Activation of Autophagy of Aggregation-prone Ubiquitinated Proteins by Timosaponin A-III

Abstract

Chemical modulators of autophagy provide useful pharmacological tools for examination of autophagic processes, and also may lead to new therapeutic agents for diseases in which control of cellular sequestration and degradation capacity are beneficial. We have identified that timosaponin A-III (TAIII), a medicinal saponin reported to exhibit anticancer properties and improve brain function, is a pronounced activator of autophagy. In this work, the salient features and functional role of TAIIIinduced autophagy were investigated. In TAIII-treated cells, autophagic flux with increased formation of autophagosomes and conversion into autolysosomes is induced in association with inhibition of mammalian target of rapamycin activity and elevation of cytosolic free calcium. The TAIII-induced autophagy is distinct from conventional induction by rapamycin, exhibiting large autophagic vacuoles that appear to contain significant contents of endosomal membranes and multivesicular bodies. Furthermore, TAIII stimulates biosynthesis of cholesterol, which is incorporated to the autophagic vacuole membranes. The TAIII-induced autophagic vacuoles capture ubiquitinated proteins, and in proteasome-inhibited cells TAIII promotes autophagy of aggregation-prone ubiquitinated proteins. Our studies demonstrate that TAIII induced a distinct form of autophagy, and one of its pharmacological actions is likely to enhance the cellular quality control capacity via autophagic clearance of otherwise accumulated ubiquitinated protein aggregates.Autophagy, or precisely referred to macroautophagy, is a distinct lysosomal degradation pathway for intracellular materials (1, 2). Although the ubiquitin proteasome system (UPS) 2 usually degrades short-lived soluble proteins, autophagy complements UPS to degrade long-lived proteins, protein aggregates, or organelles (3-5). Autophagy is essentially a regulated intracellular vacuolization process consisting of two key steps: formation of autophagosomes that capture the cytosolic substrates, and fusion of autophagosomes and lysosomes to form autolysosomes in which the autophagic content is degraded. The membrane trafficking processes in autophagy are specifically controlled by proteins encoded by highly conserved autophagy related genes, ATGs, as well as the signaling lipid phosphatidylinositol 3-phosphate that recruits effector proteins to the membranes of autophagic vacuoles (6 -8).A major function of autophagy is to recycle free amino acids, lipids, and carbohydrates for resynthesis of biomolecules or energy production, particularly under starvation conditions. In this regard, autophagy was thought to be a nonspecific process involving random sequestration and degradation of cytoplasmic components, but it is now evident that cargo can also be selectively incorporated into autophagosomes (9). Emerging evidence has shown that autophagy may play a role in selective sequestration and degradation of aggregated protein complexes and/or damaged organelles, thereby serving as a cellular quality control system for ...

Timosaponin A-III reverses multi-drug resistance in human chronic myelogenous leukemia K562/ADM cells via downregulation of MDR1 and MRP1 expression by inhibiting PI3K/Akt signaling pathway

“…TAIII, one of the main active constituents of Chinese medicinal herb Anemarrhena asphodeloides Bunge, was reported to possess a variety of pharmacological functions. There is accumulating evidence that TAIII is a pronounced activator of autophagy (21,32,33). Preclinical study reported that TAIII was efficacious in inducing autophagy in various cancer cells correlating with a potent inhibitory effect on the activity of mammalian target of rapamycin (mTOR) (31)(32)(33).…”

“…Rhizoma Anemarrhenae (Zhimu in Chinese), a wellknown traditional Chinese medicinal herb, which is officially listed in the Chinese Pharmacopoeia, has been effectively used for febrile diseases in oriental clinical practices. Timosaponin A-III (TAIII), a steroidal saponin isolated from the rhizomes of Anemarrhena asphodeloides (AA), has been credited with a wide spectrum of bioactivities, including improving learning and memory (16), inhibiting inflammation (17), suppressing allergic reaction (18), controlling hyperglycemic (19,20), activating autophagy (21) and inducing cancer cells apoptosis (22)(23)(24). However, the ability of TAIII to reverse MDR has not been reported.…”

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

“…There is a keen interest in analyzing protein aggregates for several reasons: To elucidate how a protein’s primary and secondary structure determines its propensity to associate into larger aggregates, under either physiological or pathological conditions such as in neurodegenerative diseases (Alzheimer’s, Parkinson’s and Huntington’s diseases) associated with amino acid mutations 2–5 ;To map the post-translational oxidative modifications associated with pathologies that are known to induce protein aggregation, such as chronic inflammatory, metabolic and degenerative diseases and aging 6–8 ; andTo fractionate highly purified protein aggregates of different sizes and conformation in order to analyze the different cellular machinery associated with their refolding or disposal including chaperones, autophagy-related proteins and proteasome 9–15 .A recent review 16 that discussed the current proteomics prefractionation methods highlighted the emerging role and advantages of FFF, a flow-based separation technique invented in the 1960s by Giddings 17,18 . Flow FFF uses a ‘flow’ field to accomplish the separation, which consists of a stream of mobile phase that is applied orthogonally to the elution flow.…”

“…To fractionate highly purified protein aggregates of different sizes and conformation in order to analyze the different cellular machinery associated with their refolding or disposal including chaperones, autophagy-related proteins and proteasome 9–15 .…”

Rapid and Sensitive Determination of Timosaponin AIII in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Application

“…It was reported that TA-III could ameliorate learning and memory deficits in mice [1]. Another studies showed that TA-III had remarkable anticancer activity [2,3] and anti-respiratory syncytial virus (RSV) properties [4], which were also revealed to be a pronounced activator of autophagy [5]. …”