Untargeted Metabolomic Analysis of Leaves and Roots of Jatropha curcas Genotypes with Contrasting Levels of Phorbol Esters.

AIMS: Phorbol esters (PE) are toxic diterpenoids accumulated in physic nut (Jatropha curcas L.) seed tissues. Their biosynthetic pathway remains unknown, and the participation of roots in this process may be possible. Thus, we set out to study the deposition pattern of PE and other terpenoids in roots and leaves of genotypes with detected (DPE) and not detected (NPE) phorbol esters based on previous studies. OUTLINE OF DATA RESOURCES: We analyzed physic nut leaf and root organic extracts using LC-HRMS. By an untargeted metabolomics approach, it was possible to annotate 496 and 146 metabolites in the positive and negative electrospray ionization modes, respectively. KEY RESULTS: PE were detected only in samples of the DPE genotype. Remarkably, PE were found in both leaves and roots, making this study the first report of PE in J. curcas roots. Furthermore, untargeted metabolomic analysis revealed that diterpenoids and apocarotenoids are preferentially accumulated in the DPE genotype in comparison with NPE, which may be linked to the divergence between the genotypes concerning PE biosynthesis, since sesquiterpenoids showed greater abundance in the NPE. UTILITY OF THE RESOURCE: The LC-HRMS files, publicly available in the MassIVE database (identifier MSV000092920), are valuable as they expand our understanding of PE biosynthesis, which can assist in the development of molecular strategies to reduce PE levels in toxic genotypes, making possible the food use of the seedcake, as well as its potential to contain high-quality spectral information about several other metabolites that may possess biological activity.

Growth, metabolism and digestibility of Nile tilapia fed diets with solvent and extrusion-treated Jatropha curcas cake.

Physic nut Jatropha curcas cake/meal obtained after oil extraction has a high protein content, however, the presence of antinutrients (trypsin inhibitor, lectin and phytate) and toxic compounds (phorbol esters) limit their use as an alternative feedstuff. Thus, the detoxification process in cake/meal is necessary to allow their inclusion in fish diets. The present study aimed to evaluate the effects of solvent and extrusion-treated jatropha cake (SETJC) in Nile tilapia (Oreochromis niloticus) diets on growth, body composition, nutrient utilization, metabolic and hematological responses, and digestibility of experimental diets. Five experimental diets were formulated to be isonitrogenous (28.50% digestible protein) and isoenergetic (13.39 MJ/kg digestible energy) with graded levels of SETJC (0, 3, 6, 9, and 12%). The experimental design was completely randomized with five treatments and four replicates. The detoxification treatments reduced the phorbol esters (PE) of jatropha cake by 96% (0.58 mg/g of PE before and 0.023 mg/g of PE after treatments). Increased levels of SETJC depressed growth, feed efficiency, and protein digestibility. A similar trend was observed for hematological and biochemistry parameters. Aspartate and alanine aminotransferase, as well as phosphorus and magnesium concentrations in the fillets, increased at the highest levels of SETJC. Thus, the data of the present study suggests that the residual content, different structural forms of phorbol ester and its biological activity, as well as some antinutritional factors, can influence negatively the growth, metabolism and digestibility of experimental diets for Nile tilapia.

Comparison of the ligand binding site of C1 domains: a molecular dynamics simulation study of the C1 domain-phorbol 13-acetate-membrane system.

C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 µs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCßII C1B, PKCθ C1B, Munc13-1 C1, and ßII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.

A two-subpopulation model that reflects heterogeneity of large dense core vesicles in exocytosis.

Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell excitation demonstrates large heterogeneity. However, there are currently no models that clearly reflect such heterogeneity. Here, we develop a novel model based on single vesicle release events from amperometry recordings of PC12 cells using carbon fiber microelectrode. In this model, releasable vesicles can be grouped into two subpopulations, namely, SP1 and SP2. SP1 vesicles replenish quickly, with kinetics of ~0.0368 s-1, but likely undergo slow fusion pore expansion (amperometric signals rise at ~2.5 pA/ms), while SP2 vesicles demonstrate slow replenishment (kinetics of ~0.0048 s-1) but prefer fast dilation of fusion pore, with an amperometric signal rising rate of ~9.1 pA/ms. Phorbol ester enlarges the size of SP2 partially via activation of protein kinase C and conveys SP1 vesicles into SP2. Inhibition of Rho GTPase-dependent actin rearrangement almost completely depletes SP2. We also propose that the phorbol ester-sensitive vesicle subpopulation (SP2) is analogous to the subset of superprimed synaptic vesicles in neurons. This model provides a meticulous description of the architecture of the readily releasable vesicle pool and elucidates the heterogeneity of the vesicle priming mechanism.

Molecular dynamics simulation studies on binding of activator and inhibitor to Munc13-1 C1 in the presence of membrane.

Munc13-1 is a presynaptic active zone protein that plays a critical role in priming the synaptic vesicle and releasing neurotransmitters in the brain. Munc13-1 acts as a scaffold and is activated when diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane. Our previous studies showed that bryostatin 1 activated the Munc13-1, but resveratrol inhibited the phorbol ester-induced Munc13-1 activity. To gain structural insights into the binding of the ligand into Munc13-1 C1 in the membrane, we conducted 1.0 µs molecular dynamics (MD) simulation on Munc13-1 C1-ligand-lipid ternary system using phorbol 13-acetate, bryostatin 1 and resveratrol as ligands. Munc13-1 C1 shows higher conformational stability and less mobility along membrane with phorbol 13-acetate and bryostatin 1 than with resveratrol. Bryostatin 1 and phorbol ester remained in the protein active site, but resveratrol moved out of Munc13-1 C1 during the MD simulation. While bryostatin 1-bound Munc13-1 C1 showed two different positioning in the membrane, phorbol 13-acetate and resveratrol-bound Munc13-1 C1 only showed one positioning. Phorbol 13-acetate formed hydrogen bond with Ala-574 and Gly-589. Bryostatin 1 had more hydrogen bonds with Trp-588 and Arg-592 than with other residues. Resveratrol formed hydrogen bond with Ile-590. This study suggests that different ligands control Munc13-1 C1's mobility and positioning in the membrane differently. Ligand also has a critical role in the interaction between Munc13-1 C1 and lipid membrane. Our results provide structural basis of the pharmacological activity of the ligands and highlight the importance of membrane in Munc13-1 activity.Communicated by Ramaswamy H. Sarma.

Quantitative Proteome Analysis of Jatropha curcas L. Genotypes with Contrasting Levels of Phorbol Esters.

The phorbol esters in the seeds of Jatropha curcas are a major hindrance to the full exploitation of the potential of this oil crop as a source of raw material for the production of biodiesel. Here, various quantitative proteomic strategies are used to establish the proteomes of roots, leaves, and endosperm of two genotypes of J. curcas with contrasting levels of phorbol esters in the seeds. In total 4532, 1775, and 503 proteins are identified respectively in roots, leaves, and endosperm, comprising 5068 unique proteins; of this total, 185 are differentially abundant in roots, 72 in leaves, and 20 in the endosperm. The biosynthetic pathways for flavonoids and terpenoids are well represented in roots, including the complete set of proteins for the mevalonate and non-mevalonate/Deoxyxylulose 5-Phosphate pathways, and proteins involved in the branches which lead to the synthesis tricyclic diterpenoids and gibberellins. Also, casbene synthase which catalyzes the first committed step in the biosynthesis of tigliane-type diterpenes is identified in roots of both genotypes, but not in leaves and endosperm. This dataset will be a valuable resource to explore the biochemical basis of the low toxicity of Jatropha genotypes with low concentration of phorbol esters in the seeds.

Subunit-Specific Augmentation of AMPA Receptor Ubiquitination by Phorbol Ester.

Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal plasma membrane. Activity-dependent ubiquitination of AMPA receptor (AMPAR) subunits sorts internalised receptors to late endosomes for degradation, which ultimately determines the number of AMPARs on neuronal membrane. Our recent study has demonstrated a functional cross-talk between the phosphorylation and ubiquitination of the GluA1 subunit in mammalian central neurons. However, the existence of such a cross modulation for the GluA2 subunit remains unknown. Here, we have shown that bicuculline induced GluA2 ubiquitination on the same lysine residues (Lys-870 and Lys-882) in the C-terminal as those elicited by the AMPA treatment. Interestingly, bicuculline-induced ubiquitination was markedly enhanced by the phospho-mimetic GluA2 S880E mutant. Pharmacological activation of protein kinase C (PKC) by phorbol ester, which mediates the phosphorylation of GluA2 at Ser-880, augmented bicuculline-induced ubiquitination of GluA2 in cultured neurons. This effect was specific for the GluA2 subunit because phorbol ester did not alter the level of GluA1 ubiquitination. However, phorbol ester-induced enhancement of GluA2 ubiquitination did not require Ser-880 phosphorylation. This suggests that pseudo-phosphorylation of Ser-880 is sufficient but is not necessary for the augmentation of bicuculline-induced GluA2 ubiquitination. Collectively, these data provide the first demonstration of subunit-specific modulation of AMPAR ubiquitination by the PKC-dependent signalling pathway in mammalian central neurons.

Transient receptor potential ion-channel subfamily V member 4: a potential target for cancer treatment.

The transient receptor potential ion-channel superfamily consists of nonselective cation channels located mostly on the plasma membranes of numerous animal cell types, which are closely related to sensory information transmission (e.g., vision, pain, and temperature perception), as well as regulation of intracellular Ca2+ balance and physiological activities of growth and development. Transient receptor potential ion channel subfamily V (TRPV) is one of the largest and most diverse subfamilies, including TRPV1-TRPV6 involved in the regulation of a variety of cellular functions. TRPV4 can be activated by various physical and chemical stimuli, such as heat, mechanical force, and phorbol ester derivatives participating in the maintenance of normal cellular functions. In recent years, the roles of TRPV4 in cell proliferation, differentiation, apoptosis, and migration have been extensively studied. Its abnormal expression has also been closely related to the onset and progression of multiple tumors, so TRPV4 may be a target for cancer diagnosis and treatment. In this review, we focused on the latest studies concerning the role of TRPV4 in tumorigenesis and the therapeutic potential. As evidenced by the effects on cancerogenesis, TRPV4 is a potential target for anticancer therapy.

The story of PKC: A discovery marked by unexpected twists and turns.

Protein kinase C (PKC) is activated by 1,2-diacylglycerol as a second messenger in the signaling mechanism coupled with the hydrolysis of membrane inositol phospholipids, although it was not found by screening for a 1,2-diacylglycerol-dependent enzyme. PKC is also a receptor for the tumor-promoting phorbol esters, but it was not identified by its property of binding phorbol esters, either. Instead, the discovery and characterization of PKC, now known to comprise a family with multiple isoforms, was through a circuitous voyage filled with unexpected twists and turns. This review summarizes the discovery and the initial experiments of PKC as a historical perspective of the enzyme family in the context of the progress in the studies on protein phosphorylation. © 2018 IUBMB Life, 71(6):697-705, 2019.

Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed.

Establishment of the Reference Intervals of Lymphocyte Function in Healthy Adults Based on IFN-γ Secretion Assay upon Phorbol-12-Myristate-13-Acetate/Ionomycin Stimulation.

The function of lymphocytes is the key to reflect the immune status of hosts. Evaluation of lymphocyte function is a useful tool to monitor the effect of immunosuppressive treatment and predict the prognosis of immune-mediated diseases (e.g., cancer, autoimmune diseases, and infectious diseases). As the lymphocytes have various activities, such as activation, cytotoxicity, and cytokine secretion, it is a challenge to evaluate the function of lymphocytes in clinical practice and the reference intervals (RIs) of lymphocyte function are rarely reported. The present study showed that the secretion of IFN-γ was well correlated with the activation, chemotaxis, and cytotoxicity of CD4+, CD8+ T cells, and NK cells, which suggests that IFN-γ production can be used as a symbol of lymphocyte function. We therefore created a simple method to detect the function of CD4+, CD8+ T cells, and NK cells simultaneously according to IFN-γ secretion by using whole blood instead of peripheral blood mononuclear cells. We further established the RIs of lymphocyte function (CD4+ T cells: 15.31-34.98%; CD8+ T cells: 26.11-66.59%; NK cells: 39.43-70.79%) in healthy adults. This method showed good reproducibility for the evaluation of lymphocyte function. The established RIs were suitable for use in other centers based on the validation data. We also validated the RIs in individuals with different immune status, and the results showed that kidney transplant recipients and infants (0-1 year) had a decreased lymphocyte function, whereas T cells in systemic lupus erythematosus patients exhibited an opposite trend. Overall, we have successfully established the RIs of lymphocyte function in healthy adults in a simple way, which might be of important clinical value in the diagnosis, monitoring, and prognosis of immune-related diseases.

Structural determinants of phorbol ester binding activity of the C1a and C1b domains of protein kinase C theta.

The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.

Phorbol ester (PMA)-treated U937 cells cultured on type I collagen-coated dish express a lower production of pro-inflammatory cytokines through lowered ROS levels in parallel with cell aggregate formation.

The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-like histiocytic lymphoma cell line. Differentiation of U937 cells was induced by phorbol ester (PMA) treatment. The cells were cultured on the collagen I-coated plate. PMA-stimulated U937 cells formed multicellular aggregates on collagen I-coated surface, whereas PMA-unstimulated cells kept themselves away off each other. Moreover, the levels of reactive oxygen species (ROS) and productions of pro-inflammatory cytokines such as IL-1ß, TNFα and PGE2, pro-inflammatory mediator, were down-regulated in differentiated U937 cells cultured on collagen I-coated dishes. However, collagen I did not influence the capacity of E. coli phagocytosis. Cell aggregation as well as the down-regulation of IL-1ß, TNFα and PGE2 caused by the culture on collagen I-coated surface were suppressed by ROS donor, tert-butylhydroperoxide (tBHP). The sizes of cell aggregates became bigger in differentiated U937 cells by treatment with ROS scavengers such as N-acetylcysteine (NAC), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In conclusion, collagen I-coated culture induces the differentiated U937 cells to form cell aggregates and decreases the production of pro-inflammatory cytokines through down-regulating ROS generation.

In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes.

Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP-C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP-C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo.

Molecular dynamics simulations reveal ligand-controlled positioning of a peripheral protein complex in membranes.

Bryostatin is in clinical trials for Alzheimer's disease, cancer, and HIV/AIDS eradication. It binds to protein kinase C competitively with diacylglycerol, the endogenous protein kinase C regulator, and plant-derived phorbol esters, but each ligand induces different activities. Determination of the structural origin for these differing activities by X-ray analysis has not succeeded due to difficulties in co-crystallizing protein kinase C with relevant ligands. More importantly, static, crystal-lattice bound complexes do not address the influence of the membrane on the structure and dynamics of membrane-associated proteins. To address this general problem, we performed long-timescale (400-500 µs aggregate) all-atom molecular dynamics simulations of protein kinase C-ligand-membrane complexes and observed that different protein kinase C activators differentially position the complex in the membrane due in part to their differing interactions with waters at the membrane inner leaf. These new findings enable new strategies for the design of simpler, more effective protein kinase C analogs and could also prove relevant to other peripheral protein complexes.Natural supplies of bryostatin, a compound in clinical trials for Alzheimer's disease, cancer, and HIV, are scarce. Here, the authors perform molecular dynamics simulations to understand how bryostatin interacts with membrane-bound protein kinase C, offering insights for the design of bryostatin analogs.

Protein kinase C in cancer: The top five unanswered questions.

Few kinases have been studied as extensively as protein kinase C (PKC), particularly in the context of cancer. As major cellular targets for the phorbol ester tumor promoters and diacylglycerol (DAG), a second messenger generated by stimulation of membrane receptors, PKC isozymes play major roles in the control of signaling pathways associated with proliferation, migration, invasion, tumorigenesis, and metastasis. However, despite decades of research, fundamental questions remain to be answered or are the subject of intense controversy. Primary among these unresolved issues are the role of PKC isozymes as either tumor promoter or tumor suppressor kinases and the incomplete understanding on isozyme-specific substrates and effectors. The involvement of PKC isozymes in cancer progression needs to be reassessed in the context of specific oncogenic and tumor suppressing alterations. In addition, there are still major hurdles in addressing isozyme-specific function due to the limited specificity of most pharmacological PKC modulators and the lack of validated predictive biomarkers for response, which impacts the translation of these agents to the clinic. In this review we focus on key controversial issues and upcoming challenges, with the expectation that understanding the intricacies of PKC function will help fulfill the yet unsuccessful promise of targeting PKCs for cancer therapeutics.

Bayesian Multi-Trait Analysis Reveals a Useful Tool to Increase Oil Concentration and to Decrease Toxicity in Jatropha curcas L.

The biggest challenge for jatropha breeding is to identify superior genotypes that present high seed yield and seed oil content with reduced toxicity levels. Therefore, the objective of this study was to estimate genetic parameters for three important traits (weight of 100 seed, oil seed content, and phorbol ester concentration), and to select superior genotypes to be used as progenitors in jatropha breeding. Additionally, the genotypic values and the genetic parameters estimated under the Bayesian multi-trait approach were used to evaluate different selection indices scenarios of 179 half-sib families. Three different scenarios and economic weights were considered. It was possible to simultaneously reduce toxicity and increase seed oil content and weight of 100 seed by using index selection based on genotypic value estimated by the Bayesian multi-trait approach. Indeed, we identified two families that present these characteristics by evaluating genetic diversity using the Ward clustering method, which suggested nine homogenous clusters. Future researches must integrate the Bayesian multi-trait methods with realized relationship matrix, aiming to build accurate selection indices models.

The BET inhibitor OTX015 reactivates latent HIV-1 through P-TEFb.

None of the currently used anti-HIV-1 agents can effectively eliminate latent HIV-1 reservoirs, which is a major hurdle to a complete cure for AIDS. We report here that a novel oral BET inhibitor OTX015, a thienotriazolodiazepine compound that has entered phase Ib clinical development for advanced hematologic malignancies, can effectively reactivate HIV-1 in different latency models with an EC50 value 1.95-4.34 times lower than JQ1, a known BET inhibitor that can reactivate HIV-1 latency. We also found that OTX015 was more potent when used in combination with prostratin. More importantly, OTX015 treatment induced HIV-1 full-length transcripts and viral outgrowth in resting CD4(+) T cells from infected individuals receiving suppressive antiretroviral therapy (ART), while exerting minimal toxicity and effects on T cell activation. Finally, biochemical analysis showed that OTX015-mediated activation of HIV-1 involved an increase in CDK9 occupancy and RNAP II C-terminal domain (CTD) phosphorylation. Our results suggest that the BET inhibitor OTX015 may be a candidate for anti-HIV-1-latency therapies.

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini.

The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the "long loop" recycling marker Rab11, whereas less overlap was seen with the "short loop" recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements in the intracellular N terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function.

Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study.

Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.