The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo-and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/ oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKC␣, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.Integral membrane receptors consist of an extracellular domain that binds specific ligands, a transmembrane domain (TMD) 2 that transmits signals in response to ligand binding, and a cytoplasmic domain to which the signals are transmitted by the TMD and that is thereby activated, resulting in a conformational change that causes binding or induction of enzymatic activity inside the cell (1, 2). The TMD is therefore critically important in transmitting signals from the external environment to the inside of the cell, with many recent investigations exploring the mechanism by which TMD interactions regulate cell signaling (2-6). TMDs of single-pass membrane receptors have been shown to cluster, resulting in homotypic and/or heterotypic interactions, with TMDs often forming not only homo-and/or heterodimers but higher-order oligomers in cell membranes (7-10). Most investigations of TMD interactions have analyzed homo-oligomerization in vitro and in vivo, with fewer to date assessing heterotypic TMD associations.One of the most investigated examples of hetero-oligomerization in biology involves the family of EGF receptors, also called the ErbB family (11-13). Although only four ErbB family members have been identified to date, ErbB1 (EGF receptor), ErbB2 (HER2, Neu), ErbB3 (HER3), and ErbB4 (HER4), they have been shown to form 28 homo-and heterodimers (12). These combinations are thought to result in diverse cellul...

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Ethylmercury (EtHg) is derived from the degradation of thimerosal, the most widely used organomercury compound. In this study, EtHg-induced toxicity and autophagy in the mouse kidney was observed and then the mechanism of toxicity was explored in vitro in HK-2 cells. Low doses of EtHg induced autophagy without causing any histopathological changes in mouse kidneys. However, mice treated with high doses of EtHg exhibited severe focal tubular cell necrosis of the proximal tubules with autophagy. EtHg dose-dependently increased the production of reactive oxygen species, reduced the mitochondrial membrane potential, activated the unfolded protein response, and increased cytosolic Calevels in HK-2 cells. Cell death induced by EtHg exposure was caused by autophagy and necrosis. N-acetyl cysteine and 4-phenylbutyric acid attenuated EtHg-induced stress and ameliorated the autophagic response in HK-2 cells. Furthermore, EtHg blocked autophagosome fusion with lysosomes, which was demonstrated via treatment with wortmannin and chloroquine. Low doses of EtHg and rapamycin, which resulted in minimal cytotoxicity, increased the levels of the autophagic SNARE complex STX17 (syntaxin 17)-VAMP8-SNAP29 without altering mRNA levels, but high dose of EtHg was cytotoxic. Inhibition of autophagic flux by chloroquin increased autophagosome formation and necrotic cell death in HK-2 cells. Collectively, our results show that EtHg induces autophagy via oxidative and ER stress and blockade of autophagic flux. Autophagy might play a dual role in EtHg-induced renal toxicity, being both protective following treatment with low doses of EtHg and detrimental following treatment with high doses.

Exosomes have been mainly studied for their potential applications in biomarker detection and drug delivery for diagnosis and treatment. However, in the field of forensic research, the potential value of exosomes derived from post-mortem body fluids has not been investigated to date. Here, we isolated the exosomes and exosomal RNAs from post-mortem body fluids, including cardiac blood, pericardial fluid, and urine. We also compared commercial exosome isolation kits to determine the optimal method for post-mortem exosome isolation. Transmission electron microscopy (TEM), the Agilent bioanalyzer system, and western blotting were used to evaluate the efficiencies of alternative isolation methods and the characteristics of isolated exosomes. There were no significant differences between exosomes obtained from post-mortem and ante-mortem body fluids in the expression of exosome surface markers or morphology. The exosomes were well-preserved even under simulated post-mortem conditions. Among the isolation procedures tested, the membrane affinity column-based method was the most suitable for post-mortem exosomal RNA isolation. These results suggest that exosomes are well-preserved in post-mortem body fluids and could be utilized for forensic diagnosis.