Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the ?-opioid receptor

© 2015 American Chemical Society. Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression

Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

The 26S proteasome is the major regulated protease in eukaryotes and is responsible for degrading ubiquitinated substrates. It consists of a barrel-shaped 20S core peptidase and one or two 19S regulatory particles, which recognize, unfold, and translocate substrates into the core. The regulatory particle can be further divided into two multi-subunit complexes: the base and the lid. Here we present protocols for expressing the Saccharomyces cerevisiae base and lid recombinantly in Escherichia coli and purifying the assembled subcomplexes using a tandem affinity purification method. The purified complexes can then be reconstituted with 20S core to form fully functional proteasomes. Furthermore, we describe a method for incorporating the unnatural amino acid p-azido-L-phenylalanine into the recombinant complexes at any residue position, allowing for non-disruptive site-specific modifications of these large assemblies. The use of recombinant proteins allows for complete mutational control over the proteasome regulatory particle, enabling detailed studies of the mechanism by which the proteasome processes its substrates. The ability to then specifically modify residues in the regulatory particle opens the door to a wide range of previously impossible biochemical and biophysical studies. The techniques described below for incorporating unnatural amino acids into the proteasomal subcomplexes should be widely transferable to other recombinant proteins, whether individually purified or in larger multi-subunit assemblies

The chaperone‐like protein Cdc48 regulates ubiquitin‐proteasome system in plants

International audienceThe degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analyzed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the amount of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response

Co-translational assembly of proteasome subunits in NOT1-containing assemblysomes

The assembly of large multimeric complexes in the crowded cytoplasm is challenging. Here we reveal a mechanism that ensures accurate production of the yeast proteasome, involving ribosome pausing and co-translational assembly of Rpt1 and Rpt2. Interaction of nascent Rpt1 and Rpt2 then lifts ribosome pausing. We show that the N-terminal disordered domain of Rpt1 is required to ensure efficient ribosome pausing and association of nascent Rpt1 protein complexes into heavy particles, wherein the nascent protein complexes escape ribosome quality control. Immunofluorescence and in situ hybridization studies indicate that Rpt1- and Rpt2-encoding messenger RNAs co-localize in these particles that contain, and are dependent on, Not1, the scaffold of the Ccr4-Not complex. We refer to these particles as Not1-containing assemblysomes, as they are smaller than and distinct from other RNA granules such as stress granules and GW- or P-bodies. Synthesis of Rpt1 with ribosome pausing and Not1-containing assemblysome induction is conserved from yeast to human cells

Resistance to the Proteasome Inhibitors: Lessons from Multiple Myeloma and Mantle Cell Lymphoma

International audienceSince its introduction in the clinics in early 2000s, the proteasome inhibitor bortezomib (BTZ) significantly improved the prognosis of patients with multiple myeloma (MM) and mantle cell lymphoma (MCL), two of the most challenging B-cell malignancies in western countries. However, relapses following BTZ therapy are frequent, while primary resistance to this agent remains a major limitation for further development of its therapeutic potential. In the present chapter, we recapitulate the molecular mechanisms associated with intrinsic and acquired resistance to BTZ learning from MM and MCL experience, including mutations of crucial genes and activation of pro-survival signalling pathways inherent to malignant B cells. We also outline the preclinical and clinical evaluations of some potential druggable targets associated to BTZ resistance, considering the most meaningful findings of the past 10 years. Although our understanding of BTZ resistance is far from being completed, recent discoveries are contributing to develop new approaches to treat relapsed MM and MCL patients