No abstract

PDCD5 (human programmed cell death 5) plays a significant role in apoptotic and paraptotic cell deaths. However, it was found that recombinant PDCD5 added exogenously to culture medium could also enhance programmed cell death triggered by certain stimuli. Here we show that PDCD5 has a remarkable role in intercellular transport in various cells (endogenous caveolin-1-positive and -negative cells) through a clathrin-independent endocytic pathway that originates from heparan sulfate proteoglycan binding and lipid rafts. These conclusions are supported by the studies of slow internalization kinetics of PDCD5 endosomes, by the resistance of endosomes to nonionic detergents, by the overexpression of the clathrin dominant negative mutant form, which did not block PDCD5-fluorescein isothiocyanate uptake, and by PDCD5 localization in lipid rafts by immunofluorescence, electron microscopy techniques, and sucrose density centrifugation. This is further supported by the findings that certain drugs that disrupt lipid rafts, compete with cell membrane heparan sulfate proteoglycans, or block the caveolae pathway, impair the PDCD5 internalization process. The translocation activity of PDCD5 may possess physiological significance and be a potential mechanism for its programmed cell death-promoting activity. PDCD5 protein also has the ability to drive the internalization of large protein cargo, depending on the residues 109 -115 mapped by deletion mutagenesis, and can introduce the Mdm-2 binding domain of human p53 into living cells to induce cell death in human cancer cells, indicating that PDCD5 may serve as a vehicle and thus have potential in the field of protein delivery to the cells. This is the first evidence of such findings.

The mitochondria-mediated apoptotic pathway is regulated by members of the Bcl-2 family. Epidermal growth factor (EGF) induces Bad phosphorylation at Ser112 via mitogen-activated protein kinase (MAPK), impairing its binding to Bcl-2 and Bcl-xL and interfering with their anti-apoptotic functions. In the current study, we utilized Western blot, immunofluorescence, flow cytometry, and confocal microscopy to examine the effects of CMTM8 overexpression on apoptosis. Our data indicated levels of Bad-S112 phosphorylation were lower in CMTM8-transfected cells compared to pCDB-transfected cells. Caspase-dependent and independent mediated apoptosis, induced by CMTM8 overexpression, was facilitated by the mitochondria and inhibited by knockdown of Bad or overexpression of Bcl-xL. Previous research in our laboratory also demonstrated CMTM8 attenuated EGFR-mediated signaling pathways by decreasing ERK1/2 phosphorylation levels. These data implicate CMTM8 as a negative regulator of EGF-induced signaling, with potential use as a novel therapeutic gene for EGFR-targeted anticancer gene therapy.

No abstract

CC chemokine receptor 4 (CCR4) is a kind of G-protein-coupled receptors with a characteristic seven-transmembrane structure and selectively expressed on Th2-type CD4+ T-cells, which play a pivotal role in allergic inflammation. In this study, the interactions between 2-(2-(2,4-dichloro-phenyl)-4-{[(2-methyl-3-chloro-phenyl)-1-ylmethyl]-carbamoyl}-methyl)-5-oxo-pyrrole-1-yl)-N-(3-piperidinyl-propyl)-acetamide (compound A), a known CCR4 antagonist, and ML40 were studied by CZE for the first time. Both qualitative and quantitative characterizations of the drug-peptide binding were determined. The binding constant of the interaction between the trans-diastereomer of compound A and ML40, calculated from the Scatchard plot by regression, was (1.06 +/- 0.11)x10(5)/M. Also, it was confirmed that the trans-diastereomer was more potent affinity with CCR4 than its cis-counterpart. The experimental results show that this reported method by CZE for the determination of the compound A and ML40 interactions is powerful, sensitive, and fast, requires less amounts of reagents, and further, it can be employed as one of the reliable screening methods to a series of lactam analogs in the drug discovery for allergic inflammation diseases.