TBC1D8B Affects Glucose Metabolism And Proliferation Of Tumor Cells Through Vesicle Trafficking

Vesicle transport is a conservative mechanism of intracellular transport,which is responsible for the material transport,cytokinesis,organelle biogenesis and its location in the normal situations.However under some pathological conditions,the abnormality within the signaling pathways of vesicle transport or/and the proteins involved in regulating vesicle transport are closely related to the metabolic imbalance,immune deficiency,tumor development.Rab GTPases act as an important molecular switch in vesicle transport and their activities are strictly regulated by guanine nucleotide exchange factors（GEFs）and GTPase activating proteins（GAPs）in cells:switch between the inactive form of Rab-GDP and the active form of Rab-GTP.Between them,the GAP of Rab GTPases is mainly composed of the TBC family proteins,which catalyze the hydrolysis of GTP into GDP and thus inactivate Rab.At present,more than 40 TBC proteins have been predicted within mammalian genome,and about 20 RAB substrates have been identified functioning in the specific biological processes.TBC1D8B is a widely expressed TBC protein in human tissues.However,the specific functions and molecular mechanisms of TBC1D8 B are still remained unclear.Recent studies have suggested that the hemizygous mutation of TBC1D8 B affects RAB11-dependent vesicle transport,resulting in an abnormal vesicle transport and localization of nephrin,a fissured membrane protein specifically expressed in glomerular podocytes,and leading to the occurrence of nephrotic syndrome.However,either the Human Protein Atlas database or the TCGA database has indicated that TBC1D8 B can express in other normal non-renal tissues as well as the tumor tissues such as breast cancer,cervical cancer,skin melanoma and so on.So in the other non-renal cells,does TBC1D8 B still regulates the Rab11-dependent vesicle transport?What are the cargo proteins it transports and what is its biological function? In our current study,using the Hela cells,a series of functional experiments such as Co-IP,immunofluorescence,glycolysis detection,etc.,have been carried out to identify the interacting proteins and the biological functions of TBC1D8 B.The main results of this paper are as follows:1.The involvement of TBC1D8 B in vesicle transport of JUPTo verify that TBC1D8 B should be also involved in the vesicle transport in the non-renal cells,we demonstrated that TBC1D8 B can co-locate with the membrane organelles including endoplasmic reticulum and Golgi apparatus in Hela cell line.Then,the Co-IP Combined mass spectrometry identified the two candidate proteins that interacted with TBC1D8 B,including ACAP2 and JUP.It was further confirmed that TBC1D8 B directly binds to JUP,a plakoglobin localized in the adherens junctions and desmosomes.It was also found that the increased expression of TBC1D8 B inhibited the expressional level of JUP and changed its subcellular localization,leading to a fibroblastic morphology and the reduced cellular adherion.2.The up-regulated expression of TBC1D8 B is related to the biological effects of tumor cell developmentTo study the effects of TBC1D8 B on the biological effects of tumor cells development,it was found that overexpression of TBC1D8 B increased the extracellular acidification rate,and the m RNA of glucose transporter（GLUT）,pyruvate kinase isozymes M2（PKM2）and lactate dehydrogenase A（LDHA）that are related to glucose metabolism and lactic acid production were up-regulated,suggesting that the overexpression of TBC1D8 B can induce aerobic glycolysis of tumor cells.In addition,overexpression of TBC1D8 B promotes Hela cell proliferation and accelerates the cell cycle.3.The effect of TBC1D8 B on the above biological processes depends on the GAP activityThe TBC domain is a functional domain that catalyses the hydrolysis of Rab-GTP and is also a key domain to fulfil the activity of GAP or regulate the vesicle transport.To validate whether the changes in the above biological processes caused by overexpression of TBC1D8 B depend on the vesicle transport function or GTP hydrolysis function of TBC1D8 B,we constructed cell lines expressing TBC domain functional inactivation,i.e.,active amino acid point mutation in the catalytic center TBC1D8B（RQ/AA）and TBC domain deletion mutation TBC1D8B（Del TBC domain）.Glycolysis analysis and Ed U proliferation detection of the TBC1D8 B mutants showed that the glucose metabolism remodeling and promotion of proliferation both depended on GAP activity,and the abnormal JUP and changes in cell morphology caused by overexpression of TBC1D8 B also required GAP activity.The innovation of this paper:It is firstly suggested the interaction between TBC1D8 B and JUP in Hela cell line,and TBC1D8 B is involved in the biological process of tumor cell development,such as glucose metabolism remodeling and abnormal cell proliferation.