Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

Apoptosis requires the transmission of apoptotic signals from the plasma membrane receptors to caspases. In receptor-mediated apoptosis, apoptotic initiation is due to the formation of a protein-signaling complex that involves the physical association of caspases followed by their activation (1, 2). Fas, a member of tumor necrosis factor receptor superfamily, has an intracellular death domain (DD) 1 in its cytoplasmic region (3-6). The DD is essential for the transduction of apoptotic signal. The Fas-associated death domain protein (FADD) binds to the DD of Fas through its DD in the C terminus (7,8). In addition to DD, FADD has a DED at its N terminus, another protein interaction module. Therefore, FADD can recruit caspase-8 to the DISC by homotypic interactions between the DEDs of FADD and caspase-8 (2, 9). The Fas-DISC formation is the first event that occurs during Fas-initiated cell death signaling. Following Fas-DISC formation, caspase-8 subsequently can be activated by autocleavage leading to the release of the active subunits p18 and p10 (2, 10). In addition, the activated caspase-8 activates downstream effector caspases such as caspase-3, caspase-6, and caspase-7 (11-16). In addition to FADD and caspase-8, another DED-containing protein related to DISC has been reported. Viral FLICE inhibitory protein (v-FLIP) is composed of two DEDs and binds to the Fas⅐FADD complex and inhibits the recruitment of caspase-8 to Fas-DISC. A human homolog of v-FLIP has many different names c-FLIP, FLAME, I-FLICE, Casper, CASH, usurpin, MRIT, and CLARP, respectively (17-24). FLICE-associated huge protein (FLASH) is another protein with binding activity to the DEDs of caspase-8 and FADD through its DEDlike domain and is a component of the Fas-DISC (25). In addition, FLASH enhances the activation of caspase-8 in Fas-mediated apoptosis. Thus, DED-containing proteins seem to modulate the apoptotic process.Two different cell types in Fas signaling pathways have been identified (26). Type I cells are characterized by recruitment of caspase-8 to the DISC following Fas activation, leading to direct activation of downstream caspases, including caspase-3 and caspase-7. In type I cells, the blocking of mitochondrial apoptotic function by overexpression of Bcl-2 has no effect on caspase activation. In type II cells, the amount of active caspase-8 generated in the DISC is low. In addition, DISC formation in type II cells is strongly reduced, and overexpression of Bcl-2 or Bcl-X L blocks caspase-8 and caspase-3 activation. Thus, Fas-mediated apoptosis in type II cells is dependent on mitochondrial activity.FAF1 is a Fas-associating molecule, which enhances Fasmediated apoptosis (27). In our previous work, mere intrinsic overexpression of FAF1 initiated apoptosis in the absence of extrinsic death signals in BOSC23 cells (28). This apoptotic potential required the region comprising amino acids 181-381 of FAF1. Mouse FAF1 (mFAF1), however, was able to enhance

Hepatitis C virus (HCV) infection results in liver injury and long‐term complications, such as liver cirrhosis and hepatocellular carcinoma. Liver injury in HCV infection is believed to be caused by host immune responses, not by viral cytopathic effects. Tumor necrosis factor‐alpha (TNF‐α) plays a pivotal role in the inflammatory processes of hepatitis C. TNF‐α induces cell death that can be ameliorated by nuclear factor kappaB (NF‐κB) activation. We investigated the regulation of TNF‐α signal transduction in HCV‐infected cells and identified HCV proteins responsible for sensitization to TNF‐α‐induced cell death. We studied the effect of HCV infection on TNF‐α signal transduction using an in vitro HCV infection model (JFH‐1, genotype 2a) with Huh‐7 and Huh‐7.5 cells. We found that TNF‐α‐induced cell death significantly increased in HCV‐infected cells. HCV infection diminished TNF‐α‐induced phosphorylation of IκB kinase (IKK) and inhibitor of NF‐κB (IκB), which are upstream regulators of NF‐κB activation. HCV infection also inhibited nuclear translocation of NF‐κB and expression of NF‐κB‐dependent anti‐apoptotic proteins, such as B‐cell lymphoma—extra large (Bcl‐xL), X‐linked inhibitor of apoptosis protein (XIAP), and the long form of cellular‐FLICE inhibitory protein (c‐FLIP). Decreased levels of Bcl‐xL, XIAP, and c‐FLIP messenger RNA and protein were also observed in livers with chronic hepatitis C. Transfection with plasmids encoding each HCV protein revealed that core, nonstructural protein (NS)4B, and NS5B attenuated TNF‐α‐induced NF‐κB activation and enhanced TNF‐α‐induced cell death. Conclusion: HCV infection enhances TNF‐α‐induced cell death by suppressing NF‐κB activation through the action of core, NS4B, and NS5B. This mechanism may contribute to immune‐mediated liver injury in HCV infection. (HEPATOLOGY 2012;56:831–840)

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In asthma, T helper 2 (TH2)-type cytokines such as interleukin (IL)-4, IL-5, and IL-13 are produced by activated CD4 + T cells.

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Dendritic cells play an important role in determining whether naïve T cells mature into either Th1 or Th2 cells. We determined whether heat-shock protein X (HspX) purified from Mycobacterium tuberculosis regulates the Th1/Th2 immune response in an ovalbumin (OVA)-induced murine model of asthma. HspX increased interferon-gamma, IL-17A, -12 and transforming growth factor (TGF)-β production and T-bet gene expression but reduced IL-13 production and GATA-3 gene expression. HspX also inhibited asthmatic reactions as demonstrated by an increase in the number of eosinophils in bronchoalveolar lavage fluid, inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyper-responsiveness. Furthermore, HspX enhanced OVA-induced decrease of regulatory T cells in the mediastinal lymph nodes. This study provides evidence that HspX plays critical roles in the amelioration of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of HspX with respect to its effects on a murine model of asthma. BMB Reports 2015; 48(3): 178-183]