Currently, major concerns about the safety and efficacy of RNA interference (RNAi)-based bone anabolic strategies still exist because of the lack of direct osteoblast-specific delivery systems for osteogenic siRNAs. Here we screened the aptamer CH6 by cell-SELEX, specifically targeting both rat and human osteoblasts, and then we developed CH6 aptamer–functionalized lipid nanoparticles (LNPs) encapsulating osteogenic pleckstrin homology domain-containing family O member 1 (Plekho1) siRNA (CH6-LNPs-siRNA). Our results showed that CH6 facilitated in vitro osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, and boosted in vivo osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both osteopenic and healthy rodents. These results indicate that osteoblast-specific aptamer-functionalized LNPs could act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level.
Protein-binding peptide is recently recognized as an effective artificial affinity reagent for protein assays. However, its application is hampered by the limited choices of available signal readout methods. Herein, we report a general electrochemical signal readout method for protein-binding peptides exploiting the host-guest chemistry of cucurbituril. Via the formation of supermolecules among cucurbituril, electrochemical reporter, and the peptide, a protein-binding peptide can be noncovalently coupled with the electrochemical reporter. To assay the target protein, the protein-binding peptides are first self-assembled in the sensing layer, and after the capturing of the target protein, a portion of the peptides become protein-bound. The protein-free peptides are then coupled with the electrochemical reporter to yield a signal readout inversely proportional to the amount of the captured target proteins. Since the only requirement of supermolecule formation is the incorporation of aromatic amino acids in the peptide sequence, this strategy is universally applicable to many protein-binding peptides. The generality and target specificity of the proposed method are successfully demonstrated in the assays of two kinds of target proteins: tumor necrosis factor-α and amyloid β 1-42 soluble oligomer, respectively. The feasibility of our method is also tested in the monitoring of tumor necrosis factor-α secretion activity of HL-60 cells. These results indicate that our method can have great use in protein detection in the future.
Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family regulates their ubiquitylation activity. However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that the C2-WW-HECT ligase Smurf1 is activated by neddylation. Smurf1 physically interacts with Nedd8 and Ubc12, forms a Nedd8-thioester intermediate, and then catalyses its own neddylation on multiple lysine residues. Intriguingly, this autoneddylation needs an active site at C426 in the HECT N-lobe. Neddylation of Smurf1 potently enhances ubiquitin E2 recruitment and augments the ubiquitin ligase activity of Smurf1. The regulatory role of neddylation is conserved in human Smurf1 and yeast Rsp5. Furthermore, in human colorectal cancers, the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12 correlates with cancer progression and poor prognosis. These findings provide evidence that neddylation is important in HECT ubiquitin ligase activation and shed new light on the tumour-promoting role of Smurf1.
SUMMARY Neurotransmission is ensured by a high concentration of neurotransmitter receptors at the postsynaptic membrane. This is mediated by scaffold proteins that bridge the receptors with cytoskeleton. One such protein is rapsyn (receptor-associated protein at synapse), which is essential for acetylcholine receptor (AChR) clustering and NMJ (neuromuscular junction) formation. We show that the RING domain of rapsyn contains E3 ligase activity. Mutation of the RING domain that abolishes the enzyme activity inhibits rapsyn- as well as agrin-induced AChR clustering in heterologous and muscle cells. Further biological and genetic studies support a working model where rapsyn, a classic scaffold protein, serves as an E3 ligase to induce AChR clustering and NMJ formation, possibly by regulation of AChR neddylation. This study identifies a previously unappreciated enzymatic function of rapsyn and a role of neddylation in synapse formation, and reveals a potential target of therapeutic intervention for relevant neurological disorders.
Insulin-like growth factor-I receptor (IGF-IR) has been implicated in cancer pathophysiology. Furthermore, impairment of IGF-IR signaling in various cancer cell lines caused inhibition of the transformed phenotype as determined by the inhibition of colony formation in soft agar and the inhibition of tumor formation in athymic nude mice. Thus, the IGF-IR might be an attractive target for cancer prevention. We showed that the tea polyphenol, (À)Àepigallocatechin-3-gallate (EGCG), is a small-molecule inhibitor of IGF-IR activity (IC 50 of 14 Mmol/L). EGCG abrogated anchorageindependent growth induced by IGF-IR overexpression and also prevented human breast and cervical cancer cell phenotype expression through inhibition of IGF-IR downstream signaling. Our findings are the first to show that the IGF-IR is a novel binding protein of EGCG and thus may help explain the chemopreventive effect of EGCG on cancer development. (Cancer Epidemiol Biomarkers Prev 2007;16(3):598 -605)
Yes-associated protein (Yap) is a major effector of the Hippo pathway that regulates cell proliferation and differentiation during development and restricts tissue growth in adult animals. However, its role in synapse formation remains poorly understood. In this study, we characterized Yap's role in the formation of the neuromuscular junction (NMJ). In HSA-Yap Ϫ/Ϫ mice where Yap was mutated specifically in muscle cells, AChR clusters were smaller and were distributed in a broader region in the middle of muscle fibers, suggesting that muscle Yap is necessary for the size and location of AChR clusters. In addition, HSA-Yap Ϫ/Ϫ mice also exhibited remarkable presynaptic deficits. Many AChR clusters were not or less covered by nerve terminals; miniature endplate potential frequency was reduced, which was associated with an increase in paired-pulse facilitation, indicating structural and functional defects. In addition, muscle Yap mutation prevented reinnervation of denervated muscle fibers. Together, these observations indicate a role of muscle Yap in NMJ formation and regeneration. We found that -catenin was reduced in the cytoplasm and nucleus of mutant muscles, suggesting compromised -catenin signaling. Both NMJ formation and regeneration deficits of HSA-Yap Ϫ/Ϫ mice were ameliorated by inhibiting -catenin degradation, further corroborating a role of -catenin or Wnt-dependent signaling downstream of Yap to regulate NMJ formation and regeneration.
Mucociliary epithelium lining the upper and lower respiratory tract constitutes the first line of defense of the airway and lungs against inhaled pollutants and pathogens. The concerted beating of multiciliated cells drives mucociliary clearance. Abnormalities in both the structure and function of airway cilia have been implicated in obstructive lung diseases. Emerging evidence reveals a close correlation between lung diseases and environmental stimuli such as sulfur dioxide and tobacco particles. However, the underlying mechanism remains to be described. In this review, we emphasize the importance of airway cilia in mucociliary clearance and discuss how environmental pollutants affect the structure and function of airway cilia, thus shedding light on the function of airway cilia in preventing obstructive lung diseases and revealing the negative effects of environmental pollutants on human health.
Based on small molecule-linked DNA and the nicking endonuclease-assisted amplification (NEA) strategy, a novel electrochemical method for protein detection is proposed in this work. Specifically, the small molecule-linked DNA (probe 1) can be protected from exonuclease-catalyzed digestion upon binding to the protein target of the small molecule, so the DNA strand may hybridize with another DNA strand (probe 2) that is previously immobilized onto an electrode surface. Consequently, the NEA process is triggered, resulting in continuous removal of the DNA strands from the electrode surface, and the blocking effect against the electrochemical species [Fe(CN)(6)](3-/4-) becomes increasingly lower; thus, increased electrochemical waves can be achieved. Because the whole process is activated by the target protein, an electrochemical method for protein quantification is developed. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 0.3 to 15 ng/mL with a detection limit of 0.19 ng/mL. Furthermore, because the method can be used for the assay of FR in serum samples and for the detection of other proteins such as streptavidin by simply changing the small molecule moiety of the DNA probes, this novel method is expected to have great potential applications in the future.
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