Queensland cholera incident of 1977. 1. The index case.

The first known case of cholera to be contracted in Australia during the seventh pandemic occurred in Queensland in 1977. There was no record of recent travel abroad by the patient, or of her having been in contact with persons suffering from gastroenteritis. Vibrio cholerae, biotype eltor, serotype Inaba, phage-type 2, was the causative microorganism. This case is unique in that the microbiological diagnosis was based on the identification of an isolate from venous blood. This indicates that the patient was bacteraemic, an observation not previously reported. The incident shows the importance of routine screening of faecal specimens for V. cholerae, as well as other enteropathogens.