Modulation of rat liver hydroxymethylglutaryl-CoA reductase by protein phosphatases: purification of nonspecific hydroxymethylglutaryl-CoA reductase phosphatases

Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase have been purified from rat liver cytosol through a process that involves DEAE-cellulose, aminohexyl-Sepharose-4B, and Bio-Gel A 1.5 m chromatographies. Protein phosphatase II (Mr 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the three substrates. Phosphatases I1 (Mr 180,000) and I3 (Mr 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the three substrates. In contrast, phosphatase I2 (Mr 200,000) showed only phosphorylase phosphatase activity with insignificant dephosphorylating capacity toward HMG-CoA reductase and glycogen synthase. Upon ethanol treatment at room temperature, the Mr of all phosphatases changed; protein phosphatases I2, I3, and II were brought to an Mr of 35,000, while phosphatase I1 was reduced to an Mr of 69,000. Glycogen synthase phosphatase activity was decreased in all four phosphatases. There was also a decrease in phosphatase I1 activity toward HMG-CoA reductase and phosphorylase as substrates. The HMG-CoA reductase phosphatase and phosphorylase phosphatase activities of phosphatases I2, I3, and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The four phosphatases increased their activity in the presence of Mn2+ and Mg2+. In general, Mn2+ was a better activator than Mg2+, and phosphatase I1 showed a stronger dependency on these cations than any other phosphatase. Phosphorylase was a competitive substrate in the HMG-CoA reductase phosphatase and glycogen synthase phosphatase reactions of protein phosphatases I1, I3, and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen synthase phosphatase activity presented less inhibition in the low-Mr forms. A comparison has been made with other protein phosphatases previously reported in the literature.     

Streptococcus pyogenes in human plasma: adaptive mechanisms analyzed by mass spectrometry-based proteomics

Streptococcus pyogenes is a major bacterial pathogen and a potent inducer of inflammation causing plasma leakage at the site of infection. A combination of label-free quantitative mass spectrometry-based proteomics strategies were used to measure how the intracellular proteome homeostasis of S. pyogenes is influenced by the presence of human plasma, identifying and quantifying 842 proteins. In plasma the bacterium modifies its production of 213 proteins, and the most pronounced change was the complete down-regulation of proteins required for fatty acid biosynthesis. Fatty acids are transported by albumin (HSA) in plasma. S. pyogenes expresses HSA-binding surface proteins, and HSA carrying fatty acids reduced the amount of fatty acid biosynthesis proteins to the same extent as plasma. The results clarify the function of HSA-binding proteins in S. pyogenes and underline the power of the quantitative mass spectrometry strategy used here to investigate bacterial adaptation to a given environment.     

Lipid droplets interact with mitochondria using SNAP23

Triglyceride-containing lipid droplets (LD) are dynamic organelles stored on demand in all cells. These droplets grow through a fusion process mediated by SNARE proteins, including SNAP23. The droplets have also been shown to be highly motile and interact with other cell organelles, including peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell.     

The mammalian verprolin homologue WIRE participates in receptor-mediated endocytosis and regulation of the actin filament system by distinct mechanisms

The mammalian verprolin family consists of three family members: WIP, WIRE and CR16. WIRE was recently found to bind to WASP and N-WASP and to have roles in regulating actin dynamics downstream of the platelet-derived growth factor β-receptor. In the current study, the WASP-binding domain of WIRE was identified, with the core of the binding motif encompassing amino acid residues 408–412. A stretch of aromatic amino acid residues close to the core motif also participates in WASP binding. Amino acid substitutions in each of these motifs abrogated WASP binding, suggesting that both motifs are involved in the binding of WIRE to WASP. Interestingly, WIRE mutants unable to bind WASP were still able to induce a reorganisation of the actin filament system, indicating that WASP did not participate in the signalling pathway that link WIRE to actin dynamics. In cells ectopically expressing WIRE, the endocytosis of the platelet-derived growth factor β-receptor was drastically reduced. However, in contrast to the effect on the actin filament system, the WIRE-induced ablation of the receptor endocytosis required an intact WASP-binding domain. Moreover, WIRE was more efficient than WIP in inhibiting the receptor endocytosis, implicating that these two mammalian verprolins have distinct roles in mammalian cells.     

Expression of smooth muscle MyHC B in blood vessels of hypertrophied heart in experimentally hypertensive rats

We demonstrated recently a significantly lower fraction of cardiac precapillary arterioles that expressed smooth muscle myosin heavy chain (MyHC) B (SMB) in spontaneously hypertensive rats. To clarify whether this reduction of SMB expression is of genetic origin, we investigated SMB expression in cardiac precapillary arterioles of normotensive and experimentally hypertensive rats (one clip, one kidney or ANG II minipump). We observed similar SMB expression patterns in precapillary arterioles of experimentally hypertensive rats compared with normotensive controls. These observations suggest that the downregulation of SMB in spontaneously hypertensive rats is of genetic origin rather than an adaptive response to chronically enhanced blood pressure and cardiac hypertrophy.