Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET

Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image‐based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment‐targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large‐scale experimental designs with standard instruments.     

Activity staining for glutamate and alanine dehydrogenases in polyacrylamide gels of varying porosity. A study of bovine placental enzyme forms.

A procedure has been developed for characterizing the various molecular forms of placental and liver glutamate dehydrogenases through a combination of activity staining and varying gel pore size in electrophoresis. At a concentration of 2 mg/ml, the bovine liver GDH remained associated in a very high molecular weight form, while the placental enzyme was substantially dissociated to a molecular species of near 240,000 molecular weight and several charge isomeric species of near 160,000 molecular weight. The general approach outlined here provides a means of definitely correlating the electrophoretic behavior of various dehydrogenase isozymes with both glutamate and alanine dehydrogenase activities and molecular size and should be applicable, even in crude extracts to other dehydrogenase enzymes which exhibit multiple forms or states of association.     

ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

Acute myeloid leukaemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. 4‐Amino‐2‐trifluoromethyl‐phenyl retinate (ATPR), a novel all‐trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show superior anticancer effect compared with ATRA on various cancers. However, its potential effect on AML remains largely unknown. Lactate dehydrogenase B (LDHB) is the key glycolytic enzyme that catalyses the interconversion between pyruvate and lactate. Currently, little is known about the role of LDHB in AML. In this study, we found that ATPR showed antileukaemic effects with RARα dependent in AML cells. LDHB was aberrantly overexpressed in human AML peripheral blood mononuclear cell (PBMC) and AML cell lines. A lentiviral vector expressing LDHB‐targeting shRNA was constructed to generate a stable AML cells with low expression of LDHB. The effect of LDHB knockdown on differentiation and cycle arrest of AML cells was assessed in vitro and vivo, including involvement of Raf/MEK/ERK signalling. Finally, these data suggested that ATPR showed antileukaemic effects by RARα/LDHB/ ERK‐glycolysis signalling axis. Further studies should focus on the underlying leukaemia‐promoting mechanisms and investigate LDHB as a therapeutic target.     

Tripartite motif protein 52 (TRIM52) promoted fibrosis in LX‐2 cells through PPM1A‐mediated Smad2/3 pathway

To investigate the roles of tripartite motif containing 52 (TRIM52) in human hepatic fibrosis in vitro, human hepatic stellate cell line LX‐2 cells were transfected with hepatitis B virus (HBV) replicon to establish HBV‐induced fibrosis in LX‐2 cells, and then treated with small interfering RNA‐mediated knockdown of TRIM52 (siTRIM52). LX‐2 cells without HBV replicon transfection were treated with lentiviruses‐mediated overexpression of TRIM52 and phosphatase magnesium dependent 1A (PPM1A). Fibrosis response of LX‐2 cells were assessed by the production of hydroxyproline (Hyp) and collagen I/III, as well as protein levels of α‐smooth muscle actin (α‐SMA). PPM1A and phosphorylated (p)‐Smad2/3 were measured to assess the mechanism. The correlation between TRIM52 and PPM1A was determined using co‐immunoprecipitation, and whether and how TRIM52 regulated the degradation of PPM1A were determined by ubiquitination assay. Our data confirmed HBV‐induced fibrogenesis of LX‐2 cells, as evidenced by significant increase in Hyp and collagen I/III and α‐SMA, which was associated with reduction of PPM1A and elevation of transforming growth factor‐β (TGF‐β), p‐Smad2/3, and p‐Smad3L. However, those changes induced by HBV were significantly attenuated with additional siTRIM52 treatment. Similar to HBV, overexpression of TRIM52 exerted promoted effect in the fibrosis of LX‐2 cells. Interestingly, TRIM52 induced the fibrogenesis of LX‐2 cells and the activation of TGF‐β/Smad pathway were significantly reversed by PPM1A overexpression. Furthermore, our data confirmed TRIM52 as a deubiquitinase that influenced the accumulation of PPM1A protein, and subsequently regulated the fibrogenesis of LX‐2 cells. TRIM52 was a fibrosis promoter in hepatic fibrosis in vitro, likely through PPM1A‐mediated TGF‐β/Smad pathway.     

Exosome biogenesis,secretion and function of exosomal miRNAs in skeletal muscle myogenesis

Exosomes are membrane‐bound extracellular vesicles that are produced in the endosomal compartment of most mammalian cell types and then released. Exosomes are effective carriers for the intercellular material transfer of material that can influence a series of physiological and pathological processes in recipient cells. Among loaded cargoes, non‐coding RNAs (ncRNAs) vary for the exosome‐producing cell and its homeostatic state, and characterization of the biogenesis and secretion of exosomal ncRNAs and the functions of these ncRNAs in skeletal muscle myogenesis remain preliminary. In this review, we will describe what is currently known of exosome biogenesis, release and uptake of exosomal ncRNAs, as well as the varied functions of exosomal miRNAs in skeletal muscle myogenesis.