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Screening The Interaction Proteins Of TRPV1 Channel In Rat DRG Neurons By Using The Split-ubiquitin Based Membrane Yeast Two-hybrid System

Posted on:2020-04-09Degree:MasterType:Thesis
Country:ChinaCandidate:J ZengFull Text:PDF
GTID:2480305903957269Subject:Biochemistry and Molecular Biology
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The TRPV1 channel(Transient Receptor Potential cation channel subfamily V member 1)is a tetrameric ligand-gated non-selective cation channel.Unlike the voltage-gated ion channels,the TRPV1 channel is characteristic of multiple modes gating;it could be activated by a variety of physical and chemical stimuli such as noxious heat(>42°C),acid(p H<6),bradykinin,cannabinoids,jellyfish,spider toxins,capsaicin and so on.The TRPV1 channel is highly expressed in dorsal root ganglia(DRG),trigeminal ganglion(TG)neurons and the unmyelinated C fibers.For its function in physiology and pathophysiology,the TRPV1 channel is closely related to the perception and conduction of pain signals,and is the major molecular determinant of noxious heat induced pain and inflammation-induced hyperalgesia.Notably,the TRPV1 channel knockout mice are resistant to capsaicin and noxious heat induced pain,and have a phenotype of losing thermal allodynia under inflammatory conditions.Furthermore,pharmacological inhibition of TRPV1 by omega-9 fatty acids and AMG628 also showed analgesic effects in animal pain models.These evidences suggest that the TRPV1 channel is an promising target for managing pain.On the other hand,the physiological and pathological function of TRPV1 is subtly regulated by its interacting proteins.These TRPV1 interacting proteins(TRIPs)modulate channel's capsaicin sensitivity,membrane transport,desensitization kinetics,thermo activation and so on.However,the interacting proteins network for TRPV1 has not been fully understood so far,and the identification of novel TRIPs and clarifying their mechanisms of regulating the TRPV1channel would certainly advance our understanding of the channel's function in physiological and pathophysiological conditions,and more importantly,would promote developing drugs targeting the TRPV1channel.In this study,we constructed a cDNA library of the rat DRG neurons with a capacity of approximately 1.65×10~6cfu,and screened the library with TRPV1 channel as a bait in aim to identify novel TRIPs by using the split ubiquitin based membrane yeast two-hybrid system.We finally identified 14 novel candidate TRIPs of full-length.Among them,one candidate protein,the palmitoyltransferase zDHHC4,has caught our particular interest.zDHHC4 belongs to the DHHC palmitoyltransferase family whose classical function is to modify their interacting proteins by attaching a palmitate to given cysteines in their protein sequence.We confirmed the TRPV1-zDHHC4 interaction in HEK293T cells by Co-immunoprecipitation experiment,and the acyl-biotin exchange(ABE)experiment showed that zDHHC4 enhanced TRPV1palmitoylation.More imporantly,electrophysiological study showed that zDHHC4 down-regulated the current density of TRPV1 expressed in HEK293T cells but did not change channel's sensitivity to capsaicin.The follow-up study will explore the in-situ interaction of TRPV1 and zDHHC4 in the DRG neurons and the underlying function of zDHHC4 in pain,as well as the relationship between zDHHC4 modifying and regulating TRPV1 channel.In this study,a new TRPV1 channel interaction protein--zDHHC4was reported for the first time,and it was found that zDHHC4 can enhance the palmitoylation of TRPV1 channel,which is also the first time to find the palmitoylation modification of TRPV1 channel.In the heterogenous expression system,we found that the co-expression between zDHHC4 and TRPV1 channel can significantly reduce the current density of TRPV1 channel.This will lay a foundation for the subsequent improvement of the TRPV1 channel interaction protein regulatory network,and provide new ideas for the research and development of drug targeting the TRPV1 channel.
Keywords/Search Tags:Split-ubiquitin based membrane yeast two-hybridization, TRPV1, zDHHC4, interaction, palmitoylation
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