Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum.

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane.

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11-positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245-dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97- vs. Golgin-245-dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin-mediated cell polarity and cell-cell junctions.

ADPredict: ADP-ribosylation site prediction based on physicochemical and structural descriptors.

Motivation: ADP-ribosylation is a post-translational modification (PTM) implicated in several crucial cellular processes, ranging from regulation of DNA repair and chromatin structure to cell metabolism and stress responses. To date, a complete understanding of ADP-ribosylation targets and their modification sites in different tissues and disease states is still lacking. Identification of ADP-ribosylation sites is required to discern the molecular mechanisms regulated by this modification. This motivated us to develop a computational tool for the prediction of ADP-ribosylated sites. Results: Here, we present ADPredict, the first dedicated computational tool for the prediction of ADP-ribosylated aspartic and glutamic acids. This predictive algorithm is based on (i) physicochemical properties, (ii) in-house designed secondary structure-related descriptors and (iii) three-dimensional features of a set of human ADP-ribosylated proteins that have been reported in the literature. ADPredict was developed using principal component analysis and machine learning techniques; its performance was evaluated both internally via intensive bootstrapping and in predicting two external experimental datasets. It outperformed the only other available ADP-ribosylation prediction tool, ModPred. Moreover, a novel secondary structure descriptor, HM-ratio, was introduced and successfully contributed to the model development, thus representing a promising tool for bioinformatics studies, such as PTM prediction. Availability and implementation: ADPredict is freely available at www.ADPredict.net. Supplementary information: Supplementary data are available at Bioinformatics online.

A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility.

BACKGROUND: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. METHODS: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. RESULTS: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. CONCLUSIONS: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.

An insight into the skin penetration enhancement mechanism of N-methylpyrrolidone.

This work aims to elucidate the mechanism by which N-methylpyrrolidone (NMP) enhances the skin permeation of a compound by combining experimental data with molecular dynamic (MD) simulations. The addition of 10% NMP significantly increased the propranolol (PR) permeation through the human epidermis (∼ 15 µg/cm(2) vs ∼ 30 µg/cm(2)) while resulting inefficacious on hydrocortisone (HC) diffusion. No significant alterations in the stratum corneum structure were found after the in vitro treatment of human epidermis with NMP dispersed in mineral oil or water by attenuated total reflectance Fourier transform infrared (ATR-FTIR) analyses. MD simulations revealed the formation of a complex by H-bonds and the π-π stacking interactions between the NMP's amido group and the drug's aromatic systems. The size of the depicted NMP/PR clusters was in line with the hydrodynamic radius derived by dynamic light scattering analyses (∼ 2.00 nm). Conversely, no interaction, and consequently cluster formation, between NMP and HC occurred. These results suggest that NMP is effective in enhancing the drug permeation through human epidermis by a cotransport mechanism when NMP/drug interaction occurs.

Exploring the activation mechanism of TRPM8 channel by targeted MD simulations.

The TRPM8 cation channel belongs to the superfamily of transient receptor potential (TRP) channels. It is involved in non-painful cool sensation and triggered by diverse chemical and physical stimuli whose precise activation mechanism is still unknown. The study presents a set of targeted molecular dynamics (MD) simulations involving selected complexes of the TRPM8 channel whose homology model was recently generated by some of us. More in detail, the MD simulations concerned the TRPM8 alone and in complex with agonists and antagonists. These simulations were focused on voltage sensor module and designed to validate the ligand induced activation mechanism as hypothesized in our previous study. The obtained results are in encouraging agreement with the proposed mechanism and allow a clear discrimination between agonists and antagonists. In addition, the MD runs confirm that the agonist binding triggers a set of concatenate conformational shifts which induce the approaching of the S3 segment toward the S4 segment and culminate in an extension of the latter. By introducing suitable constraints, the reported MD simulations were rendered as fast as possible in order to achieve a truly productive compromise between reliability and computational costs. The obtained results emphasize that suitably targeted MD runs can be fast enough to be systematically applied to predict the bioactivity of large datasets providing it as an useful tool in rational ligand design process.

Publisher Correction: Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

PARP1-produced poly-ADP-ribose causes the PARP12 translocation to stress granules and impairment of Golgi complex functions.

Poly-ADP-ribose-polymerases (PARPs) 1 and 2 are nuclear enzymes that catalyze the poly-ADP-ribosylation of nuclear proteins transferring poly-ADP-ribose (PAR) polymers to specific residues. PARPs and PAR intervene in diverse functions, including DNA repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Our study is focused on one of these PARPs, PARP12, a Golgi-localized mono-ADP-ribosyltransferase that under stress challenge reversibly translocates from the Golgi complex to stress granules. PARP1 activation and release of nuclear PAR drive this translocation by direct PAR binding to the PARP12-WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and the disassembly of the Golgi membranes, followed by a block in anterograde-membrane traffic. Notably, these functions can be rescued by reverting the stress condition (by drug wash-out). Altogether these data point at a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular-membrane traffic.

Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

Transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel, is the predominant mammalian cold temperature thermosensor and it is activated by cold temperatures and cooling compounds, such as menthol and icilin. Because of its role in cold allodynia, cold hyperalgesia and painful syndromes TRPM8 antagonists are currently being pursued as potential therapeutic agents for the treatment of pain hypersensitivity. Recently TRPM8 has been found in subsets of bladder sensory nerve fibres, providing an opportunity to understand and treat chronic hypersensitivity. However, most of the known TRPM8 inhibitors lack selectivity, and only three selective compounds have reached clinical trials to date. Here, we applied two virtual screening strategies to find new, clinics suitable, TRPM8 inhibitors. This strategy enabled us to identify naphthyl derivatives as a novel class of potent and selective TRPM8 inhibitors. Further characterization of the pharmacologic properties of the most potent compound identified, compound 1, confirmed that it is a selective, competitive antagonist inhibitor of TRPM8. Compound 1 also proved itself active in a overreactive bladder model in vivo. Thus, the novel naphthyl derivative compound identified here could be optimized for clinical treatment of pain hypersensitivity in bladder disorders but also in different other pathologies.

Isoxazole derivatives as potent transient receptor potential melastatin type 8 (TRPM8) agonists.

Modulation of the transient receptor potential melastatin type-8 (TRPM8), the receptor for menthol acting as the major sensor for peripheral innocuous cool temperatures, has several important applications in pharmaceutical, food and cosmetic industries. In the present study, we designed 12 isoxazole derivatives and tested their pharmacological properties both in F11 sensory neurons in vitro, and in an in vivo model of cold allodynia. In F11 sensory neurons, single-cell Ca(2+)-imaging experiments revealed that, when compared to menthol, some newly-synthesized compounds were up to 200-fold more potent, though none of them showed an increased efficacy. Some isoxazole derivatives potentiated allodynic responses elicited by acetone when administered to rats subjected to sciatic nerve ligation; when compared to menthol, these compounds were efficacious at earlier (0-2 min) but not later (7-9 or 14-16 min) time points. Docking experiments performed in a human TRPM8 receptor model revealed that newly-synthesized compounds might adopt two possible conformations, thereby allowing to distinguish "menthol-like" compounds (characterized by high efficacy/low potency), and "icillin-like" compounds (with high potency/low efficacy). Collectively, these data provide rationale structure-activity relationships for isoxazole derivatives acting as TRPM8 agonists, and suggest their potential usefulness for cold-evoked analgesia.