Construction Of Modular Artificial Transmembrane Signal Transduction Systems Based On Chemically Synthetic Receptors

In living organisms,cells are separated from the surrounding environment by lipid bilayer membranes,and sense external signals through natural transmembrane proteinmediated signal transduction systems,thereby controlling internal catalytic cascades,Unlike ion channel proteins,signal transducers do not transmit signal molecules directly,but complete transmembrane signal transduction through a series of processes such as recognition with extracellular signal molecules,conformational transformation,and activation of downstream reactions.Over the past few decades,scientists have reported a number of biomimetic systems for signaling through the exchange of matter,such as artificial ion transmembrane channels and transporters.However,reports of artificial transmembrane signal transduction systems that do not rely on material transmission for information exchange are still rare.The signal input and signal output of this artificial transmembrane signal transduction system are chemically unrelated,and harmful molecules can be excluded from the membrane to a certain extent,so it has extremely important significance in the fields of drug delivery and intelligent sensing.Chemosynthetic receptor refers to the synthetic molecule synthesized by chemical technology,which can specifically recognize active molecules on the membrane or liposome membrane and produce specific effects in the interior.It has the unique advantages of strong design and transformation,diverse stimulus response characteristics and modular engineering design.At present,natural signal transmembrane proteins are difficult to purify and denatured in vitro,which brings many difficulties for their mechanism research and in vivo and in vitro application.Based on this,groups with similar functions of transmembrane protein domains are distributed in a form similar to natural transmembrane proteins by using chemosynthetic receptors as the skeleton to realize specific recognition and efficient transmission of chemical information,which will not only help people understand the working mechanism of natural signal transduction proteins,but also is expected to replace or rescue damaged physiological systems in nature.To achieve the treatment of related diseases.In order to simulate the working principle of natural signaling systems,we seek inspiration for the construction of artificial signaling systems from the signal transmission process:Natural transmembrane protein-G protein-coupled receptors(GRCRs),relying on their orderly and efficient diversified functional domains,can specifically bind to the "first messenger" and trigger the conformational transformation of transmembrane proteins,and then activate the internal enzymatic catalytic activities;Tyrosine kinase receptors(RTKs)dimerize after recognizing external signals to form phosphatase sites,which in turn activate the internal enzyme catalytic system.Inspired by this,we believe that the design of modular receptors should fully consider the signal recognition sites,membrane anchoring sites and catalytic sites of enzyme-like effects,so that the synergistic effect of the three can more accurately identify external signals and transform them into internal catalytic activities.The core of the molecular transmembrane translocation mechanism is the change of the hydrophilic and hydrophobic state of the signal recognition "head group".Azobenzene,one of the most classic molecular switches,can not only be complexed with cyclodextrin to change its physicochemical properties(e.g.water solubility),but also to detach from the cavity of α-CD using UV light or 1-amantadine hydrochloride,which provides us with a powerful candidate for the construction of signal transduction.Based on the transmembrane translocation mechanism,this paper aims to tightly and orderly link chemical groups with similar functions of natural transmembrane protein domains through covalent and non-covalent interactions,and anchor them stably on the lipid membranes.The transmembrane transmission of signals and the activity regulation of internal cascade catalytic reactions are realized through transmembrane translocation of receptors.In addition,based on the interaction between different transmembrane receptors,we also extend the signal transduction of a single vesicle to the communication between different vesicles,and successfully construct a vesicle communication system.The research details are as follows:1.Light-controlled transmembrane signal transduction for switchable hydrolysis of an RNA model substrateLight,which is characterized by high spatial and temporal resolution and rapid response,adjustable wavelength and intensity,undoubtedly is one of the best ways to construct stimuli-response artificial signal transduction system.In this work,we use transazobenzene/α-CD(tAzo/α-CD)as the light-responsive headgroup for responding to external signaling inputs;lithocholic acid(L)as the lipid-anchored group that can trap the signaling molecule in the vesicular membrane;1,4,7-triazacyclonane(TACN)as the pro-catalyst tailgroup for Zn2+-coordination to form ribonuclease-like catalysis sites.Next,tAzo-L-TACN/α-CD was embedded in the lipid membrane and 2hydroxypropyl-4-nitrophenyl phosphate(HPNP)as a typical RNA model substrate was encapsulated inside the vesicles to construct a light-controlled transmembrane signal transduction system.It was found that the azobenzene group undergo a conformational conversion from trans-conformation to cis-conformation(cAzo)under in-situ ultraviolet light irradiation,which can trigger the decomplexation of tAzo-L-TACN/αCD and the release of cAzo-L-TACN for transmembrane translocation.Subsequently,the resulting cAzo-L-TACN moves into the vesicular membrane,and meanwhile the TACN unit is forced to enter the water phase inside the vesicle.Finally,the TACN unit catches a Zn2+to form a ribonuclease-like site for hydrolyzing RNA model substrate—HPNP.In addition,the "ON/OFF"-switchable hydrolysis behavior of the tAzo-LTACN/α-CD signaling system were also achieved by UV and visible light-controlled reversible conformational conversion.This work provides a new strategy for transmembrane control of internal catalytic activities through external signals,and is expected to play a key role in endogenous enzyme manipulation and gene regulation using exogenous signals.In addition,the modular design allows for the design of more artificial signal networks by changing the recognition "head group" and the precatalytic "tail group" to orchestrate simulation models of higher-order cells.2.Activation of multienzyme cascade reactions by a supramolecular signal transduction systemIn some natural signal transduction pathways,when the cell receptors recognize the "first messenger",it does not directly trigger the downstream cascade reaction,but induces the production of the "second messenger" as a new chemical information to act on other protein kinases to change their catalytic activity.In addition,we found that,besides light response,non-covalent reversible binding of ligand-receptor and competitive binding of different ligands to the same receptor molecule are also widespread signaling responses in nature.Inspired by this,we constructed an artificial signal transduction system based on the non-covalent reversible binding of supramolecular macrocyclic "host" molecule-β-cyclodextrin(β-CD)and its "guest"molecules,as well as the competitive binding of different "guest" molecules to β-CD.And also,we introduced functional "second messenger" into the signal transduction system to achieve the reversible control of supramolecular signal response and the controlled release of encapsulated content.The Azo-L-dapdoH2/β-CD transducer was constructed with three essential structural modules:trans-azobenzene(Azo)as the supramolecular recognition headgroup in response to input signal;lithocholic acid(L)as the membrane anchoring group capable of trapping the transducer in the membrane;pyridine-oxime(dapdoH2)as the pro-enzyme catalysis endgroup.In addition,2naphthalene formylated 8-hydroxypyrene-1,3,6-trisulfonate trisodium(HPTS)was used as the catalyst substrate inside the vesicles.Once the addition of 1adamantanamine hydrochloride(ADA)to the vesicles,Azo-L-dapdoH2/β-CD might be dissociated by competitive host-guest binding.The resulting Azo-L-dapdoH2 can translocate freely into the lipid membrane,leading to the movement of pyridine-oxime unit towards the intra-vesicular solution for Zn2+coordination to form a carboxylate ester hydrolase-like effector site to generate 2-naphthalene carboxylic acid as a functional "second messenger".2-Naphthalene carboxylic acid can enter the vesicle membrane phase to serve as a surfactant to change membrane permeability,and finally leading to the controlled transmembrane inflow of glucose outside the vesicles and the activation of multienzyme cascade reactions inside the vesicles.3.A cascade signal transduction network based on vesicle-to-vesicle sytem for switching activity of transmembrane channelsIn living organisms,natural transmembrane protein-mediated cell-to-cell communication enables collective behavior,where the functions of multiple cells are coordinated through exchanges between "Sender" cells and "Receiver" cells,allowing them to function synergistically as a multicellular organization.The construction of chemically synthetic receptors-mediated vesicle-to-vesicle communication system is a key step for the design of artificial cell communities with collective behavior.Herein,we reporter a transmembrane receptor(Ad-PEG-C)with amantane(Ad)as the signal recognition "head group",PEG as the linker,and cholesterol as the membrane anchoring group.Then,Ad-PEG-C was embedded on the vesicle membrane phase to form the " Senders" community.The vesicles embedded with Azo-L-dapdoH2/β-CD in the membrane and encapsulated with ion channel precursor molecules are called"Receivers" community.It was found that when in the presence of "Senders" and"Receivers" at the same time,Ad-PEG-C seizes the β-CD of Azo-L-dapdoH2/β-CD through host-guest competitive interaction to release free Azo-L-dapdoH2 for transmembrane translocation,and then the resulting Azo-L-dapdoH2 binds to intervesicular Cd2+to hydrolyze the ion channel precursor(T-ester)inside the"Receivers" for generating a functional "second messenger"-T.Finally,T can selfassemble into ion channels in the membrane phase to complete ion transport across the membrane.Therefore,this vesicle-to-vesicle communication system based on Senders? AD-PEG-C and Receivers? Azo-L-dapdoH2 provides a simple and effective way for the future design to control signal input,signal transmission and signal output of real cells.