The Effects Of HPIP On The Function Of Autophagy In Tumor Cells And Its Interaction With LC3B

Objective:Autophagy is a unique intracellular protein degradation phenomenon in eukaryotic cells, which is essentially the macromolecules such as organelles and proteins degraded by autophagy lysosome, providing nutrients for cell survival and metabolism. As type II programmed cell death (type II programmed cell death), autophagy is highly conserved in organic evolution. Current study found that autophagy is involved in a variety of diseases, such as pathogen infection, neurodegenerative diseases, aging and tumor. Our study is aim at the effect of autophagy on tumor, exploring the function of HPIP (hematopoietic pre-B-cell leukemia transcription factor-interacting protein) on autophagy of tumor cells in vitro.Methods:Human LC3B coding region and the segments was amplified from human mammary gland cDNA by PCR, as well as the ATG4B coding region, and was inserted into the prokaryotic expression vector pGEX-KG and pET-28a(K+) to obtain the recombinant plasmids. Autophagy inductions were given to tumor cells in vitro, observing the co-localization of HPIP and LC3B in cells through immunofluorescence experiments. The localization of HPIP and LC3B was detected by the nucleo-cytoplasmic separation experiment. The function of HPIP and LC3B was detected by GST pull-down assay.Results:The interaction of HPIP protein and LC3B protein was confirmed by immunoprecipitation assay. The co-localization of HPIP and LC3B was detected by immunofluorescence. The recombinant plasmids of different coding regions inserted into pGEX-KG (A+) and pET-28a (K+) were successfully obtained. The effect of HPIP on autophagy was detected by western blot. Proteins of HPIP and ATG4B, which have an interaction with each other may both act on LC3B.Conclusion:HPIP is involved in autophagy of tumor cells. Proteins of HPIP、LC3B and ATG4B have an interaction with one another. HPIP may play an important role in ATG8 ubiquitin conjugation system, hindering the process of LC3B precursor protein Pro-LC3B converting to LC3B-I, which is cleaved by ATG4B.