An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in Chlamydomonas reinhardtii.

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.

The NMR solution structure and function of RPA3313: a putative ribosomal transport protein from Rhodopseudomonas palustris.

Protein function elucidation often relies heavily on amino acid sequence analysis and other bioinformatics approaches. The reliance is extended to structure homology modeling for ligand docking and protein-protein interaction mapping. However, sequence analysis of RPA3313 exposes a large, unannotated class of hypothetical proteins mostly from the Rhizobiales order. In the absence of sequence and structure information, further functional elucidation of this class of proteins has been significantly hindered. A high quality NMR structure of RPA3313 reveals that the protein forms a novel split ßßαß fold with a conserved ligand binding pocket between the first ß-strand and the N-terminus of the α-helix. Conserved residue analysis and protein-protein interaction prediction analyses reveal multiple protein binding sites and conserved functional residues. Results of a mass spectrometry proteomic analysis strongly point toward interaction with the ribosome and its subunits. The combined structural and proteomic analyses suggest that RPA3313 by itself or in a larger complex may assist in the transportation of substrates to or from the ribosome for further processing. Proteins 2016; 85:93-102. © 2016 Wiley Periodicals, Inc.

Comparison of Mannose, Ethylene Glycol, and Methoxy-Terminated Diluents on Specificity and Selectivity of Electrochemical Peptide-Based Sensors.

We report the synthesis and application of three new antifouling diluents for the fabrication of an E-PB HIV sensor. Among the three thiolated antifouling diluents used in this study, the methoxy-terminated diluent (C6-MEG) is the most effective in alleviating both nonspecific binding and adsorption of matrix contaminants onto the sensor surface, especially when compared to the mannose- (C6-MAN) and ethylene-glycol-terminated (C6-EG) diluents. The sensor fabricated with C6-MEG has a specificity factor (∼13.5) substantially higher than the sensor passivated with only 6-mercapto-1-hexanol (∼1.5). It is functional even when employed directly in 25% serum, an achievement that has not been observed with this class of E-PB sensors. More importantly, incorporation of these antifouling diluents has negligible impact on other important sensor properties such as sensitivity and binding kinetics. This sensor passivation strategy is versatile and can potentially be used with other E-PB sensors, as well as surface-based sensors that utilize thiol-gold self-assembled monolayer chemistry.

Iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase in the biosynthesis of HSAF.

Antifungal HSAF (heat-stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two "clean" strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl-coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene-PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS-NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide-peptide natural products.

Development of affinity microcolumns for drug-protein binding studies in personalized medicine: interactions of sulfonylurea drugs with in vivo glycated human serum albumin.

This report used high-performance affinity microcolumns to examine the changes in binding by sulfonylurea drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 µL of plasma or serum and 6-12 nmol of protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4-8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple drugs and experimental conditions over hundreds of sample application cycles. Both the overall binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in site-specific binding that occurred between sulfonylurea drugs or as the level of HSA glycation was varied. This method is not limited to clinical samples of glycated HSA but could be adapted for work with other modified proteins of interest in personalized medicine.

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of FATP2 resulted in increases in all four classes of phospholipid, indicating little selectivity. In the case of C22:6, there were significant increases of this exogenous fatty acids being trafficking into PC and PI. Collectively, these data support the conclusion that FATP2 has a dual function in the pathways linking the transport and activation of exogenous fatty acids. We discuss the differential roles of FATP2 and its role in both fatty acid transport and fatty acid activation in the context of lipid homeostasis.

Paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus.

The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.

A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity.

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure. Here we show that the type III effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT). HopU1 suppresses plant innate immunity in a manner dependent on its ADP-RT active site. The HopU1 substrates in Arabidopsis thaliana extracts were RNA-binding proteins that possess RNA-recognition motifs (RRMs). A. thaliana knockout lines defective in the glycine-rich RNA-binding protein GRP7 (also known as AtGRP7), a HopU1 substrate, were more susceptible than wild-type plants to P. syringae. The ADP-ribosylation of GRP7 by HopU1 required two arginines within the RRM, indicating that this modification may interfere with GRP7's ability to bind RNA. Our results suggest a pathogenic strategy where the ADP-ribosylation of RNA-binding proteins quells host immunity by affecting RNA metabolism and the plant defence transcriptome.

A cytoplasm-specific activity encoded by the Trithorax-like ATX1 gene.

Eukaryotes produce multiple products from a single gene locus by alternative splicing, translation or promoter usage as mechanisms expanding the complexity of their proteome. Trithorax proteins, including the Arabidopsis Trithorax-like protein ATX1, are histone modifiers regulating gene activity. Here, we report that a novel member of the Trithorax family has a role unrelated to chromatin. It is encoded from an internal promoter in the ATX1 locus as an isoform containing only the SET domain (soloSET). It is located exclusively in the cytoplasm and its substrate is the elongation factor 1A (EF1A). Loss of SET, but not of the histone modifying ATX1-SET activity, affects cytoskeletal actin bundling illustrating that the two isoforms have distinct functions in Arabidopsis cells.

Unusual activities of the thioesterase domain for the biosynthesis of the polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes C3.

HSAF is an antifungal natural product with a new mode of action. A rare bacterial iterative PKS-NRPS assembles the HSAF skeleton. The biochemical characterization of the NRPS revealed that the thioesterase (TE) domain possesses the activities of both a protease and a peptide ligase. Active site mutagenesis, circular dichroism spectra, and homology modeling of the TE structure suggested that the TE may possess uncommon features that may lead to the unusual activities. The iterative PKS-NRPS is found in all polycyclic tetramate macrolactam gene clusters, and the unusual activities of the TE may be common to this type of hybrid PKS-NRPS.

Human fatty acid transport protein 2a/very long chain acyl-CoA synthetase 1 (FATP2a/Acsvl1) has a preference in mediating the channeling of exogenous n-3 fatty acids into phosphatidylinositol.

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M(r) 70,000) and FATP2b (M(r) 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14-C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.

Structure function analysis of an ADP-ribosyltransferase type III effector and its RNA-binding target in plant immunity.

The Pseudomonas syringae type III effector HopU1 is a mono-ADP-ribosyltransferase that is injected into plant cells by the type III protein secretion system. Inside the plant cell it suppresses immunity by modifying RNA-binding proteins including the glycine-rich RNA-binding protein GRP7. The crystal structure of HopU1 at 2.7-Å resolution reveals two unique protruding loops, L1 and L4, not found in other mono-ADP-ribosyltransferases. Site-directed mutagenesis demonstrates that these loops are essential for substrate recognition and enzymatic activity. HopU1 ADP-ribosylates the conserved arginine 49 of GRP7, and this reduces the ability of GRP7 to bind RNA in vitro. In vivo, expression of GRP7 with Arg-49 replaced with lysine does not complement the reduced immune responses of the Arabidopsis thaliana grp7-1 mutant demonstrating the importance of this residue for GRP7 function. These data provide mechanistic details how HopU1 recognizes this novel type of substrate and highlights the role of GRP7 in plant immunity.

Characterization of surface-exposed reactive cysteine residues in Saccharomyces cerevisiae.

Numerous cellular processes are subject to redox regulation, and thiol-dependent redox control, acting through reactive cysteine (Cys) residues, is among the major mechanisms of redox regulation. However, information on the sets of proteins that provide thiol-based redox regulation or are affected by it is limited. Here, we describe proteomic approaches to characterize proteins that contain reactive thiols and methods to identify redox Cys in these proteins. Using Saccharomyces cerevisiae as a eukaryotic model organism, we identified 284 proteins with exposed reactive Cys and determined the identities of 185 of these residues. We then characterized subsets of these proteins as in vitro targets of major cellular thiol oxidoreductases, thioredoxin and glutaredoxin, and found that these enzymes can control the redox state of a significant number of thiols in target proteins. We further examined common features of exposed reactive Cys and compared them with an unbiased control set of Cys using computational approaches. This analysis (i) validated the efficacy of targeting exposed Cys in proteins in their native, folded state, (ii) quantified the proportion of targets that can be redox regulated via thiol oxidoreductase systems, and (iii) revealed the theoretical range of the experimental approach with regard to protein abundance and physicochemical properties of reactive Cys. From these analyses, we estimate that approximately one-fourth of exposed Cys in the yeast proteome can be regarded as functional sites, either subject to regulation by thiol oxidoreductases or involved in structural disulfides and metal binding.

Fluoride-Promoted Ligand Exchange in Diaryliodonium Salts.

Diaryliodonium salts are shown to undergo rapid, fluoride-promoted aryl exchange reactions at room temperature in acetonitrile. Aryl exchange is shown to be exquisitely sensitive to the concentration of fluoride ion in solution; fast exchange is observed as the fluoride concentration approaches a stoichiometric amount at 50 mM substrate concentration. The reaction is slowed, but not halted if benzene is the solvent, indicating that free fluoride ion or a four-coordinate anionic I(III) species may be responsible for the exchange. The fluoride-promoted aryl exchange reaction is general and allows direct measurement of the relative stabilities of diaryliodonium salts featuring different aryl substituents. The aryl exchange reaction may be of practical use for the preparation of hitherto inaccessible diaryliodonium salts, thus it also has implications for labeling radiotracers for molecular imaging with (18)F-fluoride (t(1/2) = 109.7 min).

Expression artifact with retroviral vectors based on pBMN.

While characterizing various splice forms of p120 catenin, we observed what appeared to be a novel posttranslational modification of p120, resulting in a higher molecular weight form that was dependent on the splicing pattern. Further investigation revealed the higher molecular weight form to be a fusion protein between sequences encoded by the retroviral vector and p120. We found that the publicly available sequence of the vector we used does not agree with the experimental sequence. We caution other investigators to be aware of this potential artifact.

Platelet-derived growth factor receptor beta-mediated phosphorylation of MUC1 enhances invasiveness in pancreatic adenocarcinoma cells.

MUC1 is a heterodimeric transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in ductal adenocarcinomas. Differential phosphorylation of the MUC1 cytoplasmic tail (MUC1CT) has been associated with signaling events that influence the proliferation and metastasis of cancer cells. We identified a novel tyrosine phosphorylation site (HGRYVPP) in the MUC1CT by mass spectrometric analysis of MUC1 from human pancreatic adenocarcinoma cell lines. Analyses in vitro and in vivo showed that platelet-derived growth factor receptor beta (PDGFRbeta) catalyzed phosphorylation of this site and of tyrosine in the RDTYHPM site. Stimulation of S2-013.MUC1F cells with PDGF-BB increased nuclear colocalization of MUC1CT and beta-catenin. PDGF-BB stimulation had no significant effect on cell proliferation rate; however, it enhanced invasion in vitro through Matrigel and in vivo tumor growth and metastases. Invasive properties of the cells were significantly altered on expression of phosphorylation-abrogating or phosphorylation-mimicking mutations at these sites. We propose that interactions of MUC1 and PDGFRbeta induce signal transduction events that influence the metastatic properties of pancreatic adenocarcinoma.

Identification of guanylate cyclases and related signaling proteins in sperm tail from sea stars by mass spectrometry.

Marine invertebrates employ external fertilization to take the advantages of sexual reproduction as one of excellent survival strategies. To prevent mismatching, successful fertilization can be made only after going though strictly defined steps in the fertilization. In sea stars, the fertilization process starts with the chemotaxis of sperm followed by hyperactivation of sperm upon arriving onto the egg coat, and then sperm penetrate to the egg coat before achieving the fusion. To investigate whether the initiation of chemotaxis and the following signaling has species specificity, we conducted comparative studies in the protein level among sea stars, Asterias amurensis, A. forbesi, and Asterina pectinifera. Since transcription of messenger ribonucleic acid (mRNA) has been suppressed in gamete, the roles of sperm proteins during the fertilization cannot be investigated by examining the mRNA profile. Therefore, proteomics analysis by mass spectrometry was used in this study. In sea stars, upon receiving asteroidal sperm-activating peptide (asterosap), the receptor membrane-bound guanylate cyclases in the sperm tail trigger sperm chemotaxis. We confirmed the presence of membrane-bound guanylate cyclases in the three sea star species, and they all had the same structural domains including the extracellular domain, kinase-like domain, and guanylate cyclase domain. The majority of peptides recovered were from alpha-helices distributed on the solvent side of the protein. More peptides were recovered from the intracellular domains. The transmembrane domain has not been recovered. The functions of the receptors seemed to be conserved among the species. Furthermore, we identified proteins that may be involved in the guanylate cyclase-triggered signaling pathway.

Characterization of glycation adducts on human serum albumin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Non-enzymatic glycation of human serum albumin (HSA) is associated with the long-term complications of diabetes. We examined the structure and location of modifications on minimally-glycated HSA and considered their possible impact on the binding of drugs to this protein. METHODS: Minimally-glycated and normal HSA (used as a control) were digested with trypsin, Glu-C or Lys-C, followed by fractionation of the resulting peptides and their analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine the structures and locations of glycation adducts. RESULTS: Several specific lysine and arginine residues were identified as modification sites in minimally-glycated HSA. Residues K12, K51, K199, K205, K439 and K538 were found to be modified through the formation of fructosyl-lysine, while the modification of K159 and K286 involved the formation of pyrraline or N(epsilon)-carboxymethyl-lysine, respectively. Lysine K378 was found to give N(epsilon)-carboxyethyl-lysine in some forms of glycated HSA but fructosyl-lysine in other forms. Residues R160 and R472 produced a modification based on N(epsilon)-(5-hydro-4-imidazolon-2-yl)ornithine. Lysine R222 was modified to produce argpyrimidine, N(epsilon)-[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine or tetrahydropyrimidine. CONCLUSIONS: With the exception of K12, K199, K378, K439 and K525, all of the observed sites of modification for minimally-glycated HSA were new to this current study. The fact that many of these glycation-related modifications are located at or near known drug binding sites on HSA explains why some differences have been previously noted in the binding of certain drugs to normal vs glycated HSA.

Metabolic Investigations of the Molecular Mechanisms Associated with Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar cytoplasmic aggregates of α-synuclein (i.e., Lewy bodies) and the associated loss of dopaminergic cells in the substantia nigra. Mutations in genes such as α-synuclein (SNCA) account for only 10% of PD occurrences. Exposure to environmental toxicants including pesticides and metals (e.g., paraquat (PQ) and manganese (Mn)) is also recognized as an important PD risk factor. Thus, aging, genetic alterations, and environmental factors all contribute to the etiology of PD. In fact, both genetic and environmental factors are thought to interact in the promotion of idiopathic PD, but the mechanisms involved are still unclear. In this study, we summarize our findings to date regarding the toxic synergistic effect between α-synuclein and paraquat treatment. We identified an essential role for central carbon (glucose) metabolism in dopaminergic cell death induced by paraquat treatment that is enhanced by the overexpression of α-synuclein. PQ "hijacks" the pentose phosphate pathway (PPP) to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. PQ also stimulated an increase in glucose uptake, the translocation of glucose transporters to the plasma membrane, and AMP-activated protein kinase (AMPK) activation. The overexpression of α-synuclein further stimulated an increase in glucose uptake and AMPK activity, but impaired glucose metabolism, likely directing additional carbon to the PPP to supply paraquat redox cycling.

Glucose Metabolism and AMPK Signaling Regulate Dopaminergic Cell Death Induced by Gene (α-Synuclein)-Environment (Paraquat) Interactions.

While environmental exposures are not the single cause of Parkinson's disease (PD), their interaction with genetic alterations is thought to contribute to neuronal dopaminergic degeneration. However, the mechanisms involved in dopaminergic cell death induced by gene-environment interactions remain unclear. In this work, we have revealed for the first time the role of central carbon metabolism and metabolic dysfunction in dopaminergic cell death induced by the paraquat (PQ)-α-synuclein interaction. The toxicity of PQ in dopaminergic N27 cells was significantly reduced by glucose deprivation, inhibition of hexokinase with 2-deoxy-D-glucose (2-DG), or equimolar substitution of glucose with galactose, which evidenced the contribution of glucose metabolism to PQ-induced cell death. PQ also stimulated an increase in glucose uptake, and in the levels of glucose transporter type 4 (GLUT4) and Na+-glucose transporters isoform 1 (SGLT1) proteins, but only inhibition of GLUT-like transport with STF-31 or ascorbic acid reduced PQ-induced cell death. Importantly, while autophagy protein 5 (ATG5)/unc-51 like autophagy activating kinase 1 (ULK1)-dependent autophagy protected against PQ toxicity, the inhibitory effect of glucose deprivation on cell death progression was largely independent of autophagy or mammalian target of rapamycin (mTOR) signaling. PQ selectively induced metabolomic alterations and adenosine monophosphate-activated protein kinase (AMPK) activation in the midbrain and striatum of mice chronically treated with PQ. Inhibition of AMPK signaling led to metabolic dysfunction and an enhanced sensitivity of dopaminergic cells to PQ. In addition, activation of AMPK by PQ was prevented by inhibition of the inducible nitric oxide syntase (iNOS) with 1400W, but PQ had no effect on iNOS levels. Overexpression of wild type or A53T mutant α-synuclein stimulated glucose accumulation and PQ toxicity, and this toxic synergism was reduced by inhibition of glucose metabolism/transport and the pentose phosphate pathway (6-aminonicotinamide). These results demonstrate that glucose metabolism and AMPK regulate dopaminergic cell death induced by gene (α-synuclein)-environment (PQ) interactions.