Expression of Drosophila Trehalose-Phosphate Synthase in HEK-293 Cells Increases Hypoxia Tolerance

Abstract

Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(؊)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(؊)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(؊) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O 2 ). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O 2 . The amount of Na ؉ -K ؉ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress.The sensitivity of organisms to hypoxia varies, with most mammals being extremely vulnerable to O 2 deprivation. Irreversible injury may occur in mammalian tissues within 5-10 min of severe hypoxia or ischemia, whereas animals such as the turtle, Pseudemys scripta elegans, and invertebrates such as Drosophila melanogaster can recover from hours of experimental O 2 deprivation without a trace of injury. Some of the reasons for tolerance to low O 2 are as follows. (a) Hypoxia induces a decrease in membrane permeability (i.e. ion "channel arrest") that dramatically reduces the energy costs of ion flux (1). (b) These organisms, especially the turtle, have an unusually well developed capacity for rapid entry into, and return from, metabolically depressed steady states using anaerobic metabolism to sustain reduced rates of energy turnover during hypoxia. Clearly, the net effect of this regulated metabolic depression is that it conserves fermentable fuel, reduces deleterious endproduct formation, and extends survival time. (c) The ability to re-allocate cellular energy to...

Trehalose, a Novel mTOR-independent Autophagy Enhancer, Accelerates the Clearance of Mutant Huntingtin and α-Synuclein

“…8F). HEK-293 cells, from which the T-REx 293 cells and stable inducible trehalose cells were generated, do not metabolize intracellular trehalose (30), consistent with suggestions that HEK-293 cells are derived from a rare neuronal cells present in the HEK primary culture.…”

Trehalose Is an Important Mediator of Cap1p Oxidative Stress Response in Candida albicans

“…19,20) The nonreducing disaccharide trehalose is found in many organisms, in which it accumulates dramatically under stress conditions. [21][22][23][24] We demonstrate here a relationship between trehalose and Cap1p. Our results showed that trehalose was an important mediator of Cap1p oxidative stress response in C. albicans.…”

Dehydration-induced tps gene transcripts from an anhydrobiotic nematode contain novel spliced leaders and encode atypical GT-20 family proteins

“…Zentella et al [8] and Vogel et al [31] were unable to confirm TPP activity encoded by tps genes from S. lepidophylla and A. thaliana, respectively, however. This is perhaps not surprising, since the similarity between the plant C-terminal TPS and OtsB sequences is slight; more extensive is the relatedness between insect C-terminal TPS sequences and OtsB (data not shown), and it is likely that these TPP-related sequences are functional since the Drosophila TPS1 protein alone is able to direct trehalose synthesis in human cells [32]. No relatedness to TPP is apparent in TPS sequences from nematodes, however: BLAST searches with Aav-TPS-2 C-terminal sequences (residues 886-1303) show strong matches with the equivalent regions of C. elegans TPS proteins, but no apparent similarity with TPP sequences from micro-organisms or plants, nor with plant or insect TPS proteins (data not shown).…”