The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the β2-Adrenergic Receptor.

Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/β-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the β2-adrenergic receptor (β2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the β2AR in a ligand-independent manner. Although ARRDC3 has no effect on β2AR endocytosis or degradation, it negatively regulates β2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in β2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the β2AR

The functional roles of arrestin domain-containing protein 3 in regulating G protein-coupled receptor trafficking and signaling

Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian β-arrestin family, which is predicted to share similar tertiary structure with visual-/β-arrestins and also contains C-terminal PPxY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here we focused on characterizing the role of ARRDC3 in regulating the trafficking of the β2-adrenergic receptor (β2AR). Using confocal and total internal reflection fluorescence microscopy (TIR-FM) in fixed and live cells, we find that both overexpressed and endogenous ARRDC3 primarily localizes to EEA1-positive early endosomes, where it also interacts with the ESCRT-0 complex. While the PPxY motifs of ARRDC3 are essential for its endosomal localization, only one PPxY motif as well as the arrestin-like domains are needed for proper localization. Using three approaches, both in vitro and in cells, we found that ARRDC3 directly interacts with the β2AR in a ligand-independent manner. Functionally, while ARRDC3 has no effect on β2AR endocytosis or degradation, it negatively regulates agonist-promoted β2AR recycling from early endosomes to the plasma membrane. Further mechanistic studies, using fluorescent microscopy in both fixed and live cells, suggest that ARRDC3 negatively modulates β2AR entry into SNX27-occupied endosomal tubules by regulating the association between the β2AR and SNX27. Concomitantly, by modulating the endosomal residence time of the β2AR, ARRDC3 regulates the recruitment of Gαs to the receptor-occupied endosomes and the β2AR–dependent signaling from the endosomes. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the β2AR. Future studies should focus on identifying the interacting motifs of the β2AR and ARRDC3, as well as expanding additional interacting partners of ARRDC3, such as additional GPCRs

Technological Configuration Capability, Strategic Flexibility, and Organizational Performance in Chinese High-Tech Organizations

The purpose of this study was to investigate the moderating effect of corporate technology configuration capability on the relationship between strategic flexibility and organizational performance throughout the different stages of the technological life cycle. By empirically examining 439 Chinese high-tech organizations, we found that technological configuration capability enhances the effect of strategic flexibility on organizational performance in a complex dynamic environment. However, different impacts were observed on the different stages of the technological life cycle. In addition, we explored the strategic flexibility during the different stages of the technological life cycle based on our empirical study

Safe Scale-Up of a NBS-Involved Bromination Reaction: Thermal Safety Analysis Contributes to Process Optimization

A hazardous side reaction between N-bromosuccinimide (NBS) and 2-methyltetrahydrofuran (2-MeTHF) was identified during the thermal safety analysis towards a bromination process. The thermal behaviors of the side reaction was investigated by Calvet calorimeter C80 in a membrane mixing cell. A delay to initiate the reaction was observed, and it was owing to a preferred free-radical mechanism that the reaction could follow. The Advanced Kinetics and Technology Solutions (AKTS) software was used to study the thermo-kinetics of the side reaction, and its reaction progress under the proposed process temperature was predicted. The influence of NBS concentration on the side reaction was studied by differential scanning calorimeter (DSC). It was proved that the maximum reaction heat could be lowered by limiting the accumulation of NBS in the reaction mixture. Besides, the heat flow of the process desired reaction was simulated by reaction calorimeter (RC1), indicating the bromination could be controlled under a semi-batch mode with appropriate feeding program of NBS. On the basis of the investigation, two batches bromination process with 106 kg starting material were safely scaled up in pilot plant with expected yield

Characterization of Selective Ubiquitin and Ubiquitin-Like Protease Inhibitors Using a Fluorescence-Based Multiplex Assay Format

The reversible conjugation of ubiquitin and ubiquitin-like (UbL) proteins to protein substrates plays a critical role in the regulation of many cellular pathways. The removal of ubiquitin from target proteins is performed by ubiquitin proteases also known as deubiquitylases (DUBs). Owing to their substrate specificity and the central role ubiquitylation plays in cell signaling pathways, DUB are attractive targets for therapeutic development. The development of DUB inhibitors requires assays that are amenable to high-throughput screening and provide rapid assessment of inhibitor selectivity. Determination of inhibitor selectivity at an early stage of drug discovery will reduce drug failure in the clinic as well as reduce overall drug development costs. We have developed two novel assays, UbL-Enterokinase light chain and UbL-Granzyme B, for quantifying ubiquitin and UbL protease activity. In our quest to discover and characterize novel chemical entities, we have combined these assays with a previously developed assay in a multiplex format. This multiplex format allows for the detection of three distinct protease activities simultaneously, in a single well. We have demonstrated that the multiplex format is able to distinguish between selective and nonselective protease inhibitors. Specifically, we have used this assay format to characterize P022077, a selective ubiquitin-specific protease 7 inhibitor discovered at Progenra

Nanostructured Zeolitic Imidazolate Frameworks Derived from Nanosized Zinc Oxide Precursors

A facile method for the large scale transformation of ZnO nanocrystals into the corresponding nanocrystals of a zeolitic imidazolate framework was demonstrated. The methodology based on this nanoscale-facilitated transformation can be adapted to synthesize zeolitic imidazolate framework films on versatile substrates through the transformation from ZnO nanoscopic films derived from chemical vapor deposition (CVD) or solution growth

Application of Transition-Metal Catalysis, Biocatalysis and Flow Chemistry as State-of-the-Art Technologies in the Synthesis of LCZ696

LCZ696 is a novel treatment for patients suffering from heart failure that combines the two active pharmaceutical ingredients sacubitril and valsartan in a single chemical compound. While valsartan is an established drug substance, a new manufacturing process suitable for large-scale commercial production had to be developed for sacubitril. The use of chemocatalysis, biocatalysis, and flow chemistry as state-of-the-art technologies allowed to efficiently build up the structure of sacubitril and achieve the defined performance targets