Eukaryotic Initiation Factor 3e (eIF3e) Interacts With Huntingtin-associated Protein 1 And Regulates Outgrowth Of The Neurites

Eukaryotic initiation factors(e IFs)are proteins involved in the initiation phase of eukaryotic translation,including e IF1-e IF12,Its variety and complexity.Eukaryotic initiation factor is involved in the initiation of eukaryotic translation by interacting with each other,as well as the interaction between the eukaryotic initiation factor and the ribosome,m RNA,or the initiator t RNA.EIF3 is the largest and most complex eukaryotic initiation factor in mammals,made up of 13 subunits(a-m).eIF3 has proposed to play a critical role in regulation of protein translation initiation.Several studies have shown that a variety of eIF3 subunits such as eIF3 ba,eIF3 ha,and eIF3 m are involved in embryogenesis and neural development.The 5th subuint of eIF3,eIF3 e,is also known as INT6 for ‘mouse mammary tumor virus(MMTV)Integration site'.eIF3 e is conserved in evolution,which resides in most eukaryotes and is frequently expressed in human cells.eIF3 e has been shown to improve the level of histone translation and be involved in m RNA and protein degradation by strengthening the stability of eIF3 complex during translation.In embryonic mice,eIF3 e is located in the migrating neural crest cells,suggesting that it may affect the development of the nervous system.Huntingtin-associated protein 1(HAP1)was initially found to bind to the Huntingtin protein(Htt),encoded by the HD gene,which is widely expressed in the central nervous system and peripheral nervous system.Previous studies have showed that HAP1 was kown as a adaptin linking the microtubule motor proteins and transported substances was involved in intracelluar transport.Meanwhile,HAP1 was demonstrated to paly a significant role in occurrence and development of many nervous system diseases,brain development and neurite plasticity.Previous studies have demonstrated that there may be an interaction between eIF3 and HAP1,suggesting that eIF3 may be involved in development of neural cells by interacting with HAP1.1.eIF3 e interacting with HAP1 HAP1 can be divided into at least two isoforms HAP1 A and HAP1 B according to its difference at the C-end of the amino acid sequence.And HAP1 A exists in nerve cell body and neurite in the form of the punctiform or granular stigmoid body,while HAP1 B mainly exits in the cytoplasm in the form of diffusion.In order to further clarify whether eIF3 e interacts with HAP1,we first investigated whether eIF3 e co-located on HAP1A's stigmoid body.After we transfected the mouse neuroblastoma cells(N2a cells)and the cells expressed HAP1A-YFP and eIF3e-CFP,laser scanning confocal microscope found that a large number of eIF3 eCFP located on HAP1A-YFP stigmoid body.The expression of eIF3e-Myc and HAP1A-GFP was co transfected with HEK293 T cells,and the expression of eIF3 e was strongly binding to HAP1 A by immunoprecipitation analysis.Fluorescence resonance energy transfer(FRET)analysis demonstrated that there was obvious resonance energy transfer between the HAP1A-YFP and e JF3-CFP which were transfected and expressed in N2 a cells.And we transfected the original generation of cerebral cortex neurons in rats and the cells expressed HAP1A-YFP and eIF3e-CFP,laser scanning confocal microscope also found that a large number of eIF3e-CFP located on HAP1A-YFP stigmoid body.Immunofluorescence double staining observation was conducted on endogenous HAP1 and eIF3 e in hypothalamic slices of rat,numerous eIF3 e located on the HAP1 marked Stigmoid body.Further immune coprecipitation analysis showed that the HAP1 and eIF3 e in N2 a cells and mice hypothalamus have obvious interaction.2.Overexpression of eIF3 inhibiting the neurite growth Neurite growth is one of the signs of neurodevelopment.We observed the impact of eIF3e-CFP overexpression on neurite growth,in order to provide clues the relationship between eIF3 e and the neuron development.We transfected N2 a cells with eIF3e-CFP and induced them differentitian by serum-free mediun.The result showed that the neurites' length of N2 a cells which overexpressed eIF3e-CFP was obviously lower than that of N2 a cells only transfected control plasmid.That means,eIF3 e significantly inhibits the neurite growth of N2 a cells.we transfected the original generation of cerebral cortex neurons in rats with eIF3 e,the neurites' length which overexpressed eIF3 e was obviously lower than that only transfected control plasmid.That means,eIF3 e significantly inhibits the neurite growth.3.Overexpression of eIF3 e reducing the level of HAP1 HAP1 was demonstrated to promote the neurite growth.We measured the level of intracellular HAP1 by Western blot,in order to investigate whether eIF3 e which regulates protein translation and degradation and m RNA degradation inhibits the neurite growth by influencing the level of HAP1.The result showed that the level of HAP1 were reduced significantly in N2 a cells overexpressing eIF3 e.Further RT-PCR showed no obvious changes in the level of m RNA in N2 a cells overecpressing eIF3 e.The findings suggested that the overexpression of eIF3 e may be involved in the translation of HAP1 m RNA or the degradation of HAP1 that down-regulates the level of HAP1.4.Overexpression of HAP1 weakening the inhibitive effect of el F3 e on neurite growth We detected the impact of HAP1 overexpression on the inhibitive influence of el F3 e overexpression on neurite growth,in order to illuminate that el F3 e inhibites growth of neurites by descending the amount of HAP1.The result was that the neurites' length of N2 a cells and rat primary neurons which are cotransfected in el F3 e and HAP1 was obviously longer than that of N2 a cells and rat primary neurons which is only transfected in el F3 e.That means,HAP1 weakens the inhibitive effect of el F3 e on neurite growth,and el F3 e inhibites the growth of neurites by descending the amount of HAP1.Conclusion: This study showed that eIF3 e can interact with HAP1 which reduces the level of HAP1,and then inhibites the growth and development of neurites.