ABSTRACT
Accelerating the clearance of intracellular protein aggregates through elevation of autophagy represents a viable approach for the treatment of neurodegenerative diseases. In our earlier report, we have demonstrated the enhanced degradation of mutant huntingtin protein aggregates through autophagy process induced by europium hydroxide nanorods [EHNs: Eu(III)(OH)3], but the underlying molecular mechanism of EHNs mediated autophagy was unclear. The present report reveals that EHNs induced autophagy does not follow the classical AKT-mTOR and AMPK signaling pathways. The inhibition of ERK1/2 phosphorylation using the specific MEK inhibitor U0126 partially abrogates the autophagy as well as the clearance of mutant huntingtin protein aggregates mediated by EHNs suggesting that nanorods stimulate the activation of MEK/ERK1/2 signaling pathway during autophagy process. In contrast, another mTOR-independent autophagy inducer trehalose has been found to induce autophagy without activating ERK1/2 signaling pathway. Interestingly, the combined treatment of EHNs and trehalose leads to more degradation of mutant huntingtin protein aggregates than that obtained with single treatment of either nanorods or trehalose. Our results demonstrate the rational that further enhanced clearance of intracellular protein aggregates, needed for diverse neurodegenerative diseases, may be achieved through the combined treatment of two or more autophagy inducers, which stimulate autophagy through different signaling pathways.
Subject(s)
Europium/chemistry , Extracellular Signal-Regulated MAP Kinases/metabolism , Hydroxides/chemistry , Nanotubes/chemistry , Nerve Tissue Proteins/chemistry , Trehalose/chemistry , Adenine/analogs & derivatives , Adenine/chemistry , Androstadienes/chemistry , Animals , Autophagy , Autophagy-Related Protein 5 , Butadienes/chemistry , Cell Line, Tumor , Cell Survival , Chloroquine/chemistry , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Huntingtin Protein , Lysosomes/metabolism , Macrolides/chemistry , Mice , Microscopy, Fluorescence , Microtubule-Associated Proteins/metabolism , Neurodegenerative Diseases/embryology , Neurodegenerative Diseases/metabolism , Nitriles/chemistry , Phagosomes/chemistry , Phosphorylation , RNA, Small Interfering/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , WortmanninABSTRACT
TD1, a peptide chaperone consisting of the sequence ACSSSPHKHCG, has been shown to facilitate transdermal delivery for protein molecules via either co-administration or the fusion approach. We previously reported that a single TD1 motif, fused to the N-terminus of human epidermal growth factor (hEGF) can significantly enhance the transdermal efficiency of the recombinant EGF protein. In an effort to further increase the transdermal efficiency, we have created EGF fusion proteins harboring dual TD1 motifs: TD1-hEGF-TD1, containing one TD1 motif at both the N- and the Cterminus, and TD1-TD1-hEGF, containing two tandem TD1 motifs at the N-terminus. Both TD1-hEGF-TD1 and TD1- TD1-hEGF proteins, expressed in Escherichia coli and purified to apparent homogeneity, exhibited biological activity similar to unmodified hEGF, as revealed by their relative abilities to stimulate fibroblast growth, promote fibroblast migration, and activate the MAP kinase signaling cascade. On the other hand, both TD1-hEGF-TD1 and TD1-TD1-hEGF proteins exhibited a transdermal efficiency enhancement. The improvement was >5-fold compared to unmodified hEGF and 3-fold over the hEGF fusion protein with only one TD1 motif attached. These findings provided proof-of-concept for improving transdermal delivery of protein actives through rational protein design.
Subject(s)
Epidermal Growth Factor/administration & dosage , Epidermal Growth Factor/pharmacokinetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacokinetics , Administration, Cutaneous , Amino Acid Sequence , Animals , BALB 3T3 Cells , Epidermal Growth Factor/chemistry , Humans , Male , Mice , Peptides/administration & dosage , Peptides/chemistry , Peptides/pharmacokinetics , Rats, Sprague-Dawley , Recombinant Fusion Proteins/chemistry , Skin AbsorptionABSTRACT
Peptide chaperon TD1 was discovered to facilitate several proteins' transdermal delivery via topical co-administration. To design a practical, safe system for advanced transdermal peptide, a novel method was carried out. Human epidermal growth factor (hEGF) was selected as the model biological agent and a fusion protein: TD1-hEGF was designed. Study showed that TD1-hEGF not only had the similar bioactivity with native hEGF, but also possessed considerable higher transdermal ability than hEGF and a co-administration of TD1 and hEGF. These results provided convincing evidence for the advantages of TD1-hEGF in cosmetic and medical applications. Moreover, the fusion pattern between the cargoes and TD1 offered a new approach to facilitate other hydrophilic drugs' transdermal delivery for therapeutic application.
