Targeted genetic and small molecule disruption of N-Ras CaaX cleavage alters its localization and oncogenic potential.

Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.

Multi-Photon-Sensitive Chromophore for the Photorelease of Biologically Active Phenols.

Phenols confer bioactivity to a plethora of organic compounds. Protecting the phenolic functionality with photoremovable protecting groups (PPGs) sensitive to two-photon excitation (2PE) can block the bioactivity and provide controlled release of these compounds in a spatially and temporally restricted manner by photoactivation with IR light. To develop an efficient 2PE-sensitive PPG for releasing phenols, the (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ) chromophore was functionalized at the C4 position with methyl, morpholine, methoxy, para-tolyl, and 3,4,5-trimethoxyphenyl groups to provide 4-methyl-CyHQ (Me-CyHQ), 4-morpholino-CyHQ (Mor-CyHQ), 4-methoxy-CyHQ (MeO-CyHQ), 4-(p-tolyl)-CyHQ (pTol-CyHQ), and 4-(3,4,5-trimethoxyphenyl)-CyHQ (TMP-CyHQ) PPGs. The probes possess attributes useful for biological use, including high quantum yield (Φu), hydrolytic stability, and good aqueous solubility in physiological conditions. The MeO-CyHQ PPG enhanced the two-photon uncaging action cross section (δu) of dopamine 3.5-fold (0.85 GM) compared to CyHQ (0.24 GM) at 740 nm and 1.49 GM at 720 nm. MeO-CyHQ was used to mediate photoactivation via 2PE of serotonin, rotigotine, N-vanillyl-nonanoylamide (VNA) (a capsaicin analogue), and eugenol. The constructs except rotigotine showed excellent efficiency in 2PE with δu ranging from 0.75 to 1.01 GM at 740 nm and from 1.31 to 1.36 GM at 720 nm high yielding release of the payloads. These probes also performed well by using conventional single photon excitation (1PE). The spatially and temporally controlled release of dopamine from CyHQ-DA and MeO-CyHQ-DA and serotonin (5-HT) from MeO-CyHQ-5HT was quantified in cell culture by using genetically encoded sensors for dopamine and serotonin, respectively. Calcium imaging was employed to quantify the release of VNA and eugenol (EG) from MeO-CyHQ-VNA and MeO-CyHQ-EG, respectively. These tools will enable experiments to understand the intricate mechanisms involved in neurological signaling and the roles played by neurotransmitters, such as dopamine and serotonin, in the activation of their respective receptors.

Specific Disruption of Ras2 CAAX Proteolysis Alters Its Localization and Function.

Many CAAX proteins, such as Ras GTPase, undergo a series of posttranslational modifications at their carboxyl terminus (i.e., cysteine prenylation, endoproteolysis of AAX, and carboxylmethylation). Some CAAX proteins, however, undergo prenylation-only modification, such as Saccharomyces cerevisiae Hsp40 Ydj1. We previously observed that altering the CAAX motif of Ydj1 from prenylation-only to canonical resulted in altered Ydj1 function and localization. Here, we investigated the effects of a reciprocal change that altered the well-characterized canonical CAAX motif of S. cerevisiae Ras2 to prenylation-only. We observed that the type of CAAX motif impacted Ras2 protein levels, localization, and function. Moreover, we observed that using a prenylation-only sequence to stage hyperactive Ras2-G19V as a farnesylated and nonproteolyzed intermediate resulted in a different phenotype relative to staging by a genetic RCE1 deletion strategy that simultaneously affected many CAAX proteins. These findings suggested that a prenylation-only CAAX motif is useful for probing the specific impact of CAAX proteolysis on Ras2 under conditions where other CAAX proteins are normally modified. We propose that our strategy could be easily applied to a wide range of CAAX proteins for examining the specific impact of CAAX proteolysis on their functions. IMPORTANCE CAAX proteins are subject to multiple posttranslational modifications: cysteine prenylation, CAAX proteolysis, and carboxylmethylation. For investigations of CAAX proteolysis, this study took the novel approach of using a proteolysis-resistant CAAX sequence to stage Saccharomyces cerevisiae Ras2 GTPase in a farnesylated and nonproteolyzed state. Our approach specifically limited the effects of disrupting CAAX proteolysis to Ras2. This represented an improvement over previous methods where CAAX proteolysis was inhibited by gene knockout, small interfering RNA knockdown, or biochemical inhibition of the Rce1 CAAX protease, which can lead to pleiotropic and unclear attribution of effects due to the action of Rce1 on multiple CAAX proteins. Our approach yielded results that demonstrated specific impacts of CAAX proteolysis on the function, localization, and other properties of Ras2, highlighting the utility of this approach for investigating the impact of CAAX proteolysis in other protein contexts.

Synthesis of 5-unsubstituted dihydropyrimidinone-4-carboxylates from deep eutectic mixtures.

A facile one-pot synthesis of 5-unsubstituted dihydropyrimidinones from ß,γ-unsaturated ketoesters in low melting ʟ-(+)-tartaric acid-N,N-dimethylurea mixtures is reported. This solvent-free method is very general and provides easy access to 5-unsubstituted dihydropyrimidinone-4-carboxylate derivatives in good yields.

Photoactivatable Odorants for Chemosensory Research.

The chemosensory system of any animal relies on a vast array of detectors tuned to distinct chemical cues. Odorant receptors and the ion channels of the TRP family are all uniquely expressed in olfactory tissues in a species-specific manner. Great effort has been made to characterize the molecular and pharmacological properties of these proteins. Nevertheless, most of the natural ligands are highly hydrophobic molecules that are not amenable to controlled delivery. We sought to develop photoreleasable, biologically inactive odorants that could be delivered to the target receptor or ion channel and effectively activated by a short light pulse. Chemically distinct ligands eugenol, benzaldehyde, 2-phenethylamine, ethanethiol, butane-1-thiol, and 2,2-dimethylethane-1-thiol were modified by covalently attaching the photoremovable protecting group (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ). The CyHQ derivatives were shown to release the active odorant upon illumination with 365 and 405 nm light. We characterized their bioactivity by measuring activation of recombinant TRPV1 and TRPA1 ion channels expressed in HEK 293 cells and the electroolfactogram (EOG) response from intact mouse olfactory epithelium (OE). Illumination with 405 nm light was sufficient to robustly activate TRP channels within milliseconds of the light pulse. Photoactivation of channels was superior to activation by conventional bath application of the ligands. Photolysis of the CyHQ-protected odorants efficiently activated an EOG response in a dose-dependent manner with kinetics similar to that evoked by the vaporized odorant amyl acetate (AAc). We conclude that CyHQ-based, photoreleasable odorants can be successfully implemented in chemosensory research.

Measuring Cellular Ion Transport by Magnetoencephalography.

The cellular-level process of ion transport is known to generate a magnetic field. A noninvasive magnetoencephalography (MEG) technique was used to measure the magnetic field emanating from HeLa, HEK293, and H9c2(2-1) rat cardiac cells. The addition of a nonlethal dose of ionomycin to HeLa and capsaicin to TRPV1-expressing HEK293 cells resulted in a sudden change in the magnetic field signal consistent with Ca2+ influx, which was also observed by confocal fluorescence microscopy under the same conditions. In contrast, addition of capsaicin to TRPV1-expressing HEK293 cells containing an optimum amount of a TRPV1 antagonist (ruthenium red), resulted in no detectable magnetic or fluorescent signals. These signals confirmed that the measured MEG signals are due to cellular ion transport through the cell membrane. In general, there is evidence that ion channel/transporter activation and ionic flux are linked to cancer. Therefore, our work suggests that MEG could represent a noninvasive method for detecting cancer.

Photoactivatable Dopamine and Sulpiride to Explore the Function of Dopaminergic Neurons and Circuits.

Kinetic analysis of dopamine receptor activation and inactivation and the study of dopamine-dependent signaling requires precise simulation of the presynaptic release of the neurotransmitter dopamine and tight temporal control over the release of dopamine receptor antagonists. The 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting group was conjugated to dopamine and the dopamine receptor antagonist sulpiride to generate "caged" versions of these neuromodulators (CyHQ-O-DA and CyHQ-sulpiride, respectively) that could release their payloads with 365 or 405 nm light or through 2-photon excitation (2PE) at 740 nm. These compounds are stable under physiological conditions in the dark, yet photolyze rapidly and cleanly to yield dopamine or sulpiride and the caging remnant CyHQ-OH. CyHQ-O-DA mediated the light activation of dopamine-1 (D1) receptors on the breast cancer cell line MDA-MB-231 in culture. In mouse brain slice from the substantia nigra pars compacta, localized flash photolysis of CyHQ-O-DA accurately mimicked the natural presynaptic release of dopamine and activation of dopamine-2 (D2) receptors, causing a robust, concentration-dependent, and repeatable G protein-coupled inwardly rectifying potassium channel-mediated outward current in whole-cell voltage clamp recordings that was amplified by cocaine and blocked by sulpiride. Photolysis of CyHQ-sulpiride rapidly blocked synaptic activity, enabling measurement of the unbinding rates of dopamine and quinpirole, a D2 receptor agonist. These tools will enable more detailed study of dopamine receptors, their interactions with other GPCRs, and the physiology of dopamine signaling in the brain.

Synthesis of substituted hydantoins in low melting mixtures.

A novel domino synthesis of 1,3,5-trisubstituted hydantoin derivatives has been developed in low melting L-(+)-tartaric acid-DMU melt mixtures. The functionalized hydantoins are obtained in good yields from ß,γ-unsaturated ketoacids and urea under environmentally benign and simple reaction conditions.

Fischer indole synthesis in low melting mixtures.

Functionalized indoles are synthezised under mild conditions in a tartaric acid-dimethylurea melt. The melt serves as the solvent and as the catalyst. Under these reaction conditions, sensitive functional groups such as N-Boc, N-Cbz, or azides are stable, and indolenines are obtained regioselectively in excellent yields. The practical use of the method is demonstrated in the synthesis of the hormone melatonin.