The Warburg micro syndrome protein RAB3GAP1 modulates neuronal morphogenesis and interacts with axon elongation end ER-Golgi trafficking factors.

RAB3GAP1 is GTPase activating protein localized to the ER and Golgi compartments. In humans, mutations in RAB3GAP1 are the most common cause of Warburg Micro syndrome, a neurodevelopmental disorder associated with intellectual disability, microcephaly, and agenesis of the corpus callosum. We found that downregulation of RAB3GAP1 leads to a reduction in neurite outgrowth and complexity in human stem cell derived neurons. To further define the cellular function of RAB3GAP1, we sought to identify novel interacting proteins. We used a combination of mass spectrometry, co-immunoprecipitation and colocalization analysis and identified two novel interactors of RAB3GAP1: the axon elongation factor Dedicator of cytokinesis 7 (DOCK7) and the TATA modulatory factor 1 (TMF1) a modulator of Endoplasmic Reticulum (ER) to Golgi trafficking. To define the relationship between RAB3GAP1 and its two novel interactors, we analyzed their localization to different subcellular compartments in neuronal and non-neuronal cells with loss of RAB3GAP1. We find that RAB3GAP1 is important for the sub-cellular localization of TMF1 and DOCK7 across different compartments of the Golgi and endoplasmic reticulum. In addition, we find that loss of function mutations in RAB3GAP1 lead to dysregulation of pathways that are activated in response to the cellular stress like ATF6, MAPK, and PI3-AKT signaling. In summary, our findings suggest a novel role for RAB3GAP1 in neurite outgrowth that could encompass the regulation of proteins that control axon elongation, ER-Golgi trafficking, as well as pathways implicated in response to cellular stress.

Tryptamine Attenuates Experimental Multiple Sclerosis Through Activation of Aryl Hydrocarbon Receptor.

Tryptamine is a naturally occurring monoamine alkaloid which has been shown to act as an aryl hydrocarbon receptor (AHR) agonist. It is produced in large quantities from the catabolism of the essential amino acid tryptophan by commensal microorganisms within the gastrointestinal (GI) tract of homeothermic organisms. Previous studies have established microbiota derived AHR ligands as potent regulators of neuroinflammation, further defining the role the gut-brain axis plays in the complex etiology in multiple sclerosis (MS) progression. In the current study, we tested the ability of tryptamine to ameliorate symptoms of experimental autoimmune encephalomyelitis (EAE), a murine model of MS. We found that tryptamine administration attenuated clinical signs of paralysis in EAE mice, decreased the number of infiltrating CD4+ T cells in the CNS, Th17 cells, and RORγ T cells while increasing FoxP3+Tregs. To test if tryptamine acts through AHR, myelin oligodendrocyte glycoprotein (MOG)-sensitized T cells from wild-type or Lck-Cre AHRflox/flox mice that lacked AHR expression in T cells, and cultured with tryptamine, were transferred into wild-type mice to induce passive EAE. It was noted that in these experiments, while cells from wild-type mice treated with tryptamine caused marked decrease in paralysis and attenuated neuroinflammation in passive EAE, similar cells from Lck-Cre AHRflox/flox mice treated with tryptamine, induced significant paralysis symptoms and heightened neuroinflammation. Tryptamine treatment also caused alterations in the gut microbiota and promoted butyrate production. Together, the current study demonstrates for the first time that tryptamine administration attenuates EAE by activating AHR and suppressing neuroinflammation.

A novel rearrangement of fluorescent human thymidylate synthase inhibitor analogues in ESI tandem mass spectrometry.

Cu(I) catalyzed alkyne-azide cycloaddition reaction was employed to synthesize a series of anthracene-based human thymidylate synthase (hTS) inhibitor analogues. The triazolo-anthracene derivatives were characterized by ESI-MS/MS and a novel rearrangement reaction in ESI-MS/MS was observed. The mechanism is proposed whereby the protonated triazolo-anthracene derivative forms a carbocation, and then the carbocation electrophilically attacks an anthracene moiety resulting in formation of a rearrangement ion. Moreover, the carbocation prefers to attack the gamma position rather than the alpha or beta position of the anthracene moiety by an electrophilic substitution mechanism.

The mechanism of oxidative halophenol dehalogenation by Amphitrite ornata dehaloperoxidase is initiated by H2O2 binding and involves two consecutive one-electron steps: role of ferryl intermediates.

The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species. A significant amount of heterolytic cleavage of the O-O bond of cumene hydroperoxide, consistent with transient formation of a Compound I [Fe(IV)O/porphyrin pi-cation radical] species, is observed upon its reaction with ferric DHP. In addition, a more stable high-valent Fe(IV)O-containing DHP intermediate [Compound II (Cpd II) or Compound ES] is characterized by UV-visible absorption and magnetic circular dichroism spectroscopy. Spectral similarities are seen between this intermediate and horse heart myoglobin Cpd II. It is also shown in single-turnover experiments that the DHP Fe(IV)O intermediate is an active oxidant in halophenol oxidative dehalogenation. Furthermore, reaction of DHP with 4-chlorophenol leads to a dimeric product. The results presented herein are consistent with a normal peroxidase order of addition of the oxidant cosubstrate (H(2)O(2)) followed by organic substrate (TCP) and indicate that the enzymatic mechanism of DHP-catalyzed oxidative halophenol dehalogenation involves two consecutive one-electron steps with a dissociable radical intermediate.

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase.

Thymidylate synthase (TS) is an important target of several chemotherapeutic agents, including 5-FU and raltitrexed (Tomudex). During TS inhibition, TTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). Thus, BER initiated by uracil DNA glycosylase (UDG) activity has been hypothesized to influence the toxicity induced by TS inhibitors. In this study we created a human cell line expressing the Ugi protein inhibitor of UNG family of UDGs, which reduces cellular UDG activity by at least 45-fold. Genomic uracil incorporation was directly measured by mass spectrometry following treatment with TS inhibitors. Genomic uracil levels were increased over 4-fold following TS inhibition in the Ugi-expressing cells, but did not detectably increase in UNG proficient cells. Despite the difference in genomic uracil levels, there was no difference in toxicity between the UNG proficient and UNG-inhibited cells to folate or nucleotide-based inhibitors of TS. Cell cycle analysis showed that UNG proficient and UNG-inhibited cells arrested in early S-phase and resumed replication progression during recovery from RTX treatment almost identically. The induction of gamma-H2AX was measured following TS inhibition as a measure of whether uracil excision promoted DNA double strand break formation during S-phase arrest. Although gamma-H2AX was detectable following TS inhibition, there was no difference between UNG proficient and UNG-inhibited cells. We therefore conclude that uracil excision initiated by UNG does not adequately explain the toxicity caused by TS inhibition in this model.

Horse heart myoglobin catalyzes the H2O2-dependent oxidative dehalogenation of chlorophenols to DNA-binding radicals and quinones.

The heme-containing respiratory protein, myoglobin (Mb), best known for oxygen storage, can exhibit peroxidase-like activity under conditions of oxidative stress. Under such circumstances, the initially formed ferric state can react with H2O2 (or other peroxides) to generate a long-lived ferryl [Fe(IV)=O] Compound II (Cpd II) heme intermediate that is capable of oxidizing a variety of biomolecules. In this study, the ability of Mb Cpd II to catalyze the oxidation of carcinogenic halophenols is demonstrated. Specifically, 2,4,6-trichlorophenol (TCP) is converted to 2,6-dichloro-1,4-benzoquinone in a H2O2-dependent process. The fact that Mb Cpd II is an active oxidant in halophenol dehalogenation is consistent with a traditional peroxidase order of addition of H2O2 followed by TCP. With 4-chlorophenol, a dimerized product is formed, consistent with a mechanism involving generation of a reactive phenoxy radical intermediate by an electron transfer process. The radical nature of this process may be physiologically relevant since recent studies have revealed that phenoxy radicals and electrophilic quinones, specifically of the type described herein, covalently bind to DNA [Dai, J., Sloat, A. L., Wright, M. W., and Manderville, R. A. (2005) Chem. Res. Toxicol. 18, 771-779]. Thus, the stability of Mb Cpd II and its ability to oxidize TCP may explain why such compounds are carcinogenic. Furthermore, the initial rate of dehalogenation catalyzed by Mb Cpd II is nearly comparable to that of the same reaction carried out by turnover of the ferric state, demonstrating the potential physiological danger of this long-lived, high-valent intermediate.