Celluar Location Of Apoptosis Related Protein 3 And Its Role In Cell Apoptosis And Autophagy

Aim: To explore the subcellular location of a novel lysosomal membrane protein candidate apoptosis related protein 3(APR3), and to investigate its role in cell apoptosis and autophagy.Methods: Bioinformatics analysis was performed to predict the presence of signal peptide, transmembrane domain and glycosylation on APR3. Real-time PCR and western blot were used to detect APR3 in various mouse tissues and multiple cell lines. Lysosomes were isolated and purified from mouse liver by Percoll sucrose density gradient centrifugation technique; then western blot was performed to determined the abundance of APR3 protein amd lysosome associated membrane protein-1(LAMP1) in subcellular components and lysosomes. Using specific anti-APR3, anti-LAMP1 antibodies or Lyso-Tracker Red probe, the subcelluar location of APR3 was determined by cytoimmunofluorescence, further, a exogenous APR3 expression plasmid(pFUGW-APR3) was constructed and used to verified its subcellular location. NIH 3T3 cells were transfected with APR3 expression plasmid(pFUGW-APR3) and/or APR3 gene silencing vector(pLL3.7-APR3) for 48 hours, and then CCK-8 and xCELLigence real-time cell analyser was used to determined cell proliferation activity; flow cytometry was used to exam the progression and distribution of cell cycle and cell apoptosis; live cell lysosomal membrane permeability(LMP)/integrity acridine orange fluorescence detection kit was used to determined cell lysosomal membrane permeability, Lyso-Sensor probe to indirectly reflect lyosomal luman pH; Cell lysosome extraction was processed using ThermoScientific Lysosomal Enrichment Kit, the enriched lysosomal pelltes were solubilized using 1×detergent solution, and then GENMED Active Fluorescence Quantitative Detection Kit was used to determined the activity of a variety of lysosomal acid hydrolase, such as Cathepsin B, acid phosphatase, acid lipase and β-galactosidase; cell morphological changing was evaluated by transmission electron microscope scanning; westen blot was used to assess pro-apoptotic proteins(BAX, BAD, and cleaved caspase 3), anti-apoptotic proteins(BCL-2) and autophagy marker proteins(Beclin 1 and Map1LC3B). The influence of ARP3 on the expression of phos-mTOR and p62 after small dose rapamycin intervention was also observed.Results: Bioinformatics analysis revealed that APR3 is a single-pass trans-membane protein containing a signal peptide, 3 putative N-glycosylation sites(NxS/T) and 3 potential tyrosine-based sorting signals(NPLK, YTAI, and YKCM). APR3 was widely expressed in various mouse tissue and multiple cell lines, especially high in spleen, kidney, liver, lung, adipose tissues and some tumor cell lines such as SMMC-7221. Western blot analysis of subcellular fractions showed that endogenous APR3 shares a similar distribution pattern with LAMP1 which is a maker of the lysosomal integral membrane protein; this unique distribution pattern of APR3 and LAMP1 was further supported by co-localization of this two protein on lysosomes showed by confocal double immunofluorescence staining of APR3 and LAMP1, and APR3 and Lyso-Tracker Red; A similar subcellular location of exogenous APR3 was verified by immunofluorescence double staining of APR3 with Lyso-Tracker Red; Exogenous APR3 protein was also co-located with Lyso-Tracker Red, even though some exogenous APR3 appeared on cell membrane.APR3 eukaryotic expression vector(pFUGW-APR3) and APR3 gene silencing vector(pLL3.7-APR3) were successfully constructed and verified. In NIH 3T3 cells, APR3 overexpression significantly reduced cell proliferation activity, increased LMP, decreased lysosomal lumen pH, and enhanced lysosome acidification capacity; in contrast, APR3 gene silencing decreased LMP and increased lysosomal lumen pH; cathepsin B activity increased in APR3 overexpression cells, but not gene silencing cells; the activity of other lysosomal acid hydrolases, such as lysosomal acid phosphatase(LAP), lysosomal acid lipase(LAL) and β-galactosidase(β-gal) was increased in APR3 overexpression cells, with no change or slightly decrease in APR3 gene silencing cells.In NIH 3T3 cells, APR3 overexpression by tranfection of APR3 plasmid for 48 hours APR3 overexpression resulted in impaired cell cycle progression and cell cycle arrest at G1/S phase, and this blunted cell cycle progression was accompanied by a dramatic decrease in Cyclin D1; at the same time, APR3 over expression leaded to increased fraction of apoptotic cells as revealed ty annexin V-FITC/PI staining and flow cytometry; accordingly, pro-apoptotic proteins BAX and BAD were upregulated, and also cleaved caspase 3, in APR3 overexpressing cells, simultaneously, the autophagy-related marker proteins Beclin 1 and Map1LC3 B were also elevated in cells overexpressing APR3; transmission electron microscope scanning demonstrated a increase in the number and size of autolysosome in APR3 overexpressing cell, apart, some cells shrinked and condensed, nuclear chromatin marginated, nuclear envelope disassembled, and the cell surface formed a lot of germinal vesicle; after incubation with rapamycin(100nM) the increase of BAX, Map1LC3 B and Beclin 1 protein in APR3 overexpressing cells was much significant than cells without rapamycin; APR3 overexpression further reduced phos-mTOR and p62 expression after rapamycin stimulation.Conclusion: APR3 is a novel glycosylated integral membrane protein of lysosome; ARP3 overexpression reduces cell proliferation activity, inhibits cyclin D1 expression and results in cell cycle arrest at G1/S phase, leads to elevated level and coexistence of apoptosis and autophagy; ARP3 overexpression also increase lysosomal LMP, decrease luman pH value, and upregulate lysosomal acid hydrolases activity, such as CTSB, LAP, LAL and β-gal; APR3 overexpression also increase cell sensitivity to rapamycin stimulation which at least partly through mTOR and p62 signal pathway.