Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins

Apoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family. Here, we report that ASK1 is capable of binding to other isoforms of 14-3-3, suggesting that binding ASK1 is a general property of the 14-3-3 family. In support of this notion, mutational analysis revealed that the ASK1/14-3-3 interaction was mediated by the conserved amphipathic groove of 14-3-3 with some residue selectivity. Functionally, expression of various isoforms of 14-3-3 suppressed ASK1-induced apoptosis. To understand how 14-3-3 controls the ASK1 activity, we examined intracellular localization of ASK1 upon 14-3-3 co-expression. We found that 14-3-3 co-expression is correlated with the translocation of ASK1 from the cytoplasm to a perinuclear localization, likely the ER compartment. Consistent with this notion, ASK1(S967A), a 14-3-3 binding defective mutant of ASK, showed no change in intracellular distribution upon 14-3-3 co-expression. These data support a model that 14-3-3 proteins regulate the proapoptotic function of ASK1 in part by controlling its subcellular distribution.     

Thermoresponsive protein adsorption of poly(N-isopropylacrylamide)-modified streptavidin on polydimethylsiloxane microchannel surfaces

The control of protein adsorption on microchannel surfaces is important for biosensors. In this study, we demonstrated protein adsorption method that is controlled through temperature change, i.e., thermoresponsive protein adsorption, on polydimethylsiloxane (PDMS) microchannel surfaces using a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). To provide general protein adsorption control method, we adopted biotin-streptavidin chemistry and synthesized streptavidin covalently modified with PNIPAAm (PNIPAAm-StAv). Modification of streptavidin, a hydrophilic protein, with PNIPAAm induced successful thermoresponsive adsorption on a PDMS microchannel surfaces: PNIPAAm-StAv adsorbed at 37 degrees C and desorbed at 10 degrees C on the surfaces. We also demonstrated the thermoresponsive adsorption of biotinylated immunoglobulin G (IgG-b) using PNIPAAm-StAv. Conjugation of IgG-b with PNIPAAm-StAv induced successful thermoresponsive IgG-b adsorption on PDMS. Modification of PDMS surfaces with PNIPAAm reduced physical adsorption of the partially hydrophobic IgG-b on the surface and contributed to the high-contrast thermoresponsive adsorption of IgG-b: less than 1% of the IgG-b adsorbed at 37 degrees C was detected after the PNIPAAm-PDMS surface was washed at 10 degrees C. The controllable adsorption of this system is expected to be applied to the regeneration of biosensor chips and to on-chip protein manipulation.     

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.     

STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rbeta2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.     

Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment.