Patent Application
20220017599
This disclosure relates to composition and method for regulating immune cell migration.
The migration of immune cells is a key point of body immunity and host defense. It plays an important role in the effective immune surveillance and the homeostasis of the internal environment. It is a research hotspot and frontier subject in the field of life sciences. Immune cells migrate directionally from the blood to lymphatic organs and tissues through high endothelial venules (HEVs), including the homing of immune cells to peripheral lymphoid organs, or the directional migration to inflammation sites and pathological tissues. The homing of immune cells involves the coordination of multiple adhesion molecules on the surface of immune cells and vascular endothelial cells, consisting of a series of highly ordered adhesion events, namely, localization and rolling of immune cells on blood vessel walls, chemokines-dependent cell activation, stable adhesion, and finally transvascular endothelial migration and diapedesis. Among the many molecules that mediate the homing of immune cells, integrin is the most important homing receptor for immune cells, which facilitates precise regulation of immune cell adhesion and migration by interacting with corresponding integrin ligands expressed on the surface of blood vessel endothelium.
As an important cell adhesion molecule (CAM) expressed on the surface of cell membranes, integrin is a heterodimer composed of two subunits of α and β connected by non-covalent bonds. Integrins are widely distributed in organisms. At present, 18 different a subunits and 8 different β subunits have been found in vertebrates, which combine to form at least 24 integrins. Among them, α4 and β2 integrins, as the two main receptors that mediate the homing of immune cells, play a very important role in the adhesion, migration and diapedesis of immune cells on the vascular endothelium to secondary lymphoid organs, inflammation or tumor sites. The adhesion and migration of immune cells mediated by integrins is achieved by dynamic regulation of its ligand affinity. When the cell is not stimulated, the integrins on the cell surface are in a low-active conformation with a low ligand affinity. When the cell is stimulated, the relevant signaling pathways inside the cell are activated, and the regulatory protein inside the cell causes a series of conformational changes of integrins (from a low-activity folded conformation to a high-activity extended conformation) through inside-out signaling, leading to the activation of integrins. The activated integrins can bind to ligands with high affinity and mediate stable cell adhesion. On the other hand, the binding of extracellular ligands and the extracellular domains of integrins can also cause the overall conformational changes of integrins and the clustering of integrins on the cell surface, thereby recruiting intracellular signaling molecules and activating corresponding intracellular signaling pathways (outside-in signaling) to promote cell spreading and migration.
Fever is an evolutionarily highly conserved defense mechanism against injury. When the body is stimulated by pathogens, the temperature of the local inflammation site or the systemic temperature will increase significantly, which can effectively improve the survival of the organism under infection or inflammation. Specifically, fever is a complex physiological response of the body to external infections and stimuli (such as bacterial endotoxin, inflammation, and injury). Fever is generally induced by locally released pyrogenic cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), etc. In warm-blooded animals, these pyrogenic cytokines can affect the hypothalamus to increase the threshold of temperature regulation, and initiate a series of biochemical, physiological and behavioral responses, which eventually lead to an increase in body temperature. A large number of research reports have confirmed that fever-range thermal stress is closely related to the enhancement of the innate immunity and adaptive immunity under stress conditions. One of the important functions is to promote the migration of immune cells to secondary lymphoid organs or inflammatory sites, thereby promoting immune response and tissue repair. Scholars generally believe that the fever-range hyperthermia changes the dynamics of blood flow by dilating blood vessels to promote the recruitment of immune cells to tissues such as inflammation sites. However, recent studies have found that fever-range thermal stress plays a more active role in directing migration of immune cells to secondary lymphoid organs or inflammation sites. The main research results are: fever-range thermal stress promotes the expression of intercellular adhesion molecules ICAM-1 and chemokine CCL21 of the cells on the surface of capillary hyperendothelial veins, thereby enhancing the integrin LFA-1-dependent diapedesis of immune cells across capillary hyperendothelial veins. During this process, the IL-6/sIL6-Rα signaling pathway plays an important role in the increased expression of ICAM-1 induced by fever. In summary, current understanding of the regulation of immune cell migration by fever is limited to the vascular system, but little is known about the regulation of fever on immune cells themselves and its mechanism.
Interestingly, studies have found that fever-range thermal stress can directly act on immune cells, selectively promoting α4 integrin-mediated adhesion and migration, but does not affect the function of β2 integrin. However, these studies are limited to phenotype, but not related to the molecular mechanism.
The present disclosure provides a method of regulating the migration of immune cells, comprising the step of enhancing or weakening the interaction between Hsp90 and α4 integrin in immune cells.
In one or more embodiments, the method includes enhancing the interaction between Hsp90 and α4 integrin in immune cells by any one or more of the following means: (1) overexpressing Hsp90 protein and/or Hsp90 mutein in the immune cells, wherein, compared with the wild-type Hsp90 protein, the Hsp90 mutein is only mutated in its middle domain, or the Hsp90 mutein has a mutation causing its inability to self-dimerize; (2) overexpressing α4 integrin in the immune cells.
In one or more embodiments, the method includes weakening the interaction between Hsp90 and α4 integrin in immune cells by any one or more of the following means: (1) knocking out Hsp90 protein or knocking down its expression in immune cells; (2) knocking out α4 integrin or knocking down its expression in immune cells; (3) expressing Hsp90 mutein that has weakened or no interaction with α4 integrin in wild-type immune cells or immune cells with Hsp90 protein knocked out; (4) expressing α4 integrin mutein that has weakened or no interaction with Hsp90 protein in wild-type immune cells or immune cells with α4 integrin knocked out.
In one or more embodiments, treating immune cells with an expression vector and/or an integration vector of Hsp90 protein and/or Hsp90 mutein, and/or treating immune cells with an expression vector of α4 integrin and/or an integration vector, and/or treating immune cells with reagents that increase the transcription levels of Hsp90 and/or α4 integrin naturally occurred in immune cells, and/or placing immune cells at a fever-range hyperthermia, thereby enhancing the interaction between Hsp90 and α4 integrin in immune cells.
In one or more embodiments, the interaction between Hsp90 and α4 integrin in immune cells is weakened or destroyed through: knocking out Hsp90 protein or α4 integrin from immune cells by transferring a gene-knockout vector into the immune cells, and/or knocking out Hsp90 protein or α4 integrin from immune cells by ZFN, TALEN or CRISPR/Cas9 and the like, and/or knocking down the expression of Hsp90 protein and/or α4 integrin by interfering-RNA-mediated gene silencing, and/or integrating into the genome of immune cells an expression cassette expressing the Hsp90 mutein that has weakened or no interaction with α4 integrin and/or an expression cassette expressing the α4 integrin mutein that has weakened or no interaction with Hsp90 protein by transferring a gene-insertion vector into immune cells while knocking out the coding sequence of wild-type Hsp90 and/or α4 integrin.
In one or more embodiments, the Hsp90 mutein that has weakened or no interaction with α4 integrin lacks its N-terminal and/or C-terminal domain compared with the wild-type Hsp90 protein.
The present disclosure also provides a genetically engineered immune cell, wherein, compared with the wild-type immune cells, the genetically engineered immune cell has an enhanced or weakened interaction between Hsp90 and α4 integrin.
In one or more embodiments, the genetically engineered immune cell: (1) overexpresses Hsp90 protein and/or Hsp90 mutein, wherein, compared with wild-type Hsp90 protein, the Hsp90 mutein is only mutated in its middle domain, or the Hsp90 mutein has a mutation causing its inability to self-dimerize; (2) overexpresses α4 integrin.
In one or more embodiments, the genetically engineered immune cell: (1) does not express Hsp90 or has reduced expression level of Hsp90, or expresses Hsp90 having reduced activity, or expresses Hsp90 mutants, as compared with wild-type immune cells; and/or (2) has reduced expression level of α4 integrin, or expresses α4 integrin mutants, as compared with wild-type immune cells, thereby weakening or eliminating the interaction between Hsp90 and α4 integrin in the genetically engineered immune cell. In one or more embodiments, the Hsp90 mutant lacks its N-terminal and/or C-terminal domain compared to the wild-type Hsp90 protein. In one or more embodiments, compared with the wild-type α4 integrin, the α4 integrin mutant has one or more amino acid residues mutated in its intracellular segment other than amino acid residues 968-974, causing its interaction with Hsp90 protein weakened or eliminated; preferably, there is a mutation at at least one of R985, W989 and Y991; preferably, the mutation is a substitution mutation; more preferably, the substituted amino acid residue is alanine.
The present disclosure also provides a pharmaceutical composition containing the genetically engineered immune cell described herein.
The present disclosure also provides a mutant Hsp90 protein selected from the group consisting of:
(1) a mutant Hsp90 protein, lacking the N-terminal domain and/or the C-terminal domain of the wild-type Hsp90 protein, or having a mutation in C-terminal domain causing its inability to mediate the self-dimerization of Hsp90 protein, as compared with the wild-type Hsp90 protein; and
(2) a mutant Hsp90 protein, only having a mutation in the middle domain as compared with the wild-type Hsp90 protein while maintaining the ability to bind to α4 integrin as compared with the wild-type; and
(3) a mutant Hsp90 protein, having a mutation causing its inability to self-dimerize as compared with the wild-type Hsp90 protein; preferably, the mutation occurs in the C-terminal domain of the Hsp90 protein; more preferably, the mutation is the deletion of the last 49 amino acids of C-terminus.
The present disclosure also provides a mutant α4 integrin, the mutant α4 integrin has one or more amino acid residues mutated in its intracellular segment other than amino acid residues 968-974, causing its interaction with Hsp90 protein weakened or eliminated; preferably, there is a mutation at at least one of R985, W989 and Y991; preferably, the mutation is a substitution mutation; more preferably, the substituted amino acid residue is alanine.
The present disclosure also provides a coding sequence of the mutant Hsp90 protein and the mutant α4 integrin described herein, or complementary sequence thereof, a nucleic acid construct containing the coding sequence or complementary sequence, and a host cell containing the nucleic acid construct.
The present disclosure also provides uses selected from the group consisting of:
(1) use of a reagent that enhances the interaction between Hsp90 and α4 integrin in immune cells in the manufacture of immune cells for immunotherapy; preferably, the reagent is selected from the group consisting of: an expression vector or an integration vector for the wild-type Hsp90 protein, or the mutant Hsp90 protein being only mutated in its middle domain, or the mutant Hsp90 protein having a mutation causing its inability to self-dimerize; and an expression vector and/or integration vector for α4 integrin;
(2) use of immune cells with enhanced interaction between Hsp90 protein and α4 integrin in the manufacture of a medicament for the treatment of pathogen infections or tumors;
(3) use of a reagent that weakens the interaction between Hsp90 and α4 integrin in immune cells in the manufacture of immune cells for the treatment of sepsis, hematological tumors, chronic inflammations or autoimmune diseases; preferably, the reagent is selected from the group consisting of: a Hsp90 mutant lacking the N-terminal and/or C-terminal domain or having a mutation in the N-terminal and/or C-terminal domain causing its interaction with α4 integrin weakened or eliminated as compared with the wide-type Hsp90, and/or a targeting vector of the Hsp90 mutant; a α4 integrin mutant having a mutation in its intracellular segment which causes its interaction with Hsp90 weakened or eliminated, and/or a targeting vector of the α4 integrin mutant; and ZFN, TALEN and/or CRISPR/Cas9 reagents and/or small interfering RNA used to knock out Hsp90 and/or α4 integrin or to knock down its/their expression; and
(4) use of immune cells with weakened interaction between Hsp90 and α4 integrin in the manufacture of a medicament for the treatment of sepsis, hematological tumors, chronic inflammations or autoimmune diseases.
It should be understood that, within the scope of the present disclosure, the above technical features of the present disclosure and the technical features specifically described in the following (e.g., Examples) can be combined with each other, thereby forming preferred technical solution(s).
Fever is an evolutionarily conserved defense process for organisms to respond to infection or injury, and can effectively improve survival. However, the detailed mechanism of fever is not very clear. Through a large number of biochemical, molecular and cellular experiments, the inventors confirmed that: fever-range thermal stress can promote the directional migration of immune cells through the activation and signaling of α4 integrin mediated by Hsp90 protein. Hsp90 protein selectively binds α4 integrin, but not β2 integrin, thereby specifically enhancing α4 integrin-mediated T cell adhesion and transmigration. The specific molecular mechanism is: N-terminus and C-terminus of one Hsp90 molecule can simultaneously bind to the intracellular segments of two α4 subunits, leading to the dimerization and clustering of α4 integrin on the cell membrane and the activation of subsequent intracellular FAK-RhoA signaling pathway, and ultimately promoting the directional migration of immune cells (
Therefore, the disclosure provides a method for regulating the migration of immune cells by regulating the interaction between Hsp90 and α4 integrin. As used herein, “regulating” or “regulation” includes up-regulation and down-regulation, wherein up-regulation includes promotion, increase and/or enhancement, and down-regulation includes inhibition, reduction, weakening, destruction and/or diminishing. As used herein, “immune cells” include cells known in the art that are involved in or associated with immune responses in the animal body, especially the human body, including but not limited to: lymphocytes, dendritic cells, monocytes, macrophages, granulocytes and mast cells, etc. As used herein, “migrating” or “migration” refers to the movement of immune cells to their destinations such as lesions (inflammation sites or tumors), including but not limited to lymphocyte homing (lymphocytes directionally migrate from the blood to the lymphatic organs through the capillary high endothelial veins), and the diapedesis of lymphocytes to the inflammation sites. “Directional” or “directionally” refers to the specific migration of immune cells to secondary lymphoid organs, inflammatory sites or tumor tissues to enhance immune surveillance, maintain immune homeostasis, or promote immune response. The term “individual”, “subject” or “patient” herein refers to mammals, especially humans.
Herein, Hsp protein refers to heat shock protein, which is a heat stress protein that exists widely in mammals. When the organism is exposed to high temperature, it will be stimulated by heat to synthesize this protein to protect the organism itself. According to the size of the protein, heat shock proteins are roughly divided into six categories, namely Hsp110, Hsp90, Hsp70, Hsp60, Hsp40, and small molecule heat shock proteins (such as Hsp10). This disclosure specifically relates to Hsp90. Hsp90 protein includes Hsp90AA1 and Hsp90AB1. The amino acid sequence of murine Hsp90AA1 is shown in NP_034610.1 (SEQ ID NO:1), with its mRNA sequence in NM_010480.5 (SEQ ID NO: 2); the amino acid sequence of murine Hsp90AB1 is shown in NP_032328.2 (SEQ ID NO: 3), with its mRNA sequence in NM_008302.3 (SEQ ID NO: 4). The amino acid sequence of human Hsp90AA1 is shown in NP_005339.3 (SEQ ID NO: 5), with its mRNA sequence in NM_005348.3 (SEQ ID NO: 6); the amino acid sequence of human Hsp90AB1 is shown in NP_001258898.1 (SEQ ID NO: 7), with its mRNA sequence in NM_001271969.1 (SEQ ID NO: 8); the amino acid sequence of murine Hsp70 is shown in NP_034608.2 (SEQ ID NO: 9), with its mRNA sequence in NM_010478.2 (SEQ ID NO: 10)); the amino acid sequence of murine Hsp60 is shown in NP_001343441.1 (SEQ ID NO: 11), with its mRNA sequence in NM_001356512.1 (SEQ ID NO: 12); the amino acid sequence of murine Hsp40 is shown in NP_061278.1 (SEQ ID NO: 13), with its mRNA sequence in NM_018808.3 (SEQ ID NO: 14). It should be understood that the heat shock proteins described herein, especially Hsp90 protein, include heat shock proteins derived from various animals of interest, especially mammals.
Also included herein are Hsp90 mutants (also referred to as mutant Hsp90 protein or Hsp90 muteins). The Hsp90 protein includes an N-terminal domain, a middle domain and a C-terminal domain. The N-terminal domain of the wild-type Hsp90 protein is as shown in the amino acid residues 1-233 of the murine SEQ ID NO:1 or the amino acid residues 1-228 of the SEQ ID NO: 3, or the amino acid residues 1-233 of the human Hsp90 protein SEQ ID NO:5 or the amino acid residues 1-228 of SEQ ID NO: 7; the C-terminal domain is as shown in the amino acid residues 566-733 of the murine SEQ ID NO:1 or the amino acid residues 557-724 of the SEQ ID NO: 3, or the amino acid residues 565-732 of the human Hsp90 protein SEQ ID NO:5 or the amino acid residues 557-724 of SEQ ID NO: 7. The Hsp90 mutant herein include a mutant that retains the N-terminal domain and C-terminal domain of the wild-type Hsp90 protein, and only has a mutation in the middle domain. The mutation in the middle domain can be an insertion, a substitution or a deletion, and the middle domain can even be completely replaced by other amino acid sequences, as long as the mutation does not affect the binding of the N-terminal domain and C-terminal domain of the mutant Hsp90 protein to the α4 integrin. Such mutants retain the ability to interact with α4 integrin because they retain the N- and C-terminal domains of the wild-type Hsp90. In certain embodiments, Hsp90 mutants include mutants that have a mutation causing its inability to self-dimerize. Such a mutation may be a deletion mutation, especially a deletion mutation in the C-terminal domain, such as deletion of the last 49 amino acids of the C-terminal domain. In certain embodiments, Hsp90 mutants include mutants that lack the N-terminal domain, the C-terminal domain, or both the N-terminal domain and the C-terminal domain.
In this disclosure, α4 integrin has its well-known meaning in the art (murine a4 gene access number GeneID: 16401; human α4 gene access number GeneID: 3676). It is mainly expressed on the surface of immune cells and mediates the adhesion and migration of immune cells by binding with the vascular endothelial cell surface ligand VCAM-1 or MAdCAM-1.
It should be understood that, unless specifically mentioned herein, Hsp90 and α4 integrin generally refer to Hsp90 and α4 integrin from various sources.
The regulation of the interaction between Hsp90 protein and α4 integrin can be realized by regulating Hsp90 protein and/or regulating α4 integrin.
For example, the interaction between Hsp90 protein and α4 integrin can be improved by enhancing the expression or activity of Hsp90 protein. Methods for enhancing the expression of Hsp90 protein include, but are not limited to, high temperature treatment, overexpression of Hsp90 protein, and expression of its mutants. Specifically, in order to up-regulate the expression of Hsp90 protein, the well-known method of thermal stress can be adopted. Generally, the expression of Hsp90 in a cell can be up-regulated at a temperature of 38.5° C. or above. Of course, the high temperature should not cause the treated subject, such as the cell itself, or the animal body containing the cell to feel intense discomfort or death. Therefore, the duration of thermal stress is usually within 12 hours. Gene engineering techniques well known in the art can be used to achieve overexpression of Hsp90 protein. For example, a vector expressing Hsp90 protein can be constructed and transferred into cells of interest to obtain cells overexpressing Hsp90. The vector can be an expression vector that does not integrate into the genome of the cell; or it can also be an integration vector (or an insertion vector, one kind of targeting vectors), which can integrate the polynucleotide sequence expressing Hsp90 into the genome of the cell and achieve the expression. In certain embodiments, the interaction between Hsp90 and α4 integrin can be enhanced by expressing a mutant Hsp90 protein in cells of interest. Such mutant Hsp90 protein retains the N-terminal domain and C-terminal domain of the wild-type Hsp90 protein, while the middle domain between the N-terminal domain and the C-terminal domain of the wild-type Hsp90 protein can be replaced by other amino acid sequences. For example, the middle domain can be replaced by an amino acid sequence with the same or similar length of the middle domain of the wild-type Hsp90 protein (for example, within 10 amino acid residues difference in length). Alternatively, the mutant Hsp90 protein is the Hsp90 mutant described herein that has a mutation causing its inability to mediate the self-dimerization of the Hsp90 protein. Similarly, the vector used to express the mutant Hsp90 protein may be an expression vector or an integration vector.
In some embodiments, in addition to increasing the expression of Hsp90 protein or its mutants, the expression of α4 integrin can be increased. Similarly, genetic engineering techniques can be used to overexpress α4 integrin in cells of interest. The vector used to express α4 integrin may be an expression vector or an integration vector.
Methods of down-regulating the interaction between Hsp90 protein and α4 integrin include but are not limited to, down-regulating the expression of Hsp90 protein and/or α4 integrin in cells of interest. Methods known in the art can be used to down-regulate the expression of Hsp90 protein in immune cells. For example, genetic engineering techniques can be used to knock out or knock down the expression of Hsp90 protein and/or α4 integrin in immune cells. These methods include, but are not limited to: transferring a gene knockout vector into immune cells of interest, and knocking out the coding sequence of the Hsp90 protein and/or α4 integrin in the genome with the vector, so that immune cells do not express the Hsp90 protein and/or α4 integrin; or knocking out Hsp90 and/or α4 integrin in immune cells using ZFN, TALEN or CRISPR/Cas9. In certain embodiments, gene silencing mediated by interfering RNA (such as siRNA) can be used to knock down the expression of Hsp90 protein and/or α4 integrin. Alternatively, in some embodiments, certain biological activities of Hsp90 may be necessary. Therefore, the coding sequence for expressing the mutant Hsp90 protein can be integrated into the genome of the cell of interest using a gene knock-in vector (a replacement vector, one kind of targeting vectors), replacing the coding sequence of the wild-type Hsp90 protein, so that the cell of interest expresses the mutant Hsp90 protein, which does not bind to α4 integrin or provides a reduced binding (such as lower than 50%) as compared to the non-mutated Hsp90 protein, but retains the biological activities of Hsp90 protein. Exemplary mutant Hsp90 protein include, but are not limited to, Hsp90 mutants lacking the N-terminal domain and/or C-terminal domain of the Hsp90 protein, or Hsp90 mutants having a mutation in the N-terminal and/or C-terminal domain causing the binding of N-terminal and/or C-terminal domain to α4 integrin weakened or eliminated as compared with the wide-type.
In certain embodiments, the interaction between Hsp90 protein and α4 integrin can be down-regulated by mutating α4 integrin, thereby down-regulating the interaction. Since α4 integrin binds to Hsp90 protein through its intracellular segment, corresponding mutation(s) are made in the intracellular segment of α4 integrin (amino acid residues 968-999 of human and murine α4 integrin, wherein only residues 992 are different, with residue 992 of the human α4 integrin being isoleucine (I) and that of the murine α4 integrin being valine (V)). Mutations can be insertions, deletions or substitutions. The number of mutated amino acids is not limited. However, it is necessary to retain the KAGFFKR sequence in the intracellular segment of α4 integrin to maintain the salt bridge between α4 integrin and β integrin, to avoid destroying the salt bridge and causing abnormal activation of integrin. Exemplary mutations include, but are not limited to, mutation(s) in at least one of R985, W989, and Y991. Preferably, the mutation occurs at position R985. For example, R985 can be deleted, or R985 can be replaced by other amino acid(s), especially by amino acid residue(s) that do not belong to the same category. For example, R (arginine) is a positively charged polar amino acid (basic amino acid), with L (lysine) and H (histidine) in the same category. Therefore, any amino acid(s) other than these two amino acids can be used to replace R. The amino acid residues at the other two positions can also be replaced by other amino acid(s), preferably by amino acid(s) that do not belong to the same category. Preferably, the amino acid residue used for substitution is alanine (A). In certain embodiments, for murine α4 integrin, the mutation is the deletion of 9 amino acids residues “ENRRDSWSY” or 17 amino acid residues “ENRRDSWSYVNSKSNDD” in the intracellular segment. When α4 integrin of other species are used, the above-mentioned mutations can occur at positions corresponding to the murine α4 integrin described herein. The mutant α4 integrin is also included in the scope of this disclosure. Methods known in the art can be used to introduce mutant α4 integrin into immune cells. For example, a gene knock-in vector can be used to integrate the coding sequence of the mutant α4 integrin into the genome of the cell of interest to replace the coding sequence of wild-type α4 integrin, so that the cell of interest expresses the mutant α4 integrin.
In this disclosure, amino acids can be roughly divided into: (1) non-polar amino acids (hydrophobic amino acids), including alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp) and methionine (Met); (2) polar amino acids (hydrophilic amino acids), including (a) polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn) and glutamine (Gln); (b) positively charged polar amino acids (basic amino acids): lysine (Lys), arginine (Arg) and histidine (His); and (c) negative charged polar amino acids (acidic amino acids): aspartic acid (Asp) and glutamic acid (Glu).
The transcription level of Hsp90 protein is regulated by transcription factors such as HSF1. HSF1 can tightly bind to the heat shock element (HSE) of Hsp90 gene, and then Hsp90 gene can be transcribed by RNA polymerase. In addition to the transcriptional activation by HSF1, Hsp90AB1 is also regulated by the IL-6 transcription factor NF-IL6 (nuclear factor for IL-6) and STAT-3 (signal transducer and activator of transcription 3). In addition, Hsp90AB1 is also regulated by IFN-γ (interferon-γ) activated STAT-1 transcription. By adding certain agonists or inhibitors of related signaling pathways, the expression of these transcription factors can be promoted or inhibited to dynamically regulate the expression of Hsp90 protein.
The interaction between Hsp90 protein and α4 integrin can be up-regulated in immune cells to promote the directional migration of immune cells, enhance immune response, eliminate pathogen infection or kill tumor cells. Therefore, this disclosure also provides a method for enhancing immune response, eliminating pathogen infection or treating solid tumors, the method comprising up-regulating the interaction between Hsp90 protein and α4 integrin in immune cells of a subject. Immune cells can be treated with an expression vector and/or an integration vector of the wild-type Hsp90 protein, the mutant Hsp90 protein being only mutated in its middle domain, and/or the mutant Hsp90 protein having a mutation causing its inability to self-dimerize; and/or immune cells can be treated with an expression vector and/or an integration vector of α4 integrin; and/or immune cells can be treated with reagents that increase the transcription levels of Hsp90 and/or α4 integrin naturally occurred in immune cells, and/or immune cells can be placed at a fever-range high temperature, thereby enhancing the interaction between Hsp90 and α4 integrin in immune cells.
In this disclosure, pathogen infection is an acute local or systemic infection caused by pathogens or conditional pathogens that invade local tissues or blood circulation, grow and reproduce in large numbers, and produce toxins and other metabolites, including urogenital and gastrointestinal infections caused by Escherichia coli, gram-negative bacteria, or anaerobic bacteria, and respiratory tract infections caused by pneumococcus. In certain embodiments, the pathogen infections described herein include, but are not limited to, infections caused by Salmonella typhimurium, E. coli and the like. Herein, a tumor refers to a physiological disorder in mammals that is usually characterized by unregulated cell growth. Tumors can be divided into benign tumors and malignant tumors, and malignant tumors are also called cancers. Tumors can be divided into solid tumors or hematological tumors. Enhancing the interaction between the Hsp90 protein and α4 integrin in the subject's immune cells can promote the migration of immune cells to solid tumors, thereby ultimately killing tumor cells and inhibiting tumor growth. Examples of tumors include, but are not limited to, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver sarcoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, etc.
Alternatively, by down-regulating the expression of Hsp90 protein in immune cells or reducing the interaction between Hsp90 protein and α4 integrin, the migration of immune cells to secondary lymphoid organs or local tissues can be inhibited, thereby increasing the concentration of immune cells in the blood circulation, and promoting the elimination of blood infections (such as sepsis) or blood tumors by immune cells. Hematological tumors include lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as cancer of the spleen and cancer of the lymph nodes. Examples of hematological tumors include B-cell related cancers, including, for example, high-grade, intermediate-grade, and low-grade lymphomas, including B-cell lymphomas, such as mucosal-associated lymphoid tissue B-cell lymphoma and non-Hodgkin's lymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large cell lymphoma, follicular lymphoma, Hodgkin's lymphoma and T cell lymphoma; and leukemia, including secondary leukemia, chronic lymphocytic leukemia (CLL) such as B-cell leukemia (CD5+ B lymphocytes), myeloid leukemia such as acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia such as acute lymphocytic leukemia (ALL) and myelodysplasia; and other hematology- and/or B cell or T cell-related cancers. Hematological tumors also include cancers of other hematopoietic cells, and the hematopoietic cells include polymorphonuclear leukocytes, such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, red blood cells and natural killer cells.
On the other hand, down-regulating the interaction between Hsp90 protein and α4 integrin can be used to reduce immune response. For example, in chronic inflammation or autoimmune diseases, the interaction between Hsp90 protein and α4 integrin can be downregulated (such as inhibiting the expression of Hsp90 or destroying the interaction of Hsp90-α4 integrin) to reduce the immune response, inhibit the development of chronic inflammation or reduce autoimmune response, and maintain immune homeostasis. Chronic inflammation includes Inflammatory Bowel Disease (IBD), including Crohn's disease and ulcerative colitis, rheumatic arthritis, rheumatoid arthritis, non-specific chronic inflammation and granulomatous inflammation. Autoimmune diseases may include, but are not limited to, Systemic Lupus Erythematosus, asthma, psoriasis, multiple Sclerosis, Celiac Disease, insulin-dependent diabetes, Shaker Lien's syndrome or Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, spontaneous thrombocytopenia purpurea and aplastic anemia, etc.
The interaction between Hsp90 and α4 integrin in immune cells can be weakened or destroyed by: knocking out Hsp90 protein or α4 integrin from immune cells by transferring a gene-knockout vector into the immune cells, and/or knocking out Hsp90 protein or α4 integrin from immune cells using gene editing techniques such as ZFN, TALEN or CRISPR/Cas9 and the like, and/or knocking down the expression of Hsp90 protein and/or α4 integrin by interfering-RNA mediated gene silencing, and/or integrating into the genome of immune cells an expression cassette expressing the Hsp90 mutant that has weakened or no interaction with α4 integrin or that lacks N-terminal and/or C-terminal domains, and/or an expression cassette expressing the α4 integrin mutant that has weakened or no interaction with Hsp90 protein by transferring a gene insertion vector into immune cells while knocking out the coding sequence of wild-type Hsp90 and/or α4 integrin. The ZFN, TALEN and CRISPR/Cas9 applicable to the present disclosure are well known in the art. Each technique knocks out target genes through the combination of DNA recognition domain and endonuclease.
The regulation method described herein can be an in vitro method or an in vivo method. For example, in certain embodiments, provided herein is a method for preparing and enhancing the directional migration ability of immune cells, the method includes increasing the interaction between Hsp90 protein and α4 integrin of the immune cells as compared to the corresponding wide-type cells (i.e. immune cells directly isolated from the subject) by one or more of the following treatments on immune cells in vitro: (1) treating (such as transferring) immune cells with an expression vector and/or an integration vector of the wild-type Hsp90 protein, the mutant Hsp90 protein being only mutated in its middle domain, and/or the mutant Hsp90 protein having a mutation causing its inability to self-dimerize; (2) treating (such as transferring) immune cells with an expression vector and/or an integration vector of α4 integrin; (3) treating (such as transferring or co-incubating) immune cells with reagents that increase the transcription levels of Hsp90 and/or α4 integrin naturally occurred in immune cells; (4) treating immune cells with a fever-range high temperature (such as placing the immune cells under the high temperature). The expression vectors, integration vectors, and reagents can be the reagents described in any of the embodiments herein. The enhanced (or increased) directional migration ability refers to an increase in the directional migration ability of immune cells treated by the methods described herein, for example, by at least 10%, at least 20%, at least 30%, at least 50%, or at least 100% compared with immune cells that have not been treated by the methods described herein. A method known in the art for testing the directional migration ability of immune cells can be used to evaluate the directional migration ability of the immune cells of the present disclosure. For example, the method described in section 1.2.6 below can be used for the evaluation. Specifically, chemokine induced cell migration can be used. By counting the cells that have migrated to the underneath of the wells, the ability of cells to migrate across the membrane is relatively quantified. The more cells migrated, the stronger the ability of directional migration. Thus, for example, in certain embodiments, the immune cells treated with the methods described herein migrate across the membrane by at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.5 times or at least 2 times than immune cells not treated with the methods described herein. Thus, for example, in certain embodiments, the number of the immune cells treated with the methods described herein and migrated across the membrane is at least 1.1 folds, at least 1.2 folds, at least 1.3 folds, at least 1.5 folds or at least 2 folds of the number of the immune cells not treated with the methods described herein.
In certain embodiments, the present disclosure provides a method for down-regulating the directional migration ability of immune cells, the method comprises decreasing the interaction between Hsp90 protein and α4 integrin of the immune cells as compared to the corresponding wide-type cells by treating the immune cells with one or more of the following reagents in vitro: (1) reagents knocking out Hsp90 protein or knocking down its expression in immune cells; (2) reagents knocking out α4 integrin or knocking down its expression in immune cells; (3) reagents achieving the expression of the Hsp90 mutant that has weakened or no interaction with α4 integrin in immune cells; (4) reagents achieving the expression of α4 integrin mutant that has weakened or no interaction with Hsp90 protein in immune cells. The aforementioned expression vector, integration vector or ZFN, TALEN or CRISPR/Cas9 can be used as the reagent to achieve the knockout, knockdown or expression of the mutant Hsp90 protein and mutant α4 integrin. In some embodiments, the Hsp90 mutants are mutants lacking the N-terminal and/or C-terminal domain, or mutants having a mutation in the N-terminal and/or C-terminal domain and causing the interaction between Hsp90 and α4 integrin weakened or eliminated.
The weakened (or decreased) directional migration ability refers to an decrease in the directional migration ability of immune cells treated by the methods described herein, for example, by at least 10%, at least 20%, at least 30%, or at least 50% compared with immune cells that have not been treated by the methods described herein. As described above, any method known in the art for testing the directional migration ability of immune cells can be used to evaluate the directional migration ability of the immune cells of the present disclosure. For example, the method described in section 1.2.6 below can be used for the evaluation.
This disclosure also provides an immune cell, such as T lymphocytes, B lymphocytes, natural killer cells, monocytes, and dendritic cells. Compared with corresponding wild-type cells, the immune cell has increased or decreased interaction between Hsp90 and α4 integrin. In certain embodiments, the immune cell is a genetically engineered cell that: (1) overexpresses Hsp90 protein or Hsp90 mutant protein compared with wild-type cells, wherein, as compared with wild-type Hsp90 protein, said Hsp90 mutant only has a mutation in the middle domain, or the Hsp90 protein has a mutation causing its inability to self-dimerize; and/or (2) comprises an expression vector of α4 integrin; and/or (3) contains or expresses the Hsp90 transcription activator naturally occurred in immune cells. In some embodiments, the immune cell is a genetically engineered cell that, as compared with wild-type cells: (1) does not express Hsp90 or has reduced expression level of Hsp90, or expresses Hsp90 having reduced activity, or expresses Hsp90 mutants; and/or (2) has reduced expression level of α4 integrin, or expresses α4 integrin mutants, thereby weakening or eliminating the interaction between Hsp90 and α4 integrin in the genetically engineered immune cell. The immune cells described herein are living cells. In some embodiments, the immune cells are prepared by the method for regulating the directional migration ability of immune cells as described in any of the embodiments herein.
In certain embodiments, this disclosure also provides a pharmaceutical composition comprising the living immune cells described herein (promoting the interaction between Hsp90 and α4 integrin) and optionally a pharmaceutical acceptable carrier or excipient. Herein, the pharmaceutically acceptable carrier, excipient or stabilizer is non-toxic to the immune cells and recipients of the immune cells at specified dose and concentration, and may include those commonly used in immune cell therapy for the delivery of living immune cells. The immune cells are present in a therapeutically effective amount in the pharmaceutical composition. The therapeutically effective amount of the immune cells described herein are dependent on factors such as different types of immune cells, and age, sex and disease severity of the patient receiving the immune cells. The immune cells described herein can be administered by conventional methods of administration, for example, conventional immune cell therapy can be used to perform the retransfusion of immune cells.
The immune cells and pharmaceutical compositions described herein can be used to treat various diseases or symptoms that benefit from the natural biological functions of the immune cells. For example, the immune cells as described herein can be T cells, wherein genetically engineered cytotoxic T cells or pharmaceutical compositions thereof can be used to treat various diseases or symptoms (i.e., various diseases or symptoms caused by cell infection) that are benefit from the natural biological functions of cytotoxic T cells (such as destroying infected cells). More specifically, in certain embodiments, the immune cells described herein and pharmaceutical compositions thereof are particularly useful for the treatment of pathogen infections and tumors described herein.
Therefore, in certain embodiments, this disclosure also provides an immune cell therapy, especially tumor immunotherapy, comprising: obtaining immune cells from a subject to be treated; treating the immune cells in vitro to enhance the interaction between Hsp90 and α4 integrin, which makes the immune cells have increased migration ability compared with the corresponding wild-type cells; and retransfusing the immune cells. In certain embodiments, the immune cell therapy described herein comprises treating cells of a subject in need with high temperature stress, wherein the high temperature is 38.5° C. or above, usually not more than 40° C. The period of high temperature stress can be determined according to different subjects and different diseases. Moreover, if the temperature is higher, the stress period is usually shorter; otherwise, the stress period can be relatively longer. In other embodiments, the patient having tumor can be induced to fever by different means under medical monitoring, and the inhibitory effect of fever treatment on tumor development can be observed after a certain period of time. Although scientists have known for a long time that the fever caused by infection can cause concurrent remission of tumor patients' diseases, but its systemic mechanism is still unclear. This disclosure provides a possible mechanism that fever promotes immune cells to clear solid tumor cells by promoting the Hsp90-α4 integrin signal axis. Fever treatment under medical monitoring may become a new immunotherapy for patients suffering from cancer. The inducers that may cause fever in the body include various pathogens, metabolites of pathogenic microorganisms or their toxins, or cytokines that can cause fever in the body. Pathogens include but are not limited to bacteria, viruses, fungi, mycoplasma, chlamydia, rickettsiae, spirochetes, and plasmodium. Cytokines include interleukin-6, interleukin-1β, tumor necrosis factor-a and prostaglandin E2.
In certain embodiments, this document also provides a method for treating sepsis, hematological tumors, chronic inflammation, or autoimmune diseases, the method comprising the step of reducing the interaction between Hsp90 and α4 integrin in immune cells of the subject. In some embodiments, (1) the immune cells have knocked out or knocked down expression of Hsp90 and/or α4 integrin as compared with wild-type cells; and/or (2) the immune cells contain an expression vector of mutated Hsp90 and/or α4 integrin, wherein the mutation can inhibit the binding of endogenous Hsp90 to α4 integrin. The treatment can be achieved in a conventional manner. For example, the genetically engineer immune cells from the subject can treated with conventional immune cell therapy to reduce the interaction between Hsp90 and α4 integrin, and then the genetically engineered immune cells can be retransfused to the individual or the subject; or the subject can be administrated with targeting drugs harboring small interfering RNAs (siRNAs) or reagents required for gene editing technology, such as guide RNAs (sgRNAs) and related endonuclease (such as Cas9 protein complex), thereby reducing the expression of the target protein in the immune cells of the subject.
The present invention also includes the coding sequences and complementary sequences of the various mutants described herein, and nucleic acid constructs comprising the coding sequences or complementary sequences. Herein, a nucleic acid construct is an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue. The nucleic acid construct contains the coding sequence described herein or complementary sequence thereof, and one or more regulatory sequences operably linked to these sequences. The regulatory sequence can be a suitable promoter. The promoter sequence is usually operably linked to the coding sequence of the amino acid sequence to be expressed. The promoter can be any nucleotide sequence having transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, which can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription terminator, a sequence recognized by the host cell to terminate transcription. The terminator is connected to the 3′ terminus of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice can be used herein.
In certain embodiments, the nucleic acid construct is a vector. Specifically, the coding sequences described herein can be cloned into many types of vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector can be an expression vector or a cloning vector.
Generally, suitable vectors contain a replication origin that functions in at least one organism, promoter sequences, convenient restriction enzyme sites, and one or more selectable markers. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of phage λ; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, methanol oxidase promoters of Pichia pastoris, and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes can provide phenotypic traits for selection of transformed host cells, including but not limited to dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell, or tetracycline or ampicillin resistance for E. coli. When the polynucleotides described herein are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually are about 10 bp to 300 bp, which act on the promoter to enhance gene transcription.
Those skilled in the art know how to select appropriate vectors, promoters, enhancers and host cells. Methods well known to those skilled in the art can be used to construct expression vectors containing the polynucleotide sequences described herein and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology and so on.
Also included herein is a host cell comprising the polynucleotide sequences described herein or nucleic acid constructs thereof. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: bacterial cells such as cells of Escherichia coli, Streptomyces; Salmonella typhimurium; fungal cells such as yeast cells, filamentous fungi cells; plant cells; insect cells such as Drosophila S2 or Sf9 cells; animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells, etc.
The vectors herein can be introduced into host cells by conventional methods, including microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.
Further, the disclosure also includes the uses of various products described herein, including uses in manufacture of a medicament. Specifically, the disclosure includes: (1) use of a reagent that enhances the interaction between Hsp90 and α4 integrin in immune cells in the manufacture of immune cells for immunotherapy; preferably, the reagent is selected from the group consisting of: an expression vector or an integration vector for the wild-type Hsp90 protein, or the mutant Hsp90 protein being only mutated in its middle domain, or the mutant Hsp90 protein having a mutation causing its inability to self-dimerize; and an expression vector and/or integration vector for α4 integrin; (2) use of immune cells with enhanced interaction between Hsp90 protein and α4 integrin in the manufacture of a medicament for the treatment of pathogen infections or tumors; (3) use of a reagent that weakens the interaction between Hsp90 and α4 integrin in immune cells in the manufacture of immune cells for the treatment of sepsis, hematological tumors, chronic inflammations or autoimmune diseases; preferably, the reagent is selected from the group consisting of: a Hsp90 mutant lacking the N-terminal and/or C-terminal domain or having a mutation in the N-terminal and/or C-terminal domain causing the interaction between Hsp90 and α4 integrin weakened or eliminated as compared with the wide-type Hsp90, and/or a targeting vector of the Hsp90 mutant; a α4 integrin mutant having a mutation in its intracellular segment which causes the interaction between Hsp90 and α4 integrin weakened or eliminated, and/or a targeting vector of the α4 integrin mutant; and ZFN, TALEN and/or CRISPR/Cas9 reagents and/or small interfering RNA used to knock out Hsp90 and/or α4 integrin or to knock down its/their expression; and (4) use of immune cells with weakened interaction between Hsp90 and α4 integrin in the manufacture of a medicament for the treatment of sepsis, hematological tumors, chronic inflammations or autoimmune diseases. Reagents for ZFN, TALEN and/or CRISPR/Cas9 suitable for the present invention are well known in the art, and include but are not limited to DNA recognition domains (such as sgRNA) and endonucleases.
In certain embodiments, this disclosure also includes: Hsp90 or a mutant thereof for immune cell therapy, wherein, as compared with wild-type Hsp90 protein, said mutant only has a mutation in the middle domain, or the mutant has a mutation causing its inability to self-dimerize; α4 integrin for immune cell therapy; a Hsp90 mutant for treating sepsis, hematological tumors, chronic inflammations or autoimmune diseases, wherein the Hsp90 mutant lacks the N-terminal and/or C-terminal domain or having a mutation in the N-terminal and/or C-terminal domain causing its interaction with α4 integrin weakened or eliminated as compared with the wide-type; a α4 integrin mutant described herein for treating sepsis, hematological tumors, chronic inflammations or autoimmune diseases, wherein the α4 integrin mutant has a mutation in its intracellular segment; immune cells described herein for treating sepsis, hematological tumors, chronic inflammation or autoimmune diseases; and immune cells described herein for treating pathogen infections or tumors.
The present disclosure will be illustrated by way of specific examples below. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the materials and methods used in the examples are conventional materials and methods in the art.
A. Materials and Methods
1.1. Experimental Materials
1.1.1 Common Buffer
TBS: 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2;
Cell lysis buffer: TBS, 1% Triton X-100, 0.05% Tween 20, Complete Protease Inhibitor Cocktail Tablets, PhosSTOP Phosphatase Inhibitor Cocktail Tablets;
Immunoprecipitation buffer (IP buffer): TBS, 1% Triton X-100, 0.05% Tween 20;
2×SDS protein loading buffer: 100 mM Tris-HCl (pH6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, 10% β-Me;
Tris-glycine protein running buffer: 25 mM Tris, 250 mM glycine, 0.1% SDS;
Protein transfer buffer: 3 g Tris, 14.4 g glycine, 200 ml methanol, added Milli-Q H2O to 1 L;
TBST buffer: 8.8 g NaCl, 6 g Tris, 0.5 ml Tween-20, pH 7.5, added Milli-Q H2O to 1 L;
PBS: 8 g NaCl, 0.2 g KCl, 3.63 g Na2HPO4.3H2O, 0.24 g KH2PO4, added Milli-Q H2O to 1 L, pH 7.4, sterilizated by filtration;
HBS: 20 mM HEPES, 150 mM NaCl, added to 1 L, pH 7.4, sterilizated by filtration;
2×HBS: 8.0 g NaCl, 0.37 g KCl, 201 mg Na2HPO4.7H2O, 1.0 g glucose, 5.0 g Hepes, added Milli-Q H2O to 500 ml, pH 7.05, sterilizated by filtration, stored at 4° C.;
LB culture medium: 10 g peptone, 10 g NaCl, 5 g yeast extract;
Buffers used in the flow chamber experiment:
Ca2+&Mg2+-free HBSS: 137 mM NaCl, 5.4 mM KCl, 0.4 mM KH2PO4, 0.3 mM Na2HPO4, 4.2 mM NaHCO3, 5.6 mM glucose, pH 7.4;
Coating buffer: PBS, 10 mM NaHCO3, pH9.0;
Blocking buffer: 2% BSA in HBSS;
Washing buffer: 0.2 g BSA, 40 ml HBSS, 400 μl EDTA (0.5 M, pH 8.0);
Buffer A: 0.225 g BSA, 45 ml HBSS;
Buffers for purification of integrin intracellular domain model proteins (His-Tag):
Charge Buffer: 50 mM NiCl2;
Lysis buffer: 20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 5 mM imidazole, 1% Triton X-100, 1 μg/ml aprotinin;
Binding buffer (8M urea): 20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 5 mM imidazole, 0.2% Triton X-100, 8M urea;
Washing buffer (8M urea): 20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 60 mM imidazole, 0.2% Triton X-100, 8M urea;
Elution buffer (8M urea): 20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 1 M imidazole, 0.2% Triton X-100, 8M urea;
XT buffer: 50 mM NaCl, 10 mM Pipes, 150 mM sucrose, 50 mM NaF, 40 mM Na4P2O7.10 H2O, 25 mM imidazole, 1 mM Na3VO4, 0.5% Triton X-100, pH 6.8;
Buffers for purification of GST-Tag fusion protein:
Lysis buffer: PBS, 1% Triton X-100;
Elution buffer: 20 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0;
Buffers for purification of antibodies or Fc-Tag fusion protein:
Binding buffer: 0.1 M Tris-HCl, pH 8.0;
Elution buffer: 0.1 M glycine, pH 3.0;
Neutralization buffer: 1 M Tris-HCl, pH 8.0.
1.1.2 Basic Chemical Reagents
DMEM medium, RPMI 1640 medium, penicillin and streptomycin, L-glutamine, TEMED, etc. were purchased from Invitrogen; fetal bovine serum was purchased from Gibco; sodium dodecyl sulfonate (SDS), HEPES, DMSO, aprotinin, leupeptin, paraformaldehyde (PFA), etc. were purchased from Sigma; peptone and yeast extract were purchased from OXOID LTD;
The remaining reagents are conventional reagents, which can be available from, for example, the Sibas Company of the Institute of Biochemistry, Green Bird Technology Development Co., Ltd., Cal Biochem, Shanghai Chinese Academy of Sciences SLD Company, Shanghai Zhenxing No. 1 Chemical Factory and Shanghai No. 1 Reagent Factory, etc.
1.1.3 Antibody
1.1.4 Enzymes
1.1.5 Kits and Others
1.1.6 Main Instrument
1.2. Experimental Method
1.2.1 Plasmid Construction
I. Construction of Plasmid of Integrin Intracellular Domain Model Proteins
To study the intracellular regulatory protein of integrins, the most direct and effective method is to purify the full-length functional integrins. However, integrins have large extracellular domains and complex high-level structures. It is quite difficult to directly purify integrins with normal structure and function. To solve this problem, scientists have constructed a tail model protein that can well mimic the intracellular domain of integrins under physiological conditions. According to previous reports, an integrin intracellular domain model protein contains the following components from N-terminal to C-terminal: 1) polyhistidine tag (His-Tag) for coupling with Ni2+-NTA beads for subsequent expression and purification and pull down; 2) TEV restriction site for removal of the tag; 3) a pair of cysteine residues used to connect the expressed polypeptide chain into a dimer form; 4) a helical coiled coil consisting of four consecutive seven-amino-acid peptides, wherein the coiled-coil serves as a topological structure that fixes two model protein of integrin intracellular domains, which are arranged in parallel to simulate the transmembrane structure of integrin and mediate the dimerization of integrin in aqueous solution; 5) polyglycine linking sequence (glycine linker); 6) cDNA sequence of intracellular domains such as integrin a4, (31, (37 (
II. Construction of Eukaryotic Expression Vector of Integrin
The full length of the integrin α4 subunit was amplified by PCR into the pCDH-puro vector (System Biosciences), and the R985A mutation and the shRNA-tolerant synonymous point mutation were achieved by Quick change method. The C-terminus of the full-length integrin α4 subunit was fused to Split GFP (GFP 1-10 and GFP S11) with a long linker, and was added into the pCDH-puro vector.
III. Construction of Eukaryotic Expression Vector of Heat Shock Protein
First, using the cDNA of mouse T cells as a template, the mRNA sequences of Hsp90AA1, Hsp90AB1, Hsp70, Hsp60, and Hsp40 were respectively amplified by PCR (the amino acid sequence of Hsp90AA1 is shown in SEQ ID NO:1 with its mRNA sequence shown in SEQ ID NO: 2; the amino acid sequence of Hsp90AB1 is shown in SEQ ID NO: 3 with its mRNA sequence shown in SEQ ID NO: 4; the amino acid sequence of Hsp70 is shown in SEQ ID NO: 9 with its mRNA sequence shown in SEQ ID NO: 10; the amino acid sequence of Hsp60 is shown in SEQ ID NO: 11 with its mRNA sequence shown in SEQ ID NO: 12; the amino acid sequence of Hsp40 is shown in SEQ ID NO: 13 with its mRNA sequence shown in SEQ ID NO: 14), and added into pCDH-puro-mRuby2 or pHAGE-IRES-mcherry vector (Chen Jianfeng, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences), wherein the fusion expression of mRuby2 or mcherry red fluorescent protein indicates transfection efficiency. The ATPase inactivating mutations Hsp90AA1-D93N and Hsp90AB1-D88N of Hsp90AA1 and Hsp90AB1 were achieved by Quick change method, respectively.
The three domains of each of the two subtypes of Hsp90 are:
1) Hsp90AA1-N-terminal domain, M1-D233; middle domain, E234-K565; C-terminal domain, K566-D733;
2) Hsp90AB1-N-terminal domain, M1-D228; middle domain, E229-S 556; C-terminal domain, K557-D724.
Truncation or deletion mutations of Hsp90: Hsp90-NM (lacking C-terminal domain), Hsp90-MC (lacking N-terminal domain) and Hsp90-NC5 (lacking the last 49 amino acids of the C-terminal domain) were amplified by PCR into pCDNA3.1-(+)-HA-Tag. The HA tag is used for western blotting.
IV. Construction of Prokaryotic Expression Vector of Heat Shock Protein
The N-terminal and C-terminal domains of Hsp90AA1 and Hsp90AB1 were amplified by PCR and added into the pGEX-6P-1 vector (GE Healthcare). The target protein was fused to glutathione S-transferase tag (Glutathione S-transferase) for prokaryotic expression and purification.
V. Construction of Prokaryotic Expression Vector of Rho GTPases Effector Protein
The effector protein of Rac 1 and Cdc42 is PAK (p21 activating kinase), and the effector protein of RhoA is Rhotekin. The 67-150 amino acid sequence of PAK-PBD (PAK binding domain) was amplified from human cDNA, and the 7-89 amino acid sequence of Rhotekin-RBD (Rho binding domain) was amplified from murine cDNA, which were fused to the GST tag by a linker sequence for prokaryotic expression and purification and subsequent GST-Pulldown.
VI. Enzyme Digestion and Electrophoresis Separation and Recovery of Plasmids and PCR Products
The corresponding enzyme digestion buffer, vector DNA or PCR products, and restriction enzymes were added to a 10-20 μl reaction system, and the reaction system was incubated at 37° C. for 2-4h. After adding DNA loading buffer, the reaction products were separated by 1% agarose gel electrophoresis. After the correct PCR band was identified, the PCR product was recovered with DNA Mini Purification Kit (Tiangen), and dissolved in 30 μl deionized water.
VII. Ligation of DNA Fragments and Vectors
The ligation buffer, T4 ligase, the recovered DNA fragments and an appropriate amount of vector were added to a 10 μl reaction system, and the reaction system was incubated overnight at 16° C.
VIII. Transformation of E. coli DH5a Competent Cells with Plasmid
(1) 100 μl of competent cells stored at −80° C. was placed on ice until it melts;
(2) Add the prepared transformation products;
(3) Mix gently and place on ice for 30 minutes;
(4) 42° C. heat shock for 45s;
(5) Hold on ice for 2 minutes;
(6) Add 900 μl of liquid LB medium;
(7) Mix gently and hold in a 37° C. water bath for 30 minutes.
After the recovery was complete, the EP tube was centrifuged at 5000 rpm for 5 minutes to enrich the bacteria at the bottom of the tube. In an ultra-clean table, excess supernatant was removed, leaving about 100 μl, and the pellet was gently pipetted to obtain a suspension. The suspension was spread on a LB (Amp+) plate by glass beads and the beads were removed after spreading thoroughly. The plate was placed upside down in a 37° C. biochemical incubator and incubated overnight for 12 hours.
VIIII. Preparation of Small Amount Plasmid DNA
Transfer about 5 ml of the overnight bacteria to a 1.5 ml EP tube, centrifuge at 10,000 rpm for 1 min, remove the supernatant, and suspend the bacteria with the remaining solution in the tube on a shaker. Add 200 μl Sol I and mix well, then add 200 μl Sol II, gently invert the tube twice before adding 200 μl Sol III, invert and mix well and centrifuge at 12000 rpm for 5 min. Transfer the supernatant to a new tube, add 300 μl isopropanol, mix well for 15 min at room temperature, and centrifuge at 12000 rpm for 15 min. The supernatant was discarded, the pellet was washed with 70% ethanol, dried and dissolved in 50 μl Milli-Q H2O.
1.2.2 Cell Culture and Transfection
HEK (human embryonic kidney) 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco), 50 U/ml penicillin and 50 μg/ml streptomycin at 37° C. and 5% CO2.
I. Cell Recovery and Culture (293T Cells)
(1) 293T cells stored in liquid nitrogen were melt in a 37° C. water bath;
(2) Take 1 ml of DMEM culture medium slowly into the cell cryopreservation tube, gently pipette to mix, transfer to a 15 ml sterile tube containing 4 ml of fresh DMEM culture medium, centrifuge at 1200 rpm for 3 min, and remove the supernatant;
(3) Gently resuspend the cells by 1 ml of fresh DMEM culture medium, transfer to a 10 cm cell culture dish, and add cell culture medium to 10 ml;
(4) Incubate the dish at 37° C., 5% CO2, and saturated humidity.
II. Cell Passaging (293T Cells)
(1) After the cells were overgrown in the culture dish, discard the old culture medium;
(2) Add 3 ml 1×PBS, wash gently to remove residual culture medium, then discard the PBS;
(3) Add 1 ml of pancreatin and allow to rest for 10s, tap gently to dissociate the cells from the culture dish;
(4) Add 3 ml of DMEM complete culture medium, stop the digestion, and pipette to form single clones;
(5) Transfer the cells into a 15 ml centrifuge tube and centrifuge at 1200 rpm for 3 min;
(6) Discard the supernatant, add 10 ml culture medium and mix well by pipetting. If passaging at 1:10, add 1 ml to a petri dish containing 9 ml culture medium; if passaging at 1:3, add 3 ml;
(7) Incubate the dish at 37° C., 5% CO2, and saturated humidity.
III. Transient Transfection of 293T Cells Through Calcium Phosphate
(1) Divide the cells the day before transfection so that the cells reach a density of 60-70% before transfection;
(2) 1 h before transfection, change the medium with a culture medium comprising 25 μM chloroquine;
(3) In a 15 ml centrifuge tube, add 10 μg DNA, dilute to 1095 μL with MilliQ-H2O, and then add 155 μL 2M CaCl2. Add 1250 μL of 2×HBS dropwise and mix gently while adding. Then the mixture was directly added dropwise to the cells. Note that the addition should be completed within 1-2 min after adding 2×HBS. And the droplets need to be evenly sprinkled on the entire surface of the dish;
(4) Incubate for 7-11h, and very small, dusty particles will be observed. Wash once and change the medium with a medium free of chloroquine;
(5) Cells were collected 48-72h after transfection for later use.
IV. Infection of T Cells by Lentivirus
Calcium phosphate transfection of 293T cells was used for lentivirus packaging. The ratio of transfected plasmids was (take 10 cm dish as an example): pCDH vector 20m, psPAX2 15 μg and pMD2.G 6 μg. 72 hours after transfection, the culture solution comprising the lentiviral supernatant was collected with a 10 ml syringe, and the supernatant was filtered with a 0.45 μm filter and concentrated by ultracentrifugation to infect the target cells. The ultracentrifugation was performed at 20000 rpm, 2h, 4° C. After centrifugation, the precipitate was removed and re-dissolved in 100-200W serum-free culture medium. During infection, the cells was re-suspended with a 1:1 ratio of cell culture solution:lentivirus culture solution, while polybrene was added at a final concentration of 8m/ml to the cell supernatant. The mixture was mixed well, incubated at 37° C., and then the medium was replaced with fresh virus culture medium after 24h to continue the infection. Detection by flow cytometry was performed after 72h.
1.2.3 Protein Expression and Purification
I. Expression and Purification of Integrin Intracellular Domain Protein
(1) Transform the plasmid, pET-Duet-integrin tail model protein, into E. coli BL21 (DE3) pLysS strain, overnight at 37° C.;
(2) Select single clones into each 5 ml LB culture medium in 2 sterilized tubes, 37° C. for 12h;
(3) Transfer the culture solution to 1 L of LB, add ampicillin at a final concentration of 100 μg/ml, and incubate at 37° C. until OD600≈0.4;
(4) Add IPTG at a final concentration of 1 mM, 37° C. for 3h;
(5) Transfer 1 L of bacteria liquid to a 500 ml large centrifuge cup, 5000 rpm for 5 min to collect bacteria;
(6) Add 10 ml lysis buffer per gram (wet weight of the bacteria) and transfer the mixture to a 50 ml centrifuge tube, add 1 mg/ml lysozyme and incubate at 37° C. for 15 min;
(7) Break bacteria by ultrasound: 10 seconds of ultrasound treatment followed by 50 seconds of resting for 49 cycles, which is performed twice. 1 mM PMSF was added before each ultrasound;
(8) Transfer the mixture to a 50 ml round bottom centrifuge tube, and centrifuge at 20000 g, 4° C. for 20 min;
(9) Add 12 ml binding buffer (8M urea) to the precipitate and mix well, centrifuge at 30000 g, 4° C. for 20 min;
(10) Processing Ni-NTA beads in the column: wash with 5 column volumes of Milli-Q H2O; wash with 5 column volumes of 6M Gu-HCl; wash with 5 column volumes of Milli-Q H2O; wash with 5 column volumes of 100 mM EDTA; wash with 5 column volumes of Milli-Q H2O; wash with 5 column volumes of charging buffer; equilibrate with 5 column volumes of binding buffer (8M urea);
(11) The supernatant obtained by centrifugation was added to the column and incubated with Ni-NTA beads at 4° C. for 2h;
(12) Flow through the supernatant that does not bind to Ni-NTA beads;
(13) Wash with 1 column volume of binding buffer (8M urea);
(14) Wash with 3 column volume of washing buffer (8M urea);
(15) Washing each tube with 1 ml elution buffer (8M urea);
(16) Purity of the target protein was assayed by SDS-PAGE with a gel concentration of 15%;
(17) Add 1 mg of integrin intracellular domain model protein to 500 μl of processed Ni-NTA beads, and incubate at room temperature for 1 hour with rotation;
(18) Remove the supernatant after centrifugation at 2800 rpm for 3 min, and wash the Ni-NTA beads with 1 ml binding buffer (8M urea), binding buffer (4M urea), binding buffer (2M urea), and binding buffer (OM urea), respectively (gradually remove urea), wherein each are incubated for 1 h with rotation at room temperature;
(19) Centrifuge at 2800 rpm for 3 min to remove supernatant, wash the Ni-NTA beads with 1 ml XT buffer;
(20) Resuspend the Ni-NTA beads in 500 μl XT buffer and store at 4° C.
II. Expression and Purification of GST Tag Fusion Protein
(1) Transform the plasmid, pGEX-6P-1-target protein, into E. coli Rosetta-gami (DE3) pLysS strain, overnight at 37° C.;
(2) Select single clones into each 5 ml LB culture medium in 2 sterilized tubes, 37° C. for 12h;
(3) Transfer to 1 L LB, add ampicillin at a final concentration of 100 μg/ml, and incubate at 37° C. until OD600≈0.4;
(4) Add IPTG at a final concentration of 0.1 mM, induce at 16° C. for 3h;
(5) Transfer 1 L of bacteria liquid to a 500 ml large centrifuge cup, 5000 rpm for 5 min to collect bacteria;
(6) Add 10 ml lysis buffer per gram (wet weight of the bacteria) and transfer the mixture to a 50 ml centrifuge tube, add 1 mg/ml lysozyme and incubate at 37° C. for 15 min;
(7) Break bacteria by ultrasound: 3 seconds of ultrasound treatment followed by 7 seconds of resting for 99 cycles, which is performed 3 times. 1 mM PMSF was added before each ultrasound;
(8) Transfer the mixture to a 50 ml round bottom centrifuge tube, and centrifuge at 20000 g, 4° C. for 20 min;
(9) Equilibrate GST beads: wash 2 ml GST beads (50:50 stored in 20% ethanol) in a 15 ml centrifuge tube and centrifuge at 1200 rpm for 5 min. Repeat the washing three times to remove the ethanol in the storage solution;
(10) The supernatant was added to the equilibrated GST beads, add DTT with a final concentration of 5 mM, and incubate with rotation at 4° C. for 2h;
(11) Centrifuge at 750 g for 3 min, and remove the supernatant;
(12) Wash with PBS, PBS+0.5% Triton-X100, PBS+0.5% Triton-X100 and PBS, 5 ml each; then add the GST beads to the chromatography column with 10 ml PBS, and discard the flowed-through effluent;
(13) Elute the column with 10 ml elution buffer, 1 ml per Eppendorf tube;
(14) Purity of the target protein was assayed by SDS-PAGE with a gel concentration of 7-10%.
(15) If the result showed that protein purity was not good, perform purification by molecular sieve ÄKATA system: pre-equilibrating a Superdex 200 (10/300GL) column (GE healthcare) with PBS degassed by ultrasound, concentrating samples to 0.5 ml and loading the samples, and then collecting the eluate of each main peak. The protein purity was identified by SDS-PAGE, and the protein was aliquoted and stored at −80° C.
III. Expression and Purification of Antibody or Fc-Tag Protein
(1) Collect the culture solution of hybridoma cells or 293T expressing Fc-Tag protein by transfection, centrifuge at 2400 rpm for 3 min, and remove cell debris and other impurities;
(2) Equal volume mix the culture solution and binding buffer, with stirring while adding the binding buffer, pH 8.0;
(3) Centrifuge at 4° C. and 15000 rpm for 30 min;
(4) Load the supernatant on the Protein A or Protein G column, overnight at 4° C.;
(5) Wash the column with 15 column volumes of binding buffer;
(6) Elute with 10 column volumes of elution buffer. The collection tube was added with neutralization buffer at a ratio of 1:10 in advance;
(7) Collect the eluate, concentrate it by centrifugation in a concentration tube, change the buffer to HBS, and freeze at −80° C.
1.2.4 Detection of the Expression of Integrin by Flow Cytometry (FACS)
(1) Add 5×105 cells in a 1.5 ml Ep tube, centrifuge at 3000 rpm and 4° C. for 3 min, and wash with PBS;
(2) a. Dissolve the cell pellet directly in 300 μl PBS and place at 4° C. (Mock);
b. Dissolve the cell pellet in 50 μl PBS, add 5 μg/ml antibody, mix gently, then place at 4° C., and rest for 30 min. Add 1 ml PBS, mix well, centrifuge at 3000 rpm and 4° C. for 3 min, and discard the supernatant; then repeat once. Suspend the cells in 300 μl PBS;
(3) Transfer the cell suspension to a cell collection tube and analyze with a flow cytometer Calibur or LSRII (BD).
1.2.5 Detection of Cell Adhesion Mediated by Integrins and their Ligands by Flow Chamber System
I. Cell Processing
(1) Add T cells in a 15 ml centrifuge tube, centrifuge at 1200 rpm for 3 min;
(2) Wash twice with 5 ml washing buffer (HBSS, 5 mM EDTA, 2% BSA, pH 8.0), centrifuge at 1200 rpm for 3 min;
(3) Add 5 ml of buffer A (HBSS, 2% BSA, pH 8.0), centrifuge at 1200 rpm for 3 min, then repeat once;
(4) Dissolve the cells in 1 ml buffer A and count cell number. The final cell concentration was adjusted to 1×107 cells/ml.
II. Flow Chamber Cell Adhesion Experiment
Mechanism:
In order to study the integrin-mediated cell adhesion function, the inventers established a flow chamber system in vitro to study the interaction of α4 integrin or β2 integrin with its ligands VCAM-1, MAdCAM-1 or ICAM-1, respectively (
Steps:
(1) Coating ligand: draw a circle with a diameter of about 5 mm in the center of the bottom of a clean plastic plate, drop 20 μl of 5 μg/ml VCAM-1, MAdCAM-1 or ICAM-1, and incubate it in a humid box at 37° C. for 1 h;
(2) Block the contaminant protein: wash the plate with coating buffer three times at the marked place (the surface of the ligand should always be covered with liquid), add 20 μl of blocking buffer in the circle, and incubate at 37° C. for 1 hour;
(3) Wash twice with HBS, then install the flow cell system and spread a layer of HBS on it, pump air out by a vacuum pump and prevent air leakage;
(4) Add 50 μl of the processed cells to 450 μl of buffer A, add 2 μl of divalent cations, mix well, and detect in the flow chamber system (
1.2.6 Chemokine Induced Transwell Migration
The chemokine-induced transwell migration can simulate the process of integrin-mediated lymphocyte migration across vascular endothelial cells. Transwell chambers (5 μm well size, Millipore) were coated with 5 μg/ml VCAM-1, MAdCAM-1 or ICAM-1 respectively, incubated overnight at 4° C., and blocked with blocking buffer at 37° C. for 1 h. T cells were resuspended in serum-free RPMI 1640 culture medium at 2×106 cells/ml, and 150 μl of cells were added to the upper layer of Well. 600 μl of RPMI 1640 culture medium comprising 500 ng/ml CCL21 was added to the bottom layer of Well to ensure that the inside and outside liquid level of Well is even, and the Well was incubated at 37° C. for 4h. By counting the cells that have migrated to the underneath of the well, the ability of cells to migrate across the membrane was relatively quantified.
1.2.7 Immunofluorescence Staining of Cell Spreading and Integrin Clustering on the Cell Surface
(1) T cells were suspended in FBS-free RPMI 1640 culture medium, plated on a glass slide coated with PLL (100 μg/ml), and incubated at 37° C. for 10 minutes;
(2) Wash once with the incubated PBS, then fix with the incubated 2% paraformaldehyde/PBS (Sigma) for 15 min at room temperature;
(3) Block with PBS+10% FBS for 1 hour at room temperature;
(4) Primary antibody incubation (PS/2): 37° C. for 2h or 4° C. overnight, wash three times with 1% BSA+PBS, each for 5 minutes;
(5) Secondary antibody incubation (goat anti-rat-Cy3): 37° C. for 1 hour, then wash with 1% BSA+PBS three times, each for 5 minutes;
(6) Stain cell nuclei with DAPI: incubate 5 min at room temperature, then wash three times with PBS, mount the slide and take pictures.
1.2.8 Separation of Nuclear and Cytoplasmic Membrane
(1) Collect T cells (1×107), and wash once with TBS, centrifuge at 5000 rpm for 3 min;
(2) Resuspend the cells in 600 μl buffer (TBS+protease inhibitor), and allow the cells to rest on ice for 10 minutes;
(3) Repeatedly blow and suck 30-40 times by the insulin needle to break the cells by mechanical force, and collect the whole cell lysate (WCL);
(4) Centrifuge at low speed, 2000 rpm for 3 min to remove nuclear components and incompletely broken cells;
(5) Transfer the supernatant to a new Eppendorf tube, 13000 rpm, 30 min;
(6) The supernatant was the cell cytoplasmic fraction and the precipitate was the cell membrane fraction. Lyse the cells with cell lysate on ice for 30 min;
(7) Centrifuge at 14000 rpm for 30 min. the supernatant is the soluble fraction of the cell membrane after lysis. Proceed with the following Co-IP or collect Input samples.
1.2.9 Western Blotting
Western blotting was used to detect the relative content of a specific protein in a cell lysate or purified sample. The protein content was determined by BCA method. The protein samples with the same amount were separated by 7-9% SDS-PAGE electrophoresis: concentrated gel 80V, 30 min; separation gel 120V, 60 min. Following the electrophoresis, the protein was transferred to the nitrocellulose membrane (NC) using a wet electroporator with a current of 280 mA for 100 min. After the transfer, the NC membrane was obtained with the surface comprising the transferred protein facing up. Incubating with blocking solution (TBST comprising 5% skimmed milk powder) at room temperature for 1 hour; incubating with specific antibody (diluted in TBST comprising 2% BSA) for 2 hours at room temperature or overnight at 4° C.; washing NC membrane three times with TBST, each 5 minutes. Incubating with HRP-coupled secondary antibody (diluted in TBST) for 1 h at room temperature. After incubation, washing the NC membrane twice with TBST for 5 minutes each time. Washing the NC membrane twice with TBS for 5 minutes each time. After fluorescent development with ECL developing solution, holding the NC film in the holder, and displaying the protein bands in darkness with the developing solution and the fixing solution respectively.
1.2.10 Co-Immunoprecipitation
All steps of co-immunoprecipitation were performed on ice, and the used buffers such as TBS, Cell lysis buffer, and IP buffer needed to be pre-cooled on ice.
(1) Collect the cells in a 15 ml centrifuge tube, centrifuge in a horizontal rotor at 1200 rpm for 3 min;
(2) Discard the supernatant, add 1 ml TBS in the tube and transfer the mixture to a 1.5 ml Eppendorf tube, centrifuge at 5000 rpm for 3 min;
(3) Discard the supernatant, add 600 μl Cell lysis buffer and lyse on ice for 30 minutes with mixing during the lysis to avoid cell sedimentation;
(4) Centrifuge at 14000 rpm for 30 min, and the supernatant was used as Input (40 μl) for Western blotting;
(5) Add the remaining supernatant in a new 1.5 ml Eppendorf tube, add 50 μl Protein G beads, and pre-wash at 4° C. for 2h;
(6) Centrifuge at 2800 rpm for 3 min, transfer the supernatant into a new 1.5 ml Eppendorf tube and add 1 μg primary antibody, then incubate overnight at 4° C. on a turnplate;
(7) Add 50 μl Protein G beads and incubate at 4° C. for 2h;
(8) Centrifuge at 2,800 rpm for 3 min, wash with IP buffer for three times;
(9) Remove the supernatant using the insulin needle, add 60 μl 2×SDS loading buffer, and boil at 100° C. for 10 minutes;
(10) Centrifuge at 14000 rpm for 2 min, transfer to a new 1.5 ml Eppendorf tube by the insulin needle, and then detect the target protein by Western blotting.
1.2.11 Detection of Protein Interaction by Pull Down
The integrin intracellular domain model protein obtained by E. coli prokaryotic expression and purification was mixed with the supernatant of T cell lysate and incubated for 2h at 4° C. Wash away non-specific binding protein with IP buffer, resuspend the beads by 2× protein-loading-buffer, then boil at 100° C. for 10 minutes and collect the samples. The bands of the target protein were identified by Western blotting. In addition, the integrin intracellular domain model protein stained with Coomassie Blue was used as a loading control.
1.2.12 Detection of Protein Interaction by BiFC-Split GFP
In addition to traditional Pull down, Co-IP and other biochemical methods, protein-fragment complementation assay (PCA) can detect protein-protein interaction more convenient and intuitive, can be relatively quantitative, and are favored by scientists. PCA detects protein-protein interaction by dividing some enzyme protein (such as dihydrofolate reductase, β-galactosidase, β-lactamase, or luciferase) into two fragments, which are used as tags to fuse to target protein. PCA based on split green fluorescent protein (Split GFP) or its various mutants is called bimolecular fluorescence complementation (BiFC). Because the assembly of GFP elements is irreversible, Split GFP-based BiFC can be used to study transient protein-protein interactions and low-affinity complexes.
This disclosure used the Split GFP system to study the self-dimerization of α4 integrin. The intracellular domains of α4 subunits fused with two parts of GFP respectively, GFP 1-10 OPT and GFP S11 M3, were simultaneously expressed in cells. If the α4 integrin underwent self-dimerization, the two parts of the protein were close to each other, thereby forming a stable GFP fluorescent, which can be conveniently and directly observed by fluorescence microscope or FACS, and relative quantified. The cells expressing a4-Split GFP were subjected to fever-range thermal stress or Hsp90-overexpressing treatment. FACS could conveniently and intuitively detect the change of GFP fluorescence, which indicated the change of the self-dimerization of α4 integrin.
1.2.13 Detection of Protein Polymerization by Native-PAGE
Native-PAGE was used to assay the polymerized form of the target protein in cells, i.e., in its natural state. Native-PAGE mainly comprises:
(1) Collect the cell lysate, add 5× protein loading buffer (SDS free), place on ice without boiling at 100° C.;
(2) Load the sample directly after high-speed centrifugation without adding protein Marker;
(3) The electrophoresis buffer was the transfer buffer (methanol free) used in ordinary SDS-PAGE, and the electrophoresis process was performed on ice to prevent degradation of the target protein;
(4) Membrane Transfer and the following Western blotting were the same as ordinary SDS-PAGE.
1.2.14 Detection of Rho GTPase Activation
The key step of integrin-mediated migration of immune cells across the membrane is the rearrangement of cytoskeletal protein, which in turn changes the cell morphology, and finally the immune cells penetrate vascular endothelial cells into secondary lymphoid organs or inflammation sites. There are three main Rho GTPases that mediate the formation of cytoskeleton, namely Rac1, RhoA and Cdc42.
In order to detect the activity changes of the three GTPases, the inventors expressed and purified their respective GTP-binding effector protein in vitro (the effector protein of Rac1 and Cdc42 are both PAK-PBD, and the effector protein of RhoA is Rhotekin-RBD), each connected to agarose beads via GST tags and incubated with cell lysate. Thus, the activated GTPase (in the Rho-GTP form) can specifically bind to its corresponding effector-protein-beads in the lysate. The non-specifically bound protein was washed away by IP buffer, and finally each Rho GTPase antibody was detected by Western blotting. The brightness of the band reflected the degree of activation of the corresponding Rho GTPase.
1.2.15 Construction of Itga4R985A/14985A Knock-in Mice by CRISPR/Cas9
The mice used for integrin a4-R985A knock-in were C57BL/6J mice. The knock-in mice were constructed by Shanghai Model Organisms Center, Inc. Specifically,
(1) Requirement:
Mouse point mutation site: R1018A (corresponding to human site R985A), located in exon28.
(2) Cas9 Strategy:
Design guide RNAs for exon28 sequence.
Exon28 target sequence: (UTR is in bold, and non-bold sequence is the coding region)
ACACTGAGAGAACTGAAAAACTCAGG
TTAGGAAAAAGAAATCCTGTTCAGAA
GACCCGTCAGAATTTCTTCTTTTT
ACGCGAGAGACAGCTGGAGTTATGT
(3) CRISPR/Cas9 Process to Establish a Genetic Mutation Mouse
Cas9 protein, guide-RNAs and template DNAs were injected into mouse fertilized eggs through microinjection. After homologous recombination, the target gene in the genomic DNA of the blastocyst cells had been replaced with a mutant gene. Finally, the embryos were transplanted into the surrogate mice to give birth to FO generation mutant mice.
(4) Mechanism
Guide-RNAs guide the endonuclease Cas9 to locate on a specific sequence of the genomic DNA in the cell, and then cause a DNA double-strand break in the target fragment. At the same time, the template DNA with sequence homologous (but containing a new gene or gene mutation) to the flanks of the original gene break were added. The mutation was introduced into the genomic DNA sequence through homologous recombination.
B. Experimental Results
2.1: Fever-Range Thermal Stress Promotes α4 Integrin-Mediated Cell Adhesion and Transmigration
In order to study the effect of fever-range thermal stress on the adhesion and migration of lymphocytes, the inventor isolated T lymphocytes from the spleen of C57BL/6J mice, and treated them with physiological conditions (37° C.) or fever-range thermal stress (40° C.) for 12 hours. FACS found that fever-range thermal stress did not affect the expression of all α4 and β2 integrin subunits on the cell membrane surface (
2.2 Hsp90 Protein Specifically Binds to α4 Integrin and Promotes Cell Adhesion and Transmigration
When studying the related molecular mechanism, T cells were treated with fever-range thermal stress, and the expression of heat shock protein family members in the whole cell lysate was significantly up-regulated (
2.3 Both the N-Terminus and C-Terminus of Hsp90 can Bind to the Cytoplasmic Domain of α4 Integrin
Because integrin is a α/β heterodimer, Hsp90 protein can bind to α4 integrin by interacting with α or β subunit. The inventors purified the intracellular domain model proteins of α4, β1, and β7 integrins, and then verified that Hsp90 binds to the intracellular segment of α4 integrin by pull down (
2.4 Construction of Integrin Itga4R985A/R985A Knock-in Mice
In order to identify whether Hsp90 promotes α4-integrin-mediated cell adhesion and transmigration by binding to the intracellular segment of α4 integrin, Itga4R985A/R985A knock-in mice (KI) was constructed by CRISPR/Cas9 gene editing which destroyed the Hsp90-a4 interaction in vivo (
2.5 Disruption of Hsp90-α4 Integrin Binding Inhibits Fever-Induced T Cell Adhesion and Transmigration
As expected, R985A mutation of α4 integrin completely inhibited the fever-induced cell adhesion and transmigration of T cells on VCAM-1 and MAdCMA-1 ligands (
2.6 Hsp90-α4 Integrin Binding Induces Activation of α4 Integrin
In order to study whether α4 integrin is activated by Hsp90 upon fever-range thermal stress, the binding of T cell α4β1 and α4β7 integrins to soluble VCAM-1 and MAdCAM-1 was first detected. Being pre-treated at 40° C., WT T cells showed a significant increase in the binding to VCAM-1 and MAdCAM-1; on the contrary, the ligand binding of KI T cells did not change significantly. This confirmed that the ligand binding increased by fever depends on the binding of Hsp90-α4 integrin (
2.7 Knockdown of Talin and Kindlin-3 Inhibits the Activation of α4 Integrin
In order to further confirm the role of talin and kindlin-3 in the fever-induced α4 integrin activation, the expression of talin and kindlin-3 in T cells were knocked down by shRNA-mediated gene silencing. Upon fever-range thermal stress, the binding of talin and kindlin-3 to α4 integrin did not increase significantly (
2.8 Hsp90-α4 Integrin Binding Induces the Dimerization and Clustering of α4 Integrin on the Plasma Membrane
Given that both the N-terminal and C-terminal domains of Hsp90 can directly bind to the intracellular segment of α4 integrin, it is conjectured that one Hsp90 molecule may bind to two α4 subunits simultaneously in the cells, which mediates the dimerization of α4 integrin on the cell membrane and further activates the downstream signaling pathways of integrins. To confirm this conjecture, a bimolecular fluorescence complementation system (BiFC) was established, wherein two complementary components of GFP: GFP S1-10 and GFP S11 were fused to the C segment of the intracellular segment of α4 integrin and expressed. The dimerization of α4 integrin can induce the approach of GFP S1-10 and GFP S11, which reconstructed to form a functional GFP protein (
To further study the molecular mechanism of Hsp90-mediated dimerization of α4 integrin, Hsp90 mutants lacking different domains were constructed (
2.9 the Binding of Hsp90-α4 Integrin Activates the FAK-RhoA GTPase Signaling Pathway
The clustering of integrins on the cell membrane surface can trigger the activation of downstream signaling pathways of integrins from the outside to the inside. FAK and Rho family GTPases (RhoA, Rac1 and Cdc42) are important signal proteins activated by integrins, which can promote cell migration by regulating the rearrangement of skeletal proteins. Therefore, the effect of fever-range thermal stress on the activation of FAK and Rho GTPases was tested. According to the results, the 40° C. pre-treated WT T cells showed significantly increased phosphorylation of tyrosine at position 397 of FAK (
2.10 Disruption of Hsp90-a4 Interaction Inhibits Fever-Induced T Cell Trafficking In Vivo
To study the effect of the Hsp90-α4 integrin signal axis on the migration of lymphocytes in vivo, WT and KI mice were treated by normal temperature (core temperature 36.8±0.2° C.) or fever-range whole-body hyperthermia (WBH; core temperature 39.5±0.5° C.) for 6 hours, and T cells were separated from the mouse spleens. WT and KI mice showed similar expression levels of α4 integrin (
2.11 LPS-Induced Mild Fever does not Affect the Homing of T Cells in Mice
Next, mouse models of LPS-induced fever were established. LPS (10 μg/kg) or PBS was injected into the tail vein of WT and KI mice, and the rectal temperature of the mice was monitored every hour. Similar to the previous report, LPS can induce the body temperature of mice to rise to about 38° C. for less than 6 hours (
2.12 Disruption of Hsp90-a4 Interaction Impairs the Clearance of Bacterial Infection
In order to further study the role of the Hsp90-α4 integrin signal axis in the pathological process accompanied by high temperature and fever, Salmonella typhimurium infected mouse models were established. High-dose Salmonella typhimurium (SL1344) was injected by oral gavage to induce gastroenteritis and typhoid fever in mice. After injection of Salmonella typhimurium, WT and KI mice developed fever on the second day, and reached a maximum temperature of about 40° C. on the 4th day (
2.13 Disruption of Hsp90-a4 Interaction Inhibits Monocyte Recruitment to Draining Lymph Nodes in Mice During S. typhimurium Infection
Since α4 integrin is also expressed on the surface of some innate immune cells (such as monocytes), it can be conjectured that monocyte migration is also regulated by the Hsp90-a4 signal axis induced by fever. When mice were injected with S. typhimurium by oral gavage for 3 days, the increase of monocytes that migrated to PLNs, MLNs and PPs in KI mice was significantly less than that in WT mice (
2.14 Fever Promotes T Cell Trafficking Via a Thermal Sensory Hsp90-α4 Integrin Pathway
During fever, thermal stress up-regulates the expression of heat shock protein Hsp90 in immune cells. Hsp90 can specifically bind to α4 integrin and activate α4 integrin through inside-out signaling pathways. Moreover, the N-terminal and C-terminal domains of one Hsp90 molecule can directly bind to the intracellular regions of two α4 integrins simultaneously, thereby promoting the dimerization and clustering of α4 integrin on the cell surface and activating downstream FAK-RhoA pathway to regulate cytoskeleton rearrangement and promote immune cell migration (
| Number | Date | Country | Kind |
|---|---|---|---|
| 201811378994.5 | Nov 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/119171 | 11/19/2019 | WO | 00 |
