Targeted drug delivery using nanoparticles can minimize the side effects of conventional pharmaceutical agents and enhance their efficacy. However, translating nanoparticle-based agents into clinical applications still remains a challenge due to the difficulty in regulating interactions on the interfaces between nanoparticles and biological systems. Here, we present a targeting strategy for nanoparticles incorporated with a supramolecularly pre-coated recombinant fusion protein in which HER2-binding affibody combines with glutathione-S-transferase. Once thermodynamically stabilized in preferred orientations on the nanoparticles, the adsorbed fusion proteins as a corona minimize interactions with serum proteins to prevent the clearance of nanoparticles by macrophages, while ensuring systematic targeting functions in vitro and in vivo. This study provides insight into the use of the supramolecularly built protein corona shield as a targeting agent through regulating the interfaces between nanoparticles and biological systems.

Protein inactivation by reactive oxygen species (ROS) such as singlet oxygen ((1)O2) and superoxide radical (O2(•-)) is considered to trigger cell death pathways associated with protein dysfunction; however, the detailed mechanisms and direct involvement in photodynamic therapy (PDT) have not been revealed. Herein, we report Ir(III) complexes designed for ROS generation through a rational strategy to investigate protein modifications by ROS. The Ir(III) complexes are effective as PDT agents at low concentrations with low-energy irradiation (≤ 1 J cm(-2)) because of the relatively high (1)O2 quantum yield (> 0.78), even with two-photon activation. Furthermore, two types of protein modifications (protein oxidation and photo-cross-linking) involved in PDT were characterized by mass spectrometry. These modifications were generated primarily in the endoplasmic reticulum and mitochondria, producing a significant effect for cancer cell death. Consequently, we present a plausible biologically applicable PDT modality that utilizes rationally designed photoactivatable Ir(III) complexes.

The inner mitochondrial membrane (IMM) proteome plays a central role in maintaining mitochondrial physiology and cellular metabolism. Various important biochemical reactions such as oxidative phosphorylation, metabolite production, and mitochondrial biogenesis are conducted by the IMM proteome, and mitochondria-targeted therapeutics have been developed for IMM proteins, which is deeply related for various human metabolic diseases including cancer and neurodegenerative diseases. However, the membrane topology of the IMM proteome remains largely unclear because of the lack of methods to evaluate it in live cells in a high-throughput manner. In this article, we reveal the in vivo topological direction of 135 IMM proteins, using an in situ-generated radical probe with genetically targeted peroxidase (APEX). Owing to the short lifetime of phenoxyl radicals generated in situ by submitochondrial targeted APEX and the impermeability of the IMM to small molecules, the solvent-exposed tyrosine residues of both the matrix and intermembrane space (IMS) sides of IMM proteins were exclusively labeled with the radical probe in live cells by Matrix-APEX and IMS-APEX, respectively and identified by mass spectrometry. From this analysis, we confirmed 58 IMM protein topologies and we could determine the topological direction of 77 IMM proteins whose topology at the IMM has not been fully characterized. We also found several IMM proteins (e.g., LETM1 and OXA1) whose topological information should be revised on the basis of our results. Overall, our identification of structural information on the mitochondrial inner-membrane proteome can provide valuable insights for the architecture and connectome of the IMM proteome in live cells.

No abstract

Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks many Gram-negative human pathogens. A serious drawback of this strain, however, is its ineffectiveness against Gram-positive strains, such as the human pathogen Staphylococcus aureus. Here we demonstrate that the extracellular proteases produced by a host-independent B. bacteriovorus (HIB) effectively degrade/inhibit the formation of S. aureus biofilms and reduce its virulence. A 10% addition of HIB supernatant caused a 75% or greater reduction in S. aureus biofilm formation as well as 75% dispersal of pre-formed biofilms. LC-MS-MS analyses identified various B. bacteriovorus proteases within the supernatant, including the serine proteases Bd2269 and Bd2321. Tests with AEBSF confirmed that serine proteases were active in the supernatant and that they impacted S. aureus biofilm formation. The supernatant also possessed a slight DNAse activity. Furthermore, treatment of planktonic S. aureus with the supernatant diminished its ability to invade MCF-10a epithelial cells by 5-fold but did not affect the MCF-10a viability. In conclusion, this study illustrates the hitherto unknown ability of B. bacteriovorus to disperse Gram-positive pathogenic biofilms and mitigate their virulence.

AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been shown to regulate the differentiation of preadipocytes and osteoblasts. However, the role of AMPK in stem cell differentiation is largely unknown. Using in vitro culture models, the present study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We observed that expression and phosphorylation of AMPK were increased during osteogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSC). To elucidate the role of AMPK in osteogenic differentiation, we investigated the effect of AMPK inhibition or knockdown on mineralization of hAMSC. Compound C, an AMPK inhibitor, reduced mineralized matrix deposition and suppressed the expression of osteoblast-specific genes, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition, inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation, which is required for osteogenesis. Interestingly, inhibition of AMPK induced adipogenic differentiation of hAMSC, even in osteogenic induction medium (OIM). These results provide a potential mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or inhibition of AMPK, respectively.

Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases. In this study, we employed a new proximity biotin-labeling method using promiscuous biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain in living cells. FKBP25 showed the strongest biotin labeling by FRB–pBirA in the presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB–rapamycin–FKBP25 was determined at 1.67-Å resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where there is a methionine-rich space, and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling.

Communication between osteoblasts and endothelial cells is essential for bone fracture repair, but the molecular identities of such communicating factors are not well defined. Here we identify DJ-1 as a novel mediator of the cross-talk between osteoblasts and endothelial cells through an unbiased screening of molecules secreted from human mesenchymal stem cells during osteogenesis. We show that DJ-1 stimulates the differentiation of human mesenchymal stem cells to osteoblasts and that DJ-1 induces angiogenesis in endothelial cells through activation of fibroblast growth factor receptor-1 signalling. In a rodent model of bone fracture repair, extracellular application of DJ-1 enhances bone regeneration in vivo by stimulating the formation of blood vessels and new bones. Both these effects are blocked by antagonizing fibroblast growth factor receptor-1 signalling. These findings uncover previously undefined extracellular roles of DJ-1 to promote angiogenesis and osteogenesis, suggesting DJ-1 may have therapeutic potential to stimulate bone regeneration.