SecG, a subunit of the protein translocon, undergoes a cycle of topology inversion. To further examine the role of this topology inversion, we analyzed the activity of membrane vesicles carrying a SecG-PhoA fusion protein (SecG-PhoA inverted membrane vesicles (IMVs)). In the absence of externally added SecA, SecG-PhoA IMVs were as active in protein translocation as SecG ؉ IMVs per SecA. Consistent with this observation, insertion of membrane-bound SecA into SecG-PhoA IMVs was normally observed. On the other hand, externally added SecA did not affect the activity of SecG-PhoA IMVs, but it caused >10-fold stimulation of the translocation activity of SecG ؉ IMVs, indicating that the topology inversion of SecG, which cannot occur in SecG-PhoA IMVs, is essential for cytosolic SecA-dependent stimulation of protein translocation. SecG-PhoA IMVs generated a 46-kDa fragment of SecA upon trypsin treatment. The accumulation of this membrane-inserted SecA in the SecGPhoA IMVs was responsible for the loss of the soluble SecA-dependent stimulation. Moreover, fixation of the inverted SecG topology was found to be dependent on soluble SecA. The dual functions of SecG in protein translocation will be discussed.Most secretory proteins of Escherichia coli are synthesized with an N-terminally extended signal sequence and then posttranslationally translocated across the cytoplasmic membrane via the SecYEG translocon using the driving force provided by translocation ATPase SecA (see recent reviews in Refs. 1 and 2). SecA plays a central role in protein translocation by interacting with many ligands, including presecretory proteins, membrane lipids, SecYEG, and SecDF, and it undergoes a dynamic structure change coupled with protein translocation. SecA, which can be purified as a soluble protein, is inserted deep into the membrane upon the binding of both ATP and a precursor protein, which causes protein translocation of a segment of 20 -30 amino acids (3-5). Thus the translocation of the precursor is thought to be driven by the repeat of the membrane insertiondeinsertion cycle of SecA and to proceed in a stepwise manner. Based on the crystal structures of the translocon and SecA (see Refs. 1 and 2, and references therein), detailed mechanisms of protein translocation are proposed; however, they are derived from a limited number of snapshots of the catalytic cycle. Therefore, many issues, including the oligomeric state of each subunit, structure changes upon protein translocation, mode of action of the translocon, and so on, remain to be clarified.The SecG subunit of the SecYEG translocon possesses two transmembrane stretches with N and C termini exposed to the periplasm (6, 7). We have reported that SecG undergoes a cycle of topology inversion, which couples the SecA cycle with protein translocation (7-10). These phenomena were demonstrated by the translocation-dependent changes in proteinase K (PK) 2 sensitivity of the C-terminal region of SecG and by the chemical labeling of the cysteine-containing SecG mutants, using in vitro and i...

Sonodynamic therapy (SDT) is a minimally invasive anticancer therapy involving a chemical sonosensitizer and high-intensity focused ultrasound (HIFU). SDT enables the reduction of drug dose and HIFU irradiation power compared to those of conventional monotherapies. In our previous study, mouse models of colon and pancreatic cancer were used to confirm the effectiveness of SDT vs. drug-only or HIFU-only therapy. To validate its usefulness, we performed a clinical trial of SDT using an anticancer micelle (NC-6300) and our HIFU system in four pet dogs with spontaneous tumors, including chondrosarcoma, osteosarcoma, hepatocellular cancer, and prostate cancer. The fact that no adverse events were observed, suggests the usefulness of SDT.

The combinatory use of high-intensity focused ultrasound (HIFU) and epirubicin (EPI)-conjugated polymeric micellar nanoparticles (NC-6300) is thought to be a less invasive and more efficient method of cancer therapy. To investigate the mechanism underlying the combination effect, we examined the effect of trigger-pulsed HIFU (TP-HIFU) and NC-6300 from the perspective of reactive oxygen species (ROS) generation, which is considered the primary function of sonodynamic therapy (SDT), and changes in drug characteristics. TP-HIFU is an effective sequence for generating hydroxyl radicals to kill cancer cells. EPI was susceptible to degradation by TP-HIFU through the production of hydroxyl radicals. In contrast, EPI degradation of NC-6300 was suppressed by the hydrophilic shell of the micelles. NC-6300 also exhibited a sonosensitizer function, which promoted the generation of superoxide anions by TP-HIFU irradiation. The amount of ROS produced by TP-HIFU reached a level that caused structural changes to the cellular membrane. In conclusion, drug-conjugated micellar nanoparticles are more desirable for SDT because of accelerated ROS production and drug protection from ROS. Furthermore, a combination of NC-6300 and TP-HIFU is useful for minimally invasive cancer therapy with cooperative effects of HIFU-derived features, antitumor activity of EPI, and increased ROS generation to cause damage to cancer cells.