Screening The Interaction Proteins Of Cytoglobin By Yeast Two-hybrid System

Cytoglobin (CYGB) is a globin protein, and it was discovered more than a decade ago in a proteomic screen of fibrotic liver by a group of researchers in Japan and was originally named STAP (Stellate Activating Protein). In the light of its part similarity to other globins (Mb and Ngb) and currently available reports, several potential functions of CYGB have been considered in line with respiratory and stress-related activities. These include oxygen storage, transport, sensor, terminal oxidase activity, nitricxide dioxygenase activity and reactive oxygen species (ROS) scavenging activity. Additionally, CYGB is also thought to protect cells from ROS/RNS and deal with hypoxic conditions and oxidative stress in the cells. CYGB is associated with various diseases, such as organ fibrosis in liver, kidney, and pancreas, glaucoma, gastroesophageal reflux disease and several neurodegenerative disorders. Emerging new and intriguing roles of cytoglobin (CYGB) have attracted considerable attention of cancer researchers in recent years. Moreover, CYGB may act as a tumor suppressor in certain cancers and as an oncogene in others such as E-cadherin, which performs contradicting functions in different types of cancers.Cytoglobin (CYGB) has numerous biological functions. Our previous studys show that recombinant human cytoglobin can reverse the formation and development of liver fibrosis and atherosclerosis effectively. Moreover, plenty of evidences suggest that CYGB may function as a tumor suppressor, which is one of the highly explored areas in CYGB-related research recently. However, the molecular mechanisms of CYGB have not thoroughly been elucidated. To provide new clues for exploring the function Of CYGB, we performed yeast two-hybrid screening to identify proteins interacting with CYGB using Mate & PlateTM Library-Universal Human (Normalized). Seventeen novel CYGB interacting proteins including Homo sapiens ring finger protein 2 (RNF2) were obtained. The interaction of cytoglobin (CYGB) and ring finger protein 2(RNF2) was further confirmed in Y2H Gold yeast strain by a co-transformation of RNF2 and CYGB and in mammalian cells by a co-immunoprecipitation assay. Ring finger protein 2 (RNF2) is reportedly highly expressed in many different tumors and is important for cell proliferation, and it is an E3 ligase that targets p53 for p53 regulation through proteasome-dependent degradation in HCT116, and the negative Rnf2-mediated regulation of p53 affects cell cycle progression and apoptosis controlled by p53. Thus, we speculate that the interaction between CYGB and RNF2 may haxe an effect on tumor.In this study, the fine four parts, the first part is the construction of pGBKT7-CYGB bait recombinant plasmid and the detection of its expression in Y2H Gold yeast strain, and testing bait for autoactivation and toxicity in Y2H Gold yeast strain. The second part is screening proteins interacting with CYGB in Mate & Plate TMLibrary-Universal Human (Normalized) by using a yeast two-hybrid assay. The third part is verification the interaction of cytoglobin (CYGB) and ring finger protein 2(RNF2) in Y2H Gold yeast strain by a co-transformation of RNF2 and CYGB and in mammalian cells by a co-immunoprecipitation assay. The fourth part is the detection of gene relative mRNA levels in CYGB overexpression and control groups.In the first part, we used the pET28a-CYGB plasmid as a template and amplified CYGB coding sequence by PCR method, and the pGBKT7-CYGB recombinant plasmid was verified by digestion, PCR and sequencing the positive clones. And then, the expression of bait protein in Y2H Gold yeast strain was detected by Western-blot. The pGBKT7-CYGB and pGBKT7 were transformed into Y2H Gold yeast strain respectively, and the transformed products were spreaded onto SD/-Trp agar plates to test the bait for toxicity. Meanwhile, the transformed products pGBKT7-CYGB were spreaded onto SD/-Trp/X-a-Gal and SD/-Trp/X-a-Gal/AbA agar plates to test the bait for autoactivation.The results show that the pGBKT7-CYGB bait recombinant plasmid was successfully verified by digestion, PCR and sequencing. The expression of bait protein in Y2H Gold yeast strain was detected by Western-blot using an anti-MYC tag antibody, and the molecular weight is the same as expected which indicates the CYGB bait protein expresses in Y2H Gold yeast strain successfully. There was no difference in the number, size and growth rate between the yeast colony of pGBKT7-CYGB and pGBKT7 transformed yeast on SD/-Trp agar plates, which indicates the CYGB bait protein has no toxicity to Y2H Gold yeast strain. The yeast colony of pGBKT7-CYGB transformed yeast did not turn blue on SD/-Trp/X-a-Gal agar plates and could not grow on SD/-Trp/X-a-Gal/AbA agar plates, indicating that the CYGB bait protein can not autoactivate the report genes. In a word, the pGBKT7-CYGB bait recombinant plasmid is available for screening proteins interacting with CYGB in Mate & PlateTM Library-Universal Human (Normalized) by using a yeast two-hybrid assay.In the second part, we performed a control mating before we begin screening the library to familiarize ourself with the procedures and expected results of a two-hybrid assay. Positive Control Mating: Y2HGold [pGBKT7-53] and Y187 [pGADT7-T]. Negative Control Mating:Y2HGold [pGBKT7-Lam] and Y187 [pGADT7-T]. pGBKT7-CYGB bait recombinant plasmid is used for screening proteins interacting with CYGB in Mate & PlateTM Library-Universal Human (Normalized) by using a yeast two-hybrid assay. After that, we rescued the plasmid from yeast cells grown on QDO/X/A and amplified the insert fragments of pray library plasmids in positive colonys by PCR. And then, we analyzed the sequencing and blast results of the PCR products.The results show that the positive control colonies could grow and turn blue on SD/-Leu/-Trp/-His/-Ade/X-a-Gal/AbA(QDO/X/A) selective plates, while the negative colonies could not grow on SD/-Leu/-Trp/-His/-Ade/X-a-Gal/AbA(QDO) selective plates, which indicats the yeast two-hybrid system runs normally and is available for screening proteins interacting with CYGB in Mate & PlateTM Library-Universal Human (Normalized). The Y2H Gold yeast strain pGBKT7-CYGB was mated with the Mate & PlateTM Library-Universal Human (Normalized). After three times screening on the SD/-Leu/-Trp/-His/-Ade/X-a-Gal/AbA(QDO/X/A) plates,32 positive clones were detected and selected. After amplifying the insert fragments of pray library plasmids in positive colonys by PCR, we analyzed the sequencing and blast results of the PCR products.17 different Homo sapiens genes were identified by using the yeast two-hybrid analysis using CYGB as bait for the prey of the Mate & PlateTM Library-Universal Human (Normalized). RNF2 is one of them. Ring finger protein 2 (RNF2) is reportedly highly expressed in many different tumors and is important for cell proliferation, and it is an E3 ligase that targets p53 for p53 degradation, and its E3 ligase activity requires Bmi1 protein,a component of the polycomb group (PcG) complex. Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability. These results provide a possible mechanism explaining the oncogenic function of RNF2, and because RNF2 is important for cancer cell survival and proliferation, it might be an ideal target for cancer therapy or prevention.The third part is verification the interaction of cytoglobin (CYGB) and ring finger protein 2(RNF2) in Y2H Gold yeast strain by a co-transformation of RNF2 and CYGB and in mammalian cells by a co-immunoprecipitation assay. Using the small-scale transformation procedure according to Matchmaker Gold Yeast Two-Hybrid System User Manual, co-transformed 100 ng of each of the following pairs of vectors into Y2HGold Competent Cells: pGBKT7-CYGB+pGADT7-RNF2, Empty pGBKT7+pGADT7-RNF2, pGBKT7-53+pGADT7-T as a positive control, pGBKT7-lam+pGADT7-T as a negative control. And then the transformed Y2HGold Cells were plated on SD/-Leu/-Trp/X-a-Gal Agar and SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA agar. We used the pET28a-CYGB plasmid as a template and amplified CYGB coding sequence by PCR method, and the pCMV-MYC-CYGB recombinant plasmid was verified by digestion, PCR and sequencing the positive clones. HEK 293T cells were co-transfected using pENTER-FLAG-HIS-RNF2 together with pCMV-MYC-CYGB or pCMV-MYC vectors. Following transient transfection for 48 h, the cells were lysed in a mild lysis buffer. The supernatant was collected and incubated with ANTI-MYC M2 Affinity Gel overnight at 4℃, and then centrifuged for 30 sec at 7500g. The precipitates were washed with 100μl IP washing buffer four times. After that, the precipitates were eluted with 40μl 2×sample buffer and boiled for three minutes. Finally, the samples were centrifuged for 30 sec at 7500g and the supernatant was prepared for loading on SDS-PAGE and immunoblotting.The results show that all the Y2H Gold cells grew on SD/-Leu/-Trp agar plate, thus indicating all the vector pairs were successfully co-transformed into Y2H Gold Competent Cells. On the SD/-Leu/-Trp/X-a-Gal agar plate, only the yeast strain co-transformed with pGBKT7-CYGB+pGADT7-RNF2 turned blue, while the yeast strain co-transformed with pGBKT7+pGADT7-RNF2 did not. On the SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA agar plate, only the Y2H Gold cells co-transformed with pGBKT7-CYGB and pGADT7-RNF2 and positive control could grow and turn blue, indicating that RNF2 interacted with CYGB in Y2H Gold yeast strain.The results show that FLAG-tagged RNF2 protein was detected in the precipitates from cell lysates co-transfected with both proteins, but not from cell lysates co-transfected with FLAG-tagged RNF2 protein and MYC empty vector. In the summary, FLAG-tagged RNF2 protein was efficiently percipitated by MYC-tagged-CYGB fusion protein but not by MYC-tag alone, indicating that RNF2 interacts with CYGB in mammalian cells.In the fourth part, we detect the relative mRNA levels of CYGB/RNF2/P53/P21 in CYGB overexpression and control groups.The results show that, compared with control group, the relative mRNA levels of CYGB and RNF2 in CYGB overexpression group are 2.1592±0.32988 and 6.2408±0.82304,(P<0.001), which indicates that the mRNA levels of CYGB and RNF2 are elevated in CYGB overexpression group. Compared with control group, the relative mRNA levels of P53 and P21 in CYGB overexpression group are 0.4503±0.09536 and 0.4237±0.07985(P<0.001), which indicates that the mRNA levels of P53 and P21 are reduced in CYGB overexpression group.These results concluded that:1.We construct pGBKT7-CYGB bait recombinant plasmidand successfully, and the CYGB bait protein can express in Y2H Gold yeast strain. The CYGB bait protein has no toxicity to Y2H Gold yeast strain and can not autoactivate the report genes. In a word, the pGBKT7-CYGB bait recombinant plasmid is available for screening proteins interacting with CYGB in Mate & PlateTM Library-Universal Human (Normalized) by using a yeast two-hybrid assay.2.17 different Homo sapiens genes were identified by using the yeast two-hybrid analysis using CYGB as bait for the prey of the Mate & PlateTM Library-Universal Human (Normalized). RNF2 is one of them.3.We verify the interaction of cytoglobin (CYGB) and ring finger protein 2(RNF2) in Y2H Gold yeast strain by a co-transformation of RNF2 and CYGB and in mammalian cells by a co-immunoprecipitation assay successfully.4.In HCT116 cells, the overexpression of CYGB elevates RNF2 mRNA level, and reduces P53/P21 mRNA levels, which indicates that CYGB may play a negative regulation role on RNF2.