The Role Of An Antifreeze Protein In The Formation Of Stress Granules In Cells Under Cold Stress

The Antarctic fishes evolved a series of strategies to adapt to the freezing environment, and among which, the antifreeze proteins(AFPs) are essential for their survival. The AFP protein family in fishes are diverse in composition and structure. Typically, the Type III AFPs are molecules contain 64 amino acids with a globular structure. They are abundantly expressed in Antarctic eelpouts. In past years, our laboratory identified a 2.87 Kb cDNA cloning, ld12 which encode a protein with 12 domains of the regular AFPIII. We then cloned a type III AFP gene that encodes 4 AFPIII molecules linked in tandem by a regular 9-aa peptide. Previous researches indicated that this protein are capable of conferring cold tolerance to tobacco at 4oC when AFPs are not supposed to function.Fish AFPs are known to function by a mechanism termed as ‘absorption-inhibition’, by which the antifreeze molecules bind with ice and inhibit ice-growth. In the conventional sense, antifreeze proteins protect the fishes from freezing damage by a physical interaction with the ice, rather through any biochemical functions in the physiology. The mechanisms by which LD4 confers cold tolerance to other organisms at a cold but non-freezing temperature is unclear.Recent researches reported that low temperatures induce cells to form stress granules, which assume a protective effect under cold stress. We thus asked whether LD4 plays a role in stress granule formation.We cloned ld4 into an expression vector and transfected the plasmid into HeLa cells(human cervical cancer cell). Through Reverse Transcription PCR(RT-PCR) and Western Blot, we verify that ld4 was successfully expressed in HeLa cells. We surveyed the survival rate of the transfected cells under cold stress(10℃) by trypan blue staining, and found that transfected cells are higher in survival rate than the control cells. Subsequently, we assayed the extent of stress granule formation and showed that compared with the To reveal the mechanisms underlie the reduced stress granule formation in Ld4 expressing cells, we checked the levels of phosphorylated eIF2 a, a critical trigger for stress granule formation in cells. We found that the ld4 expressing cells had significantly reduced levels of phosphorylated eIF2 a under the cold stress, suggesting that LD4 functions to reduce stress formation by reducing phosphorylation of e IF2 a. The results implied that LD4 may have raised the threshold of stress granule formation in cells exposed to cold stress and thus promoting the regular growth and proliferation of the cells.