Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (N⌬150 and N⌬150/ C⌬24) using isopropyl ␤-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH 2 -terminal regulatory domain. The solubility of hTPH2, N⌬150, and N⌬150/ C⌬24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t1 ⁄ 2 at 37°C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, N⌬150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH 2 -terminal domain and the COOHterminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH 2 -terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V max values. These data identify the NH 2 -terminal regulatory domain as the source of hTPH2 instability and reduced solubility.Tryptophan hydroxylase (TPH 2 ; EC 1.14.16.4), a tetrahydropterin (BH 4 )-dependent amino acid hydroxylase, is the key regulator of serotonin (5-hydroxytryptamine) biosynthesis. Utilizing BH 4 and O 2 as co-substrates and Fe 2ϩ as a cofactor, TPH hydroxylates tryptophan to 5-hydroxytryptophan ( Fig. 1). Subsequent decarboxylation of 5-hydroxytryptophan by amino acid decarboxylase generates serotonin (1-3). This essential monoamine has been found in a variety of tissues and implicated in a wide range of physiological functions. In the central nervous system, serotonin is synthesized primarily in the dorsal raphe nucleus and acts as a neurotransmitter; in the pineal gland, it serves as a precursor for melatonin biosynthesis (4). In the periphery, serotonin constricts large blood vessels and regulates platelet adhesion (5-7). Serotonin, produced by the enterochromaffin cells of the intestinal system, initiates peristaltic and secretory reflexes (8). Altered activity of serotonin is associated with various disorders such as depression, impulsive behaviors, aggression, suicide, drug abuse and alcoholism, sleep disorders, gastrointestinal diseases (such as irritable bowel syndrome), and cardiovascular dysfunction leading to heart failure (9).Recently, Walther et al. (10) discovered the gene responsible for nervous system TPH (TPH2). Unlike TPH1 now known to be the peripheral enzyme (11), TPH2 is neuron-specific and expressed predominantly in serotonergic neurons of the raphe nuclei (10, 12) and in the peripheral myenteric neurons in the gut (13). Human TPH1 and TPH2 display 72% sequence h...

In this work, we report a drug delivery system based on the pH-responsive self-assembly and -disassembly behaviors of peptides. Here, a systematically designed histidine-rich lipidated peptide (NP1) is presented to encapsulate and deliver an anticancer drug ellipticine (EPT) into two model cells: non-small-cell lung carcinoma and Chinese hamster ovary cells. The mechanism of pH-responsive peptide self-assembly and -disassembly involved in the drug encapsulation and release process are extensively investigated. We found that NP1 could self-assemble as a spherical nanocomplex (diameter = 34.43 nm) in a neutral pH environment with EPT encapsulated and positively charged arginine amino acids aligned outward and EPT is released in an acidic environment due to the pH-triggered disassembly. Furthermore, the EPT-encapsulating peptide could achieve a mass loading ability of 18% (mass of loaded-EPT/mass of NP1) with optimization. More importantly, it is revealed that the positively charged arginine on the periphery of the NP1 peptides could greatly facilitate their direct translocation through the negatively charged plasma membrane via electrostatic interaction, instead of via endocytosis, which provides a more efficient uptake pathway.

This study demonstrates a novel and unexpected role in cytokinesis for leukemia-associated RhoGEF (LARG). Depletion of LARG results in delayed abscission, and thus LARG is the first RhoGEF to be implicated in late cytokinesis.