Jae Sung Yi

Amyloid β oligomerization is induced by brain lipid rafts

Detergent‐resistant lipid rafts are required for the generation of Aβ as they concentrate not only amyloid precursor protein (APP), but also the β‐ and γ‐secretase that convert APP to Aβ. Recently, Aβ has been shown to be oligomerized, which results in neuronal cytotoxicity and synaptic failure. In this study, we have demonstrated that Aβ oligomers appeared immediately after the incubation of Aβ with lipid rafts isolated from the brain tissues of rats, and were converted into few Aβ fibrils, even after longer periods of incubation. The oligomerization of Aβ was not abolished after the brain lipid rafts were treated with heat, or with protease K, implying that the lipid raft proteins were determined not to be prerequisites for Aβ oligomerization. The cholesterol present in the lipid rafts might not be essential to Aβ oligomerization because Aβ oligomerization was not prevented after the cholesterol was removed from the lipid rafts with methyl‐β‐cyclodextrin (MβCD). The Aβ oligomerization was accelerated by the application of lipid rafts isolated from ganglioside‐rich cells, C2C12 cells, whereas this was not observed with the lipid rafts isolated from ganglioside‐poor cells SK‐N‐MC and HeLa cells. In addition, lipid raft‐induced Aβ oligomerization was shown to be inhibited in CHO‐K1 cells which were defective with regard to ganglioside biosynthesis. This indicates that Aβ oligomerization requires gangliosides that are enriched in the lipid rafts. J. Cell. Biochem. 99: 878–889, 2006.

MG53-induced IRS-1 ubiquitination negatively regulates skeletal myogenesis and insulin signalling

Mitsugumin 53 (MG53) negatively regulates skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1). Here, we show that MG53 is an ubiquitin E3 ligase that induces IRS-1 ubiquitination with the help of an E2-conjugating enzyme, UBE2H. Molecular manipulations that disrupt the E3-ligase function of MG53 abolish IRS-1 ubiquitination and enhance skeletal myogenesis. Skeletal muscles derived from the MG53-/- mice show an elevated IRS-1 level with enhanced insulin signalling, which protects the MG53-/- mice from developing insulin resistance when challenged with a high-fat/high-sucrose diet. Muscle samples derived from human diabetic patients and mice with insulin resistance show normal expression of MG53, indicating that altered MG53 expression does not serve as a causative factor for the development of metabolic disorders. Thus, therapeutic interventions that target the interaction between MG53 and IRS-1 may be a novel approach for the treatment of metabolic diseases that are associated with insulin resistance.

TRIM72 negatively regulates myogenesis via targeting insulin receptor substrate-1.

Lipid rafts have been known to be platforms to initiate cellular signal transduction of insulin-like growth factor (IGF) inducing skeletal muscle differentiation and hypertrophy. Here, tripartite motif 72 (TRIM72), with a really interesting new gene (RING)-finger domain, a B-box, two coiled-coil domains, and a SPRY (SPla and RYanodine receptor) domain, was revealed to be predominantly expressed in the sarcolemma lipid rafts of skeletal and cardiac muscles. Adenoviral TRIM72 overexpression prevented but RNAi-mediated TRIM72 silencing enhanced C2C12 myogenesis by modulating the IGF-induced insulin receptor substrate-1 (IRS-1) activation through the molecular association of TRIM72 with IRS-1. Furthermore, myogenic activity was highly enhanced with increased IGF-induced Akt activation in the satellite cells of TRIM72−/− mice, compared to those of TRIM72+/+ mice. Because TRIM72 promoter analysis shows that two proximal E-boxes in TRIM72 promoter were essential for MyoD- and Akt-dependent TRIM72 transcription, we can conclude that TRIM72 is a novel antagonist of IRS-1, and is essential as a negative regulator of IGF-induced muscle differentiation.

Cell-surface Receptor for Complement Component C1q (gC1qR) Is a Key Regulator for Lamellipodia Formation and Cancer Metastasis

We previously demonstrated that the receptor for the complement component C1q (gC1qR) is a lipid raft protein that is indispensable for adipogenesis and insulin signaling. Here, we provide the first report that gC1qR is an essential component of lamellipodia in human lung carcinoma A549 cells. Cell-surface gC1qR was concentrated in the lamellipodia along with CD44, monosialoganglioside, actin, and phosphorylated focal adhesion kinase in cells stimulated with insulin, IGF-1, EGF, or serum. The growth factor-induced lamellipodia formation and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases. Moreover, the gC1qR-depleted cells exhibited a reduced proliferation rate in culture as well as diminished tumorigenic and metastatic activities in grafted mice. We therefore conclude that cell-surface gC1qR regulates lamellipodia formation and metastasis via receptor tyrosine kinase activation.

Ginsenoside Rh2 induces ligand-independent Fas activation via lipid raft disruption

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity, caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.

PTRF/cavin-1 is essential for multidrug resistance in cancer cells

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts.

Mitsugumin 53 (MG53) Ligase Ubiquitinates Focal Adhesion Kinase during Skeletal Myogenesis

Background: The FAK protein level decreases, but its mRNA level remains constant, during skeletal myogenesis, suggesting that an E3 ligase could induce FAK ubiquitination. Results: The E3 ligase MG53 induces FAK ubiquitination and degradation. Conclusion: MG53-mediated FAK ubiquitination and degradation is induced during myogenesis. Significance: This work provides a molecular mechanism for the negative feedback regulation of skeletal myogenesis. The striated muscle-specific mitsugumin 53 (MG53) is a novel E3 ligase that induces the ubiquitination of insulin receptor substrate 1 (IRS-1) during skeletal myogenesis, negatively regulating insulin-like growth factor and insulin signaling. Here we show that focal adhesion kinase (FAK) is the second target of MG53 during skeletal myogenesis. The FAK protein level gradually decreased, whereas its mRNA level was constant during myogenesis in C2C12 cells and MyoD-overexpressing mouse embryonic fibroblasts. The FAK protein was associated with the E2 enzyme UBE2H and the E3 enzyme MG53 in endogenous and exogenous immunoprecipitation experiments. FAK ubiquitination and degradation was induced by MG53 overexpression in myoblasts but abolished by MG53 or UBE2H knockdown in myotubes. Because RING-disrupted MG53 mutants (C14A and ΔR) did not induce FAK ubiquitination and degradation, the RING domain was determined to be required for MG53-induced FAK ubiquitination. Taken together, these data indicate that MG53 induces FAK ubiquitination with the aid of UBE2H during skeletal myogenesis.

Crystal structure of PRY‐SPRY domain of human TRIM72

Tripartite motif-containing (TRIM) family proteins consist of multimodular domains including a relatively conserved N-terminal RBCC domain consisting of a RING finger for E3 ubiquitin ligase activity, a zinc-bound B-box for protein–protein interaction, one or two coiledcoil domains for oligomerization, and a variable C-terminal domain. In some cases, however, TRIM proteins have a PRY-SPRY domain (PRY segment followed SPRY domain identified in a Dictyostelium discoidueum kinase splA and mammalian Ca-release channels ryanodine receptors) at their C-terminus, which has been identified as a targeting module.1,2 More than 70 members of this family have been identified and characterized, and show a very similar domain architecture; however, their cellular functions are extremely diverse, and include roles in cell proliferation, differentiation, development, oncogenesis, apoptosis, and retroviral replication.1,2 The E3 ligase activity of several TRIM proteins has been previously demonstrated, as they usually harbor a RING domain at the N-terminal region. Each TRIM protein interacts with distinct targets, which are critical in the aforementioned cellular processes.3–8 Therefore, relatively newly and incompletely characterized C-terminal domains, including the PRY-SPRY domain, are believed to be a central mediator for selective interaction with their partners. Well-studied members of the TRIM family include the following: TRIM1, TRIM5a, TRIM19, and TRIM22, which target retroviruses and prevent their replication inside cells3,6; TRIM18/MID1 and TRIM20/pyrin, which are linked to Opitz G/BBB syndrome and familial Mediterranean fever, respectively2,9; TRIM21/Ro52, which is a major autoantigen in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Sjorgen’s syndrome10,11; and TRIM63/Murf1, TRIM55/ Murf2, TRIM41, and TRIM32, which function in muscle cells.1,12 Recently, another TRIM family protein, TRIM72/MG53 has been shown to be expressed specifically in skeletal muscle and heart, and also been demonstrated to perform a critical function in membrane repair following acute muscle injury.13–15 Human TRIM72 consists of 477 amino acid residues with the standard domain organization of the TRIM family [Fig. 1(A)]. By way of contrast with the RBCC domain, which is predictive of its molecular function, very little is currently known regarding the PRY-SPRY domain, or how it has evolved to mediate diverse functions in each TRIM protein. Thus far, two structures of the SPRY domain have been determined in the complex state; however, their interac-

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host–pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Mitochondrial oxidative phosphorylation system is recruited to detergent‐resistant lipid rafts during myogenesis

Since detergent‐resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent‐resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface‐biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH‐dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.