Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Showa University School of Pharmacy

Cholesterol metabolism is tightly regulated by both cholesterol and its metabolites in the mammalian liver, but the regulatory mechanism of triacylglycerol (TG) synthesis remains to be elucidated. Lipin, which catalyzes the conversion of phosphatidate to diacylglycerol, is a key enzyme involved in de novo TG synthesis in the liver via the glycerol-3-phosphate (G3P) pathway. However, the regulatory mechanisms for the expression of lipin in the liver are not well understood.

Dietary cholesterol reduces plasma triacylglycerol in apolipoprotein E-null mice: suppression of lipin-1 and -2 in the glycerol-3-phosphate pathway.

Cytosolic lipid droplets (LDs), which are now recognized as multifunctional organelles, accumulate in leukocytes under various inflammatory conditions. However, little is known about the characteristic features of LDs in neutrophils. In this study, we show that perilipin-3 (PLIN3; formerly called TIP47) is involved in LD formation and the inflammatory response in HL-60-derived neutrophils. HL-60, a promyelocytic cell line, was differentiated into neutrophils via treatment with all-trans retinoic acid. After differentiation, cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g-LPS), a major pathogen in adult periodontitis. When HL-60-derived neutrophils were stimulated with P.g-LPS, LDs increased in both number and size. In the differentiated cells, PLIN3 was induced while PLIN1, PLIN2 and PLIN5 were not detected. PGE2 production and the PLIN3 protein level were increased by the P.g-LPS treatment of the cells in a dose-dependent manner. When PLIN3 was down-regulated with siRNA treatment, LDs essentially disappeared and the level of PGE2 secreted in the cell culture medium decreased by 65%. In addition, the suppression of PLIN3 repressed the PGE2 producing enzymes; i.e., microsomal PGE synthase-1, -2 and cyclooxygenase-2. These findings indicate that PLIN3 has a pivotal role in LD-biogenesis in HL-60-derived neutrophils, and that PLIN3 is associated with the synthesis and secretion of PGE2. 

Crucial role of perilipin-3 (TIP47) in formation of lipid droplets and PGE2 production in HL-60-derived neutrophils.

Oral health conditions have a significant relationship with diabetes mellitus (DM) as well as dyslipidemia. In this study, we investigated the levels of apolipoprotein B (apoB) and oxidized low-density lipoprotein (oxLDL) in the gingival crevicular fluid (GCF) from patients with DM.

The apolipoprotein B concentration in gingival crevicular fluid increases in patients with diabetes mellitus.

Oxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced.

Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein.

Lipid droplets (LDs) are functional subcellular organelles involved in multiple intracellular processes. LDs are found in nearly all types of eukaryotic cells, but their properties are highly variable in different types of tissues. Steroidogenic cells synthesize steroid hormones de novo from the cholesterol deposited in cytosolic LDs. However, the roles of LD proteins in steroidogenesis under pituitary hormone stimulation have not been well elucidated. The protein profile of isolated LDs from the mouse Leydig tumor cell line MLTC-1 was distinct from that of hepatic cells or macrophages. By proteomic analysis of the components using mass spectrometry, two enzymes for steroidogenesis, 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) and 17 β-hydroxysteroid dehydrogenase type 11 (17βHSD11), were identified in two strong bands in the LD fractions. The LD fraction of MLTC-1 cells also included CYP11A1 and CYP17, suggesting that the LDs contain all the enzymes needed for testosterone synthesis. The steroidogenesis in Leydig cells is activated by luteinizing hormone through a PKA-dependent pathway. Stimulation of MLTC-1 cells with luteinizing hormone or 8-bromo-cAMP caused drastic changes in the morphology of the LDs in the MLTC-1 cells. Upon stimulation, large perinuclear LDs are turned into much smaller LDs and dispersed throughout the cytosol. These results raise the possibility that LDs are involved in a regulatory pathway of steroidogenesis, not just by serving as a storage depot for cholesterol esters, but also by providing enzymes and generating sites for enzymatic activity. 

Characterization of lipid droplets in steroidogenic MLTC-1 Leydig cells: Protein profiles and the morphological change induced by hormone stimulation.

Second generation antipsychotics are useful for the treatment of schizophrenia, but concerns have been raised about the side effects of diabetes mellitus and obesity. Olanzapine, especially, is associated with more weight gain than the others. It has been reported that olanzapine promotes adipocyte-differentiation in rodents both in vivo and in vitro. In this study the effects of antipsychotics on human adipocytes were investigated by using human mesenchymal stem cells (hMSCs). When hMSCs were differentiated and treated with various antipsychotics, olanzapine and clozapine increased intracellular lipids. Olanzapine induced lipid accumulation in a dose-dependent manner. Proteomic analysis revealed that PLIN4 and several enzymes for lipid metabolism were increased in the hMSCs after olanzapine treatment. During adipocyte differentiation, olanzapine increased the protein expression of PLIN1, PLIN2 and PLIN4. These proteins are known to be associated with the initial stage of lipid droplet formation. Immunocytochemistry showed that olanzapine increased and enlarged the lipid droplets coated with PLIN1 and PLIN2 while PLIN4 was largely distributed in the cytosol. mRNA expression of PLIN2, but not PLIN1 or PLIN4, was increased by olanzapine. On the other hand, olanzapine did not alter the mRNA level of transcription regulators involved in adipocyte-differentiation or adipokines. The present study shows that olanzapine induced transient PLIN2 expression in hMSCs that could result in an accumulation of lipid droplets and overexpression of PLIN1 and PLIN4, providing information of possible interest for olanzapine-induced weight gain. 

Olanzapine promotes the accumulation of lipid droplets and the expression of multiple perilipins in human adipocytes.

Gingivitis commonly progresses to periodontitis in permanent dentition but rarely in deciduous teeth. Little is known about the biochemical differences between gingiva of deciduous and permanent teeth. Here, we compared the protein profiles of gingival crevicular fluids (GCF) from the gingiva of deciduous and permanent teeth.

Quantitative proteomic analysis of gingival crevicular fluids from deciduous and permanent teeth.

Oxidatively modified low-density lipoprotein (oxLDL) is known to be involved in various diseases, including cardiovascular diseases. The presence of oxLDL in the human circulatory system and in atherosclerotic lesions has been demonstrated using monoclonal antibodies. Studies have shown the significance of circulating oxLDL in various systemic diseases, including acute myocardial infarction and diabetic mellitus. Several different enzyme-linked immunosorbent assay (ELISA) procedures to measure oxLDL were utilized. Evidence has been accumulating that reveals changes in oxLDL levels under certain pathological conditions. Since oxLDL concentration tends to correlate with low-density lipoprotein (LDL)-cholesterol, the ratio of ox-LDL and LDL rather than oxLDL concentration alone has also been focused. In addition to circulating plasma, LDL and oxLDL are found in gingival crevicular fluid (GCF), where the ratio of oxLDL to LDL in GCF is much higher than in plasma. LDL and oxLDL levels in GCF show an increase in diabetic patients and periodontal patients, suggesting that GCF might be useful in examining systemic conditions. GCF oxLDL increased when the teeth were affected by periodontitis. It is likely that oxLDL levels in plasma and GCF could reflect oxidative stress and transfer efficacy in the circulatory system.

The Significance of Oxidized Low-Density Lipoprotein in Body Fluids as a Marker Related to Diseased Conditions.

The events that trigger early onset of atherosclerotic lesion formation are poorly understood. Initially, microscopic atherosclerotic lesions appear in the aortic root in 10-week-old apoE-knockout mice that are fed normal chow. Using proteome and immunohistochemical analyses, we investigated proteins in aortic media whose expression changes in athero-prone regions at the beginning of lesion formation. Protein profiles of the root/arch and thoracic/abdominal regions of aortas in 10-week-old apoE-knockout mice were analyzed using 2D-gel electrophoresis. Proteins in 81 spots with different abundance were identified. Among them, we focused on proteins related to oxidative stress and smooth muscle cells (SMCs). The level of peroxiredoxin 2 (Prx2), a major cellular antioxidant enzyme that reduces hydrogen peroxide, was lower in aortic root/arch compared with thoracic/abdominal aorta. Immunohistochemical staining demonstrated that Prx2 expression in SMCs in the aortic root was high at 4 weeks and decreased at 10 weeks in apoE-knockout mice, while Prx2 expression in the aorta was unchanged in wild-type mice. The level of Prx2 expression correlated positively with the SMC differentiation markers, α-smooth muscle actin and transgelin, suggesting that a decline in Prx2 expression accompanies SMC dedifferentiation. Accumulated acrolein-modified proteins and the infiltration of macrophages in aortic media were observed in areas with low Prx2 expression. These results showed that Prx2 expression declines in athero-prone aortic root before lesion formation, and this reduction in Prx2 expression correlates with lipid peroxidation, SMC dedifferentiation, and macrophage recruitment.

Takashi Obama

Division of Biological Chemistry,
Department of Molecular Biology,
Division of Biological Chemistry, Department of Molecular Biology

Showa University School of Pharmacy

Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique

Takashi Obama

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Biological Chemistry,Department of Molecular Biology,Division of Biological Chemistry, Department of Molecular Biology

Showa University School of Pharmacy

Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique

Division of Biological Chemistry,
Department of Molecular Biology,
Division of Biological Chemistry, Department of Molecular Biology