CN114736306A - Chemically regulated protease tool and matched substrate thereof - Google Patents
Chemically regulated protease tool and matched substrate thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and particularly provides a protease tool under chemical regulation and control and a matched substrate thereof. The protease tool comprises an HIV-1 protease mutant and a DmrB domain; the DmrB domain is connected with the HIV-1 protease mutant through a flexible peptide fragment; the HIV-1 protease mutants are obtained by mutating amino acid sites that mediate HIV-1 protease dimerization under natural conditions. The invention changes the protease tool from a heterodimerization form to a homodimerization form, reduces two protease tool elements required to be transferred into the cell experiment from one protease tool element to two protease tool elements, shortens the length of the amino acid sequence of the protease tool, and has a simpler structure. In addition, when the protease tool provided by the invention is used together with a matched substrate, the control of signal paths of artificial signal input in mammalian cells can be realized.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a protease tool under chemical regulation and control and a matched substrate thereof.
Background
The artificial intervention is carried out on the signal path in the cell by a synthetic biology means, and the artificial control on the cell behavior is realized, so that the artificial control has important clinical diagnosis and treatment values. Proteases have the function of altering the state of a protein substrate by hydrolyzing peptide bonds, including inactivating the hydrolyzed protein substrate and removing inhibitory domains originally present in the substrate to confer downstream activities on the protein substrate. Thus, proteases may be used as an effective molecular tool in the intervention of intracellular signal transduction. In order to artificially control the hydrolysis activity of the protease, engineering the protease is a relatively common technical means. The current general strategy is to cleave the intact protease into two domains and then to link the two protease domains separately to inducible heterodimerization domains in a flexible manner. The six split inducible proteases which have been published at present are respectively modified by the following proteases: tobacco etch virus protease (TEVp), turnip mosaic virus protease (TUMVp), tobacco vein mottle virus protease (TVMVp), sunflower mild mosaic virus protease (SuMMVp), soybean mosaic virus protease (SbMVp), and plum pox virus protease (PPVp).
Artificial signal input into the cell induces heterodimerization of the heterodimerization domains, which in turn allows the reassortment of the cleaved protease domains into active intact protease structures, restoring the proteolytic activity of the protease, and thus opening the downstream signaling process. The above-mentioned viral proteases are all endonucleases, and have accurate amino acid recognition cleavage sequences. The corresponding protease recognition cleavage sequence is introduced into the protease target substrate to form an artificial protease substrate, and the artificial protease substrate is matched with the corresponding protease to realize a specific intracellular signal control effect.
The existing protease tools which can respond to artificial signal regulation are realized based on the technical route of 'split protein'. This means that both of these regulatable protease tools must be activated in a heterodimeric fashion. In constructing such a protease tool, it is necessary to construct and optimize the split two-part protease domain separately, and in subsequent use, it is necessary to simultaneously transfect both heterologous two-part protease elements into mammalian cells. Therefore, the existing technical means for regulating and controlling the protease has certain limitations: firstly, the fact that the split protease is in a heterodimeric form limits the application of some efficient homodimeric protein domains to the construction of protease tools; and secondly, two protein domains need to be constructed, optimized and transfected simultaneously when each protease tool is constructed, so that the workload of the construction process and the effective packaging capacity occupied on the virus transfection vector are increased.
Based on the situation, the invention develops a homodimerization type protease tool which can be manually regulated by using a technical route based on the mutant natural homodimerization protease, and simultaneously develops a series of protease substrates which can be matched with the novel protease to realize different regulation functions in cells.
Disclosure of Invention
The invention aims to overcome the problem that the prior controllable protease technical means has certain limitation.
To this end, the present invention provides a chemically regulated protease tool comprising an HIV-1 protease mutant and a DmrB domain; the DmrB domain is connected with the HIV-1 protease mutant through a flexible peptide fragment; the HIV-1 protease mutants are obtained by mutating amino acid positions that mediate HIV-1 protease dimerization under natural conditions. The protease is subjected to dimerization activation after being induced by a chemical small molecule AP 20187.
Furthermore, in order to weaken the homodimerization activity of the natural HIV-1 protease and ensure that the mutant protease can restore the catalytic activity under the dimerization mediated by the connected chemical induction dimerization domain, the HIV-1 protease mutant is a T96A mutant or an N98D mutant; wherein the T96A mutant is a mutant obtained by mutating threonine at position 96 of HIV-1 protease into alanine; the N98D mutant is a mutant obtained by mutating 98 th asparagine of HIV-1 protease into aspartic acid.
Further, the amino acid sequence of the flexible peptide fragment is SGGGSGGGSGGG.
Furthermore, the DmrB structural domain can be connected to the most N end of the HIV-1 protease mutant and can also be connected to the most C end of the HIV-1 protease mutant, protease activities with different strengths can be generated by the two connection modes, and when the DmrB structural domain is connected to the N end of the HIV-1 mutant, the induced protease activity is weaker, and the background activity is low in the non-induced state; the protease activity after induction is stronger when the protease is connected to the C terminal, but the protease shows higher background activity in a non-induced state. When the Dmrb structural domain is connected to the N terminal of the HIV-1 mutant, the protease tool shows a better ratio of change of protease activity intensity before and after induction. Therefore, the direction of the connection of the DmrB domain and the HIV-1 protease mutant can be selected according to the requirement of the intensity of the proteolytic activity in practical application.
In order to realize the control of intracellular signal processes by the protease tool, the invention also provides a matched substrate of the protease tool, which comprises a Ub (R) -dK degradation tag, a protease tool recognition cleavage site, a target effector protein, a protease tool recognition cleavage site and 4 tandem PEST motifs which are sequentially connected from the N end to the C end.
The invention also provides a matched substrate of the protease tool, wherein the matched substrate comprises a substrate protein, and a protease tool recognition cleavage site is inserted into a natural protease recognition cleavage site of the substrate protein; the substrate protein is a protein which is naturally activated after being hydrolyzed by protease.
The invention also provides a matched substrate of the protease tool, wherein the matched substrate comprises a target effector protein, a protease tool recognition cleavage site and a CAAX sequence which are sequentially connected from an N end to a C end; wherein the CAAX sequence is a CAAX amino acid motif from the C-terminal end of the RAS2 protein.
Specifically, because HIV-1 protease has a plurality of different recognition cleavage sites, in order to realize higher hydrolysis efficiency of the protease on a substrate, the HIV-1 protease recognition sequence used in the substrate construction process is optimized and tested, the optimal recognition cleavage site is selected, and the amino acid sequence of the recognition cleavage site of the protease tool is VSFNFPQITL.
Specifically, the target effector protein is one of a fluorescent protein, a transcription factor with downstream transcription regulation activity, or a protein kinase with an interference effect on a cell endogenous signal transduction pathway.
Specifically, the substrate protein is Caspase3 protein or Gasderm D protein.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the chemically regulated protease tool provided by the invention changes the protease tool which can be regulated by an artificial signal from a heterodimerization form to a homodimerization form, so that the number of protease tool elements required to be transferred into cells is reduced from two to one during cell experiments, and the length of the amino acid sequence of the protease tool is also shortened. In comparison with the currently most commonly used heterodimeric protease tool developed based on tobacco etch virus protease (TEVp), the complete chemically inducible TEVp protease tool consists of two parts, an N-terminal TEV protease linked to the FKBP protein and a C-terminal TEV protease linked to the Frb protein, which have 244 and 230 amino acids in amino acid number, respectively, so that a total of 474 amino acid-sized proteins need to be transfected in order to use the TEV protease tool completely. The homodimeric protease tool constructed in the invention only needs to transfect one structural domain when in use, and the size of the chemical regulation type protease tool is only 222 amino acids. The more compact structure and smaller protein size allow the protease tool to occupy less available packaging capacity of the vector when used, and the protease tool provided by the invention is more advantageous in some complex artificial signaling pathway construction tasks that require intracellular transfection of multiple protein elements. When the protease tool provided by the invention is used together with various different specific substrates matched with the protease tool, various different intracellular signal processes such as intracellular fluorescent protein stability, fluorescent protein subcellular localization change, downstream target gene expression started by specific transcription factors, apoptosis, cell apoptosis and the like can be regulated and controlled through the chemical micromolecules AP20187, so that the control of signal paths in mammalian cells by manual signal input is realized.
The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a flow chart of screening different combinations of degradation factors in example 1 of the present invention.
FIG. 2 is a schematic diagram showing the protein sequence of the fluorescent protein reporter substrate constructed in example 1 of the present invention.
FIG. 3 is a flow chart of HIV-1 protease mutant screening and chemically regulated protease tool construction in example 1 of the present invention.
FIG. 4 is a schematic representation of the protein sequence of the chemically regulated protease tool of example 1 of the present invention; wherein, a is a schematic diagram when the DmrB structural domain is connected to the N terminal of the HIV-1 protease mutant, and b is a schematic diagram when the DmrB structural domain is connected to the C terminal of the HIV-1 protease mutant.
FIG. 5 is a schematic diagram of a substrate kit designed based on a protein naturally activated by proteolysis in example 1 of the present invention.
FIG. 6 is a schematic diagram of a complementary substrate designed based on the CAAX amino acid motif from the C-terminal end of RAS2 protein in example 1 of the present invention.
Reference numerals: 1. ub (R) -dK degradation tag; 2. a recognition cleavage site for a protease tool; 3. a target effector protein; 4. a PEST motif; 5. a DmrB domain; 6 flexible peptide segment; 7. HIV-1 protease mutants; 8. a CAAX amino acid motif.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made thereto without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The technical scheme of the invention specifically comprises two major parts, namely a homodimerization type chemical regulation protease tool which can be subjected to dimerization activation after being induced by a chemical micromolecule AP20187 and a series of matched protease substrates which can be hydrolyzed by the protease tool to generate state change. The substrates in the invention are three types, namely substrates which are hydrolyzed by protease and then converted from a degradable state into a stable state, substrates which are hydrolyzed by protease and then converted from a cytoplasmic membrane to a cytoplasmic matrix in subcellular localization, and cell fate control substrates which are hydrolyzed by protease and activated to trigger cell apoptosis or cell scorching.
The effects of the chemically regulated protease tool and its associated substrate of the present invention are investigated by the following specific examples.
Example 1:
among the natural viral proteases, the viral protease derived from HIV-1 (human immunodeficiency virus type 1) (HIV-1 protease) is a homodimeric protease, and the hydrolytic activity is generated by homodimerization of two identical subunits to form a complete catalytic center, and such natural structure meets the requirement of constructing a homodimeric protease tool, and the present example develops a protease molecular tool which can be artificially regulated based on the HIV-1 protease.
First, construct a fluorescent protein reporter substrate that can be used to reflect the intracellular activity of HIV-1 protease
In order to reflect the level of hydrolytic activity in mammalian cells in the subsequent protease tool construction process, it is first necessary to construct a reporter substrate that can express HIV-1 protease activity in mammalian cells. The development of such substrates based on fluorescent proteins would facilitate the quantitative analysis of signals produced by living cells using flow cytometry techniques or fluorescence microscopy imaging techniques. Therefore, in the embodiment, a fluorescent protein design connected with a degradation label is used, degradation factors are added at the N end or/and the C end of the fluorescent protein and are connected by HIV-1 protease cleavage sites, when HIV-1 protease is inactive, the degradation factors guide the degradation of the fluorescent protein, and when HIV-1 protease plays an active role, the degradation factors at two ends of a fluorescent protein substrate can be cut off, and the fluorescent protein can be stably existed in cells. In order to obtain the best reporting effect, the present example screens different degradation factor combinations, and the screening process is shown in fig. 1.
The experimental result shows that the combination of the N-terminal Ub (R) -dK degradation label and the C-terminal 4 tandem PEST motifs derived from I kappa B has the largest difference in the proportion of the activity of the cellular fluorescent protein before and after HIV-1 proteolysis, so that the degradation factor combination is selected as the basis for designing a substrate which is hydrolyzed by a protease tool and then is converted from a readily degradable state to a stable state, and the protein sequence composition of the fluorescent protein reporter substrate is shown in FIG. 2.
The fluorescent protein reporter substrate is a substrate which is converted from a degradable state into a stable state after being hydrolyzed by a protease tool, and a target effector protein in the substrate can be the fluorescent protein playing a reporting role, can also be a transcription factor with downstream transcription regulation activity, such as Tet3G, Gal4 and the like, and can also be protein kinase with an interference effect on a cell endogenous signal transduction pathway. In actual use, the required target effector protein can be replaced based on the substrate design principle provided by the invention according to the regulation function required to be realized, and the process of controlling the intracellular target effector protein to be converted from the off state to the on state through a protease tool is realized.
II, connecting the HIV-1 protease mutant with a chemical small molecule induced dimerization domain to screen the HIV-1 protease mutant and construct a chemically regulated protease tool
In the complete HIV-1 protease structure, the N-terminal and C-terminal amino acids of the two subunits form four antiparallel, interdigitated beta-sheet structures that form the terminal dimerization plane of HIV-1 protease, and thus the N-terminal and C-terminal of the subunits are the key regions that determine the spontaneous dimerization activity of the HIV-1 protease subunits under natural conditions. In order to construct a protease that can be regulated by artificial signal input, mutations or combinatorial mutations at key amino acid positions that mediate HIV-1 protease dimerization under natural conditions are required, thereby disrupting the natural dimerization of HIV-1 protease and disrupting the natural hydrolytic activity of HIV-1 protease. For this purpose, 12 HIV-1 protease mutants were screened in this example, as shown in Table 1.
TABLE 1 HIV-1 protease mutants screened during protease tool construction
| Name of mutation | Mutation position | The primary amino acid | Post-mutation amino acid |
| D25N | 25 | Aspartic acid (D) | Asparagine (N) |
| |
1 | Proline (P) | Alanine (A) |
| |
2 | Glutamine (Q) | Alanine (A) |
| |
3 | Isoleucine (I) | Alanine (A) |
| T96A | 96 | Threonine (T) | Alanine (A) |
| L97A | 97 | Leucine (L) | Alanine (A) |
| N98D | 98 | Asparagine (N) | Aspartic acid (D) |
| F99A | 99 | Phenylalanine (F) | Alanine (A) |
| 97&99A | 97 and 99 | Leucine and phenylalanine | Alanine (A) |
| Δ99 | 99 | Phenylalanine (F) | |
| I3A&Δ99 | |||
| 3 and 99 | Isoleucine and phenylalanine | Alanine at |
As shown in figure 3, the mutant HIV-1 protease is connected with a homodimerization protein structure domain DmrB induced by a chemical small molecule AP20187 through a flexible peptide segment to construct a chemical regulation and control protease tool to be screened, and the amino acid sequence connected with the flexible peptide segment is SGGGSGGGSGGG. The protease tool to be screened and the fluorescent protein reporter substrate are transfected into a human embryonic kidney cell line (HEK-293T cell line) together, and the change of the hydrolysis activity of the tool with different HIV-1 protease mutants before and after induction is carried out by adding chemical micromolecules through flow cytometry.
In order to attenuate the homodimerization activity of the native HIV-1 protease, while ensuring that the mutant protease can regain catalytic activity under dimerization mediated by the attached chemically-induced dimerization domain. According to the screening results of the HIV-1 protease mutant, the T96A mutant and the N98D mutant are determined as the optimal HIV-1 protease mutants for constructing a protease tool. The T96A mutant is a mutant for mutating threonine at the 96 th position of the HIV-1 protease into alanine, and the N98D mutant is a mutant for mutating asparagine at the 98 th position of the HIV-1 protease into aspartic acid.
Because of the spatial differences in the N-and C-termini of the two monomeric subunits upon dimerization of the HIV-1 protease, the two chemically inducible protease tools, which differ in the direction of attachment of the DmrB domains, will differ in hydrolytic activity. After determining the HIV-1 protease mutant used for constructing the protease tool under chemical regulation, the hydrolysis activity of the protease tool formed by connecting the protease mutant and a chemical induction homodimerization domain DmrB (DmrB domain is connected to the N end or the C end of the HIV-1 protease mutant before and after induction by chemical small molecules) is tested.
Experiments prove that the protease tool connecting DmrB to the C terminal of the HIV-1 protease mutant has relatively higher background hydrolytic activity under the condition of no chemical micromolecule induction, and also shows higher hydrolytic activity after induction by the chemical micromolecules. The protease tool connecting DmrB to the N terminal of the HIV-1 protease mutant shows very low background activity, the hydrolysis activity after induction is relatively low, and the ratio of the hydrolysis activity before and after induction of the chemical small molecules is integrally higher than that of the protease tool connecting DmrB to the C terminal.
The protein sequence organization of the chemically regulated protease tool is shown in FIG. 4.
Substrate matched with chemically regulated protease tool
In addition to the above-described fluorescent protein reporter substrates, this example also developed two types of matched substrates for chemically regulated protease tools.
1. Based on proteins naturally activated by proteolytic hydrolysis
The protein which is naturally activated after being hydrolyzed by protease mainly exists in signal processes of apoptosis, cell scorching and the like, generally, the protein has two subunits which take effect on response and inhibition, and a specific protease hydrolysis cleavage site is arranged between the two subunits. When hydrolyzed by upstream proteases, the inhibitory subunit separates from the effector subunit, the inhibitory effect is released, and the protein produces downstream effects.
Therefore, the recognition cleavage site of the protease tool (HIV-1 protease cleavage site) is inserted into the natural protease recognition cleavage site of the substrate protein, and the natural protease recognition cleavage site of the substrate is destroyed, and simultaneously, the substrate can be activated under the regulation of the artificially designed protease tool. This example contains two such types of complementary substrates, the structure of which is shown in FIG. 5.
2. Based on the "CAAX" amino acid motif from the C-terminal end of RAS2 protein
A protein containing a "CAAX" amino acid motif from the C-terminus end of the RAS2 protein in the C-terminus is recognized by farnesyl transferase and geranyl transferase after translation in mammalian cells, resulting in prenylation modifications from the cysteine sites on the motif, and the prenylated modified protein is anchored to the plasma membrane. In order to realize the regulation and control of the subcellular localization of target proteins in cells by a protease tool, the design of the matched substrates is as follows: a "CAAX" amino acid motif is added to the C-terminus of a particular target protein and is linked to the original protein via a recognition cleavage site of a protease tool (HIV-1 protease cleavage site). The protein substrate thus designed is localized to the cell membrane when the protease tool is not activated, and by artificially controlling the activation of the protease tool, it is possible to achieve a change in the subcellular localization of the target substrate released from the cell membrane into the cytoplasm or nucleus (if the target protein contains a nuclear localization sequence). The design of such a substrate is schematically shown in FIG. 6.
Fourth, recognition cleavage site optimization of protease tool
Because HIV-1 protease has a plurality of different recognition cleavage sites, in order to realize higher hydrolysis efficiency of the protease on a substrate, the HIV-1 protease recognition sequence used in the substrate construction process is optimized and tested, and the optimal recognition cleavage site with the amino acid sequence of VSFNFPQITL is selected.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims.
Claims (10)
1. A chemically regulated protease tool characterized by: the protease tool comprises an HIV-1 protease mutant and a DmrB domain; the DmrB domain is connected with the HIV-1 protease mutant through a flexible peptide fragment; the HIV-1 protease mutants are obtained by mutating amino acid sites that mediate HIV-1 protease dimerization under natural conditions.
2. The chemically modulated protease tool of claim 1, wherein: the HIV-1 protease mutant is a T96A mutant or an N98D mutant; wherein the T96A mutant is a mutant obtained by mutating threonine at position 96 of HIV-1 protease into alanine; the N98D mutant is a mutant obtained by mutating 98 th asparagine of HIV-1 protease into aspartic acid.
3. The chemically modulated protease tool of claim 1, wherein: the amino acid sequence of the flexible peptide fragment is SGGGSGGGSGGG.
4. The chemically modulated protease tool of claim 1, wherein: the DmrB structural domain is connected to the N end or the C end of the HIV-1 protease mutant through a flexible peptide segment.
5. A substrate for a kit according to any one of claims 1 to 4, wherein: the matched substrate comprises a Ub (R) -dK degradation label, a protease tool recognition and cleavage site, a target effector protein, a protease tool recognition and cleavage site and 4 tandem PEST motifs which are connected in sequence from the N end to the C end.
6. A substrate for a kit according to any one of claims 1 to 4, wherein: the matched substrate comprises a substrate protein, and a protease tool recognition cleavage site is inserted into a natural protease recognition cleavage site of the substrate protein; the substrate protein is a protein which is naturally activated after being hydrolyzed by protease.
7. A substrate for a kit according to any one of claims 1 to 4, wherein: the matched substrate comprises a target effector protein, a protease tool recognition cleavage site and a CAAX sequence which are sequentially connected from an N end to a C end; wherein the CAAX sequence is a CAAX amino acid motif from the C-terminal end of the RAS2 protein.
8. The substrate kit according to any one of claims 5 to 7, wherein: the amino acid sequence of the protease tool recognition cleavage site is VSFNFPQITL.
9. The substrate kit for a protease tool according to claim 5 or 7, wherein: the target effector protein is one of fluorescent protein, transcription factor with downstream transcription regulation activity or protein kinase with interference effect on cell endogenous signal transduction pathway.
10. The substrate kit for a protease tool according to claim 6, wherein: the substrate protein is Caspase3 protein or Gasderm D protein.
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