Moreover, immunoprecipitation indicates that FAM83H, SCRIB, and β-catenin, form a complex, and knock-down of either FAM83H or SCRIB accelerated proteasomal degradation of β-catenin. In conclusion, this study has found that the individual and combined expression patterns of nuclear FAM83H and SCRIB are prognostic indicators of gastric carcinomas and further suggests that FAM83H and SCRIB are involved in the progression of gastric carcinomas by stabilizing β-catenin.FAM83H primarily is known for its function in tooth development. Recently, a role for FAM83H in tumorigenesis, conjunction with MYC and β-catenin, has been suggested. Analysis of public data indicates that FAM83H expression is closely associated with SCRIB expression in human gastric cancers. Therefore, this study investigated the roles of FAM83H and SCRIB in 200 human gastric cancers and gastric cancer cells. In human gastric carcinomas, both the individual and combined expression patterns of the nuclear FAM83H and SCRIB were independent indicators of shorter survival of gastric carcinoma patients. In MKN-45 and NCI-N87 gastric cancer cells, the expression of FAM83H and SCRIB were associated with proliferation and invasiveness of cells. FAM83H-mediated  Howe cancers , 7. More cancers , 8–10. T cancers  and high cancers , liver [ cancers , kidney  cancers , and bon cancers . TherefoSCRIB (scribble) is a polarity protein expressed on the basolateral side of epithelial cells and is important in maintaining tight junctions . The losGastric cancer is ranked fifth in incidence  and thirhttp://gepia.cancer-pku.cn. Accessed 10 January 2020)  . The cutry 2020) . With thry 2020) . FAM83H-ry 2020) . SCRIB-Nry 2020) . SCRIB-Cry 2020) . Positivry 2020) .P = 0.002, RFS; P = 0.002) and CA19-9 , tumor stage , lymph node metastasis , venous invasion , tumor invasion , and the expression of FAM83H-N , FAM83H-C , SCRIB-N , SCRIB-C , and β-catenin-N   and relapse-free survival (RFS) were preoperative serum level of CEA  . The pro= 0.017) . In 150 = 0.017) .-/SCRIB-N-, FAM83H-N-/SCRIB-N+, FAM83H-N+/SCRIB-N-, and FAM83H-N+/SCRIB-N+ subgroups, the FAM83H-N-/SCRIB-N- subgroup had the longest survival and the FAM83H-N+/SCRIB-N+ subgroup had the shortest survival , intermediate (FAM83H-N+/SCRIB-N-), and poor (FAM83H-N+/SCRIB-N+) subgroups. In-between these subgroups, there was a significant difference in OS and RFS. The 5- and 10-year OS rates of the favorable (FAM83H-N-/SCRIB-N- or FAM83H-N-/SCRIB-N+) subgroup were 87% and 82%, respectively. The 5- and 10-year OS rates of the poor subgroup with FAM83H-N+ and SCRIB-N+ were 25% and 16%, respectively. The 5- and 10-year OS rates of the intermediate (FAM83H-N+/SCRIB-N-) subgroup were 60% and 39%, respectively   .Based on the prognostic significance of the expression of FAM83H and SCRIB in gastric carcinoma patients, we performed proliferation and migration/invasion assays after inducing knock-down or overexpression of FAM83H and SCRIB in gastric cancer cells. In MKN-45 and NCI-N87 gastric cancer cells, the proliferation of cells was inhibited with knock-down of FAM83H and increased with overexpression of FAM83H . In addiSCRIB also stimulated the proliferation and migration/invasion activity of MKN-45 and NCI-N87 gastric cancer cells –6D. The in vivo growth of NCI-N87 cells and loss of SCRIB suppressed in vivo tumor growth compared with controls transfected with empty vectors  [+/SCRIB-N+ subgroup of gastric carcinoma had the shortest survival. Moreover, FAM83H and SCRIB stimulated the proliferation and invasion of gastric cancer cells, and FAM83H overexpression-stimulated proliferation of gastric cancer cells was attenuated with a knock-down of SCRIB in vitro and in vivo. The expression of mRNA and protein of SCRIB was significantly altered according to FAM83H expression. However, the expression of FAM83H was not influenced by SCRIB alteration. These findings suggest that FAM83H and SCRIB are closely related to the progression of gastric carcinomas, and that there are tumorigenic roles of FAM83H in mediating SCRIB; furthermore, the expression of SCRIB was dependent on FAM83H. However, despite the close correlation between FAM83H and SCRIB, the knock-down of SCRIB could not completely attenuate the effect of FAM83H overexpression in In the cBioPortal public database, there was a strong correlation between the expression of FAM83H mRNA and SCRIB mRNA in gastric cancers  , 7. In aConcerning the role of FAM83H in conjunction with SCRIB in tumorigenesis, the oncogene MYC might be closely associated. In our previous study, we demonstrated that FAM83H was an intermediate of the oncogene MYC on the MYC-mediated proliferation and invasiveness of liver cancer cells . The expvia interaction with β-TrCP and USP47. However, despite direct interaction between SCRIB and β-catenin, the localization of β-catenin was not affected by SCRIB [In this study, the effect of FAM83H and SCRIB on the proliferation of gastric cancer cells was associated with the canonical Wnt/β-catenin pathway. The reduced expression of both FAM83H and SCRIB decreased the protein level of β-catenin and the expression of cyclin D1. When considering the important role of the Wnt/β-catenin pathway on cellular proliferation, FAM83H and SCRIB had an effect on cellular proliferation through the regulation of the Wnt/β-catenin pathway by stabilizing the β-catenin protein. In our results, FAM83H and SCRIB were shown to bind to β-catenin and prevent its proteasomal degradation. Consistently, the possibility of FAM83H-mediated stabilization of β-catenin has been suggested in colorectal cancers  and osteby SCRIB . Moreoveby SCRIB . Therefovia a regulation of EMT that accompanies disruption of cellular adhesion. Supportively, the cytoplasmic expression of SCRIB is inversely correlated with the membranous expression of E-cadherin [via transfecting with mutant SCRIB promoted hepatic and mammary tumorigenesis by activating PI3K-Akt signaling [In gastric cancer cells, FAM83H and SCRIB stimulated the invasiveness of cancer cells and FAM83H induced pulmonary metastasis in mice transplanted cancer cells with FAM83H overexpression. Acquiring invasive and metastatic potential is important in the progression of cancers, and EMT is an important phenotype of advanced carcinomas , 29, 30.cadherin . In addicadherin . Our resignaling , 24. In ignaling . In addiignaling , 35. WheIn conclusion, we demonstrate that FAM83H and SCRIB cooperatively activate the progression of gastric carcinoma by stabilizing β-catenin. Moreover, individual and co-expression patterns of the nuclear FAM83H and SCRIB might be used as novel prognostic markers of gastric cancers. Therefore, the FAM83H-SCRIB-β-catenin pathway might be a novel therapeutic target for the poor prognostic subgroups of gastric cancer, which express high levels of FAM83H and SCRIB.th edition of the American Joint Committee Cancer Staging System [th edition of the American Joint Committee Cancer Staging System [To investigate the clinicopathological significance of the expression of FAM83H and SCRIB in human gastric carcinoma, this study evaluated 200 gastric carcinomas from patients operated on between January 1997 and December 2005 at Jeonbuk National University Hospital. In addition, tissue samples from 20 cases of non-neoplastic gastric mucosa unrelated with gastric cancer, and 40 cases of gastric dysplasia were included in this study. At first evaluation, the cases were staged according to the 6g System , and 50 g System  and re-sg System . InformaThe expression of FAM83H, SCRIB, and β-catenin in human tissue samples were evaluated by immunohistochemical staining of tissue microarray sections. The tissue microarray was constructed from one 3.0 mm core area composed primarily of intact cells without degeneration. The tissue sections from the tissue microarray blocks were boiled with a microwave oven in pH 6.0 antigen retrieval solution  for 20 minutes. Immunohistochemical staining was performed with primary antibodies for the FAM83H , SCRIB , and β-catenin . Immunohistochemical staining for FAM83H, SCRIB, and β-catenin were evaluated separately for nuclear and cytoplasmic expression by two pathologists (KYJ and KMK) with consensus. The staining intensity was scored as negative (score 0), weak (score 1), intermediate (score 2), and strong (score 3) staining and the staining area was scored as no staining (score 0), ~1% (score 1), 2~10% (score 2), 11~33% (score 3), 34~66% (score 4), and 67~100% (score 5) staining area , 38–40. ® 2000 DNA transfection reagent  was used for transfection.Two human gastric cancer cell lines, MKN-45  and NCI-N87  were purchased from the Korean Cell Line Bank . The indicated cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum  and streptomycin and penicillin (100 U/ml). The knock-down of FAM83H was induced with shRNA for FAM83H . The FAM83H duplex had the sense and antisense sequences 5′-CACCGCTCATCTTC AGCACGTCACATTCAAGAGATGTGACGTGCTGAAGATGAGCTTTTTTG-3′ and 5′-GATCCAAA AAAGCTCATCTTCAGCACGTCACATCTCTTGAATGTGACGTGCTGAAGATGAGC-3′, respectively. To induce knock-down of SCRIB, we used hSCRIB CRISPR/Cas9 KO plasmid . The overexpression vector for SCRIB  and FAM83H  were purchased from GeneCopoeia . Lipofectamine3 cells in a 6-well plate and counting with a hemocytometer after 24, 48, and 72 hours. The MTT assay was performed by seeding 3x103 cells per well in 96 well plates for 24, 48, and 72 hours. The colony-forming assay was performed by plating 1x103 cells per well in 6-well culture dishes in triplicates and allowing them to grow and form colonies for ten days. The culture plates were fixed with cold methanol and stained with 0.01% crystal violet for 1 hour; then, the number of colonies was counted with GeneTools analysis software .Cell proliferation was evaluated by counting cells, a 3--2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay, and a colony-forming assay. Cell counts were performed by seeding 3x10in vitro, a 24-transwell migration or an invasion chamber  was used. The migration assay was performed by seeding 1x105 cells in serum-free medium in the upper chamber with 8 μm-pore filters. The invasion assay was performed by seeding 2x105 cells in serum-free medium in the upper 8μm-pore Matrigel Invasion Chamber . The bottom chamber consisted of 10% FBS containing RPMI-1640 for both migration and invasion assays. The migration and invasion chambers were incubated for 48 hours at 37 °C. The un-migrated and un-invaded cells on the upper surface of the chamber were removed using a cotton swab and the migrated and invaded cells on the underside of the insert were stained with DIFF-Quik staining solutions . The migrated or invaded cells were counted in five microscopic fields (magnification x200) per well.To evaluate migration- and invasion-activity of cells via SDS-PAGE and immunoblotted. Immunoprecipitation was performed using a Dynabeads-Protein A kit  and 10-Tube Magnet . The protein of interest was precipitated by pre-incubated 2 μg of primary antibodies with 1.5 mg of Dynabeads Protein A  at room temperature. Then, 500 μg of diluted cell lysate was added and incubated with Dynabead-antibodies complex at 4°C overnight. The proteins in the antibody-bound complex were pelleted, washed three times with ice-cold protein extraction solution NP-40  and denatured with 2x Laemmli sample buffer at 95 °C for 10 minutes. The eluted proteins were detected by immunoblotting.To obtain protein, cells were lysed in ice-cold PRO-PREP Protein Extraction Solution . The primary antibodies used in this study were against FAM83H , SCRIB , β-catenin , active (dephosphorylated) β-catenin , cyclin D1 , E-cadherin , N-cadherin , TGF-β1 , snail , vimentin , MMP2 , MMP9 , β-TrCP , USP47 , GSK3β , phosphorylated GSK3β (Ser9) , lamin B1 , GAPDH , and actin . Subcellular protein fractionation was performed using a Subcellular Protein Fractionation Kit  according to the manufacturer’s protocol. The fractionated samples were resolved Total RNA was obtained with an RNeasy Mini Kit  and reverse transcription was performed on 1.5 μg RNA with TaqMan Reverse Transcription Reagents . A quantitative reverse-transcription polymerase chain reaction was performed with Applied Biosystems Prism 7900HT sequence Detection System and SYBR Green PCR Master Mix . All experiments were carried out in triplicate, and the results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene. The primer sequences for the polymerase chain reaction are listed in Renilla luciferase plasmid DNA  in addition to empty vectors, shRNA for FAM83H, hSCRIB CRISPR/Cas9 KO plasmid, overexpression vector for FAM83H, or overexpression vector for SCRIB by using Lipofectamine® 2000 . Twelve hours after transfection, cells were lysed with 1x Passive Lysis buffer using Dual-Glo Assay . The firefly luciferase signals were monitored from 10 μl of each protein lysate by using the Dual-Luciferase Reporter Assay System , and normalized to the signals obtained from positive controls of co-transfected Renilla luciferase expression. The assay was performed in quadruplicate and repeated three times.For cell-based luciferase reporter assay, gastric cancer NCI-N87 and MKN-45 cells were plated in 6-well plates at ~50% confluency. The cells were co-transfected with 100 ng/well TOPflash or FOPflash plasmid DNA and 2 ng/well pRL-TK 6 NCI-N87 cells were injected subcutaneously on the back. The size of the implanted tumor was measured every week, and the tumor volume was calculated by the equation: tumor volume = length x width x height x 0.52. At five weeks after tumor cell inoculation, the mice were euthanized after anesthetizing with sodium pentobarbital and evaluated for the tumors. All animal experiments were performed with the approval of the institutional animal care and use committee of Jeonbuk National University .Eight-week-old male FoxnN.Cg/c nude mice  were used to establish the xenografted gastric cancer model. The mice were randomly divided into four groups with five mice in each group. According to the experimental groups, NCI-N87 cells were transfected with empty vector, FAM83H-overexpressed vector, vectors to induce knockdown of SCRIB, or both of FAM83H-overexpression vector and vector for knockdown of SCRIB. After that, 2x10MKN-45 gastric cancer cells were transfected with empty vector, shRNA for FAM83H, or hSCRIB CRISPR/Cas9 KO plasmid. After 24 hours post-transfection, the cells were treated with 30 μM cycloheximide  or 30 μM MG132  for 30 minutes to two hours. Western blotting for β-catenin  and actin  was performed on whole protein lysates. In addition, to evaluate the ubiquitination of β-catenin with knock-down of FAM83H or SCRIB, western blotting with anti-ubiquitin antibody after immunoprecipitation with anti-β-catenin antibody was performed. For this evaluation, MKN-45 cells were transfected with empty vector, shRNA for FAM83H, or hSCRIB CRISPR/Cas9 KO plasmid, and the cells were treated with 30 μM MG132 for two hours. The total cell lysates were immunoprecipitated with anti-β-catenin antibodies and blotted with anti-Ubiquitin antibody .t-test, and one-way ANOVA analysis. All experiments were done in triplicate, and the representative data are presented. All statistical analysis was performed using SPSS software . P values less than 0.05 were considered statistically significant.The positivity for the immunohistochemical expression of FAM83H, SCRIB, and β-catenin were determined by receiver operating characteristic curve analysis , 41, 42.


CD4+ T cell responses to cytomegalovirus (CMV) pp65 restricted by single HLA class II allotype defined in 45 healthy donors. The average magnitude of CD4+ T cell responses by HLA-DR allotypes was higher than HLA-DQ and HLA-DP allotypes. CD4+ T cell responses by DRA*01:01/DRB1*04:06, DQA1*01:02/DQB1*06:02, DPA1*02:02/DPB1*05:01 were higher among the other alleles in each HLA-DR, -DQ, and -DP locus. Interestingly, the frequencies of HLA-DR alleles and the positivity of specific allotypes showed an inverse correlation. One allotype within individuals is dominantly used in CD4+ T cell response in 49% of donors, and two allotypes showed that in 7% of donors, and any positive response was detected in 44% of donors. Even if one individual had several dominant alleles, CD4+ T cell responses tended to be restricted by only one of them. Furthermore, CD8+ and CD4+ T cell responses by HLA class I and class II were correlated. Our results demonstrate that the CD4+ T cell preferentially use a few dominant HLA class II allotypes within individuals, similar to CD8+ T cell response to CMV pp65.Within an individual, six different HLA class II heterodimers are expressed co-dominantly by two alleles of HLA-DR, -DQ, and -DP loci. However, it remained unclear which HLA allotypes were used in T cell responses to a given antigen. For the measurement of the CD4 The diversity of human leucocyte antigen (HLA) molecules is generated by mutations, driven and maintained by purifying selection or balancing selection, such as negative frequency-dependent selection or heterozygote advantage –5. High + T cells and CD4+ T cells respectively recognize endogenous and exogenous antigens presented by major histocompatibility complex (MHC) class I and II molecules on antigen-presenting cells for the initiation and maintenance of adaptive immune response  expressing a single HLA allele to enumerate the responses restricted by a single HLA allotype. Previously, allele dominance was defined that one or two alleles restricted CMV pp65-specific CD8+ T cell responses out of six HLA class I alleles within individuals  were purified by density gradient centrifugation using the Ficoll-Hypaque . PBMCs were then cryopreserved in liquid nitrogen suspended in FBS (Gibco) containing 10% dimethyl sulfoxide (Mylan) and 50% RPMI 1640 medium (Lonza). HLA typing was performed by Catholic Hematopoietic Stem Cell Bank , using polymerase chain reaction-sequencing based typing and next-generation sequencing  with genomic DNA isolated from red blood cells and granulocytes (Tiangen Biotech Corporation), as previously described . ResearccDNAs of HLA-DRA allele, 21 HLA-DRB1 alleles, 10 HLA-DQA1 alleles, 11 HLA-DQB1 alleles, three HLA-DPA1 alleles, 11 HLA-DPB1 alleles were obtained from lymphoblastoid cell lines of donors . Because of the SNPs near start codon and stop codon, cDNAs were amplified using each primer with short consensus sequence at the coding region and the sequence at 5′ UTR, 3′ UTR with mixed bases without a change in reading frames, and cloned into pCDH lentivector  without any tag using Gibson assembly . The sequences were verified frequently.6 293TN producer cells  were co-transfected  with 1.3pmol psPAX2 (RRID : Addgene_12260), 0.72pmol pMD.2G (RRID : Addgene_12259) and 1.64pmol single HLA class II allele-encoding plasmid, and cultured with two days in 10ml complete media. The aAPCs, which do not express HLA class I, HLA-DR, -DQ, and -DP, were transduced with alpha chain and beta chain specific lentivirus. Specifically, HLA-DRA*01:01 only transduced monoclonal aAPCs were isolated by limiting dilution and used to determine the titer of single HLA-DRB1 allotype-specific lentivirus. The 635 ul ± 277 ul HLA-DRB1 allotype-specific lentivirus is used to transduce 1×104 aAPC at an MOI of 20. The aAPCs expressing HLA-DRA*01:01 were transduced for each HLA-DRB1 allotype-specific lentivirus and expressed 67% ± 24% HLA-DR. The aAPCs expressing HLA-DQ and HLA-DP were established by transduction of 1ml alpha chain allele-specific lentivirus and 1ml beta chain allele-specific lentivirus to 1×104 aAPCs.The 5×10After the transduction of each allele-specific lentivirus, HLA-DR  or HLA-DQ , HLA-DP  positive aAPCs were isolated by flow cytometry-guided sorting . The aAPCs expressing HLA class II were cultured in RPMI 1640 supplemented with 2 mM L-glutamine,100 U/ml Penicillin-Streptomycin-Amphotericin B Mixture (Lonza), and 10% fetal bovine serum (Gibco). The aAPCs were cryopreserved after HLA class II expression was confirmed by using flow cytometry .4) expressing HLA were loaded with 60 nM of 15 amino acid peptides spanning the entire CMV pp65 protein with 11 amino acid overlap (JPT Peptide Technologies) for 3 h in serum-free media in a 96-well plate. The aAPCs loaded peptide pool were washed three times with serum-free media using centrifuge and microplate washer .Thawed aAPCs . The purity of CD4+ T cells was confirmed by flow cytometry (97% ± 2%).For the precise comparison of the frequency of antigen-specific CD4+ T cell responses restricted by a single HLA class II allotype were measured by IFNγ ELISPOT assay as described previously (4 antigen-pulsed aAPCs and 5×105 CD4+ T cells were incubated for 20h at 37°C. The spot forming cells were counted using an AID ELISPOT Reader System (AID Diagnostika GmbH). The magnitude of HLA-restricted antigen-specific CD4+ T cell response was calculated as [(response to aAPCs expressing HLA pulsed with peptide pools) – (response to aAPCs expressing HLA)] – [(response to aAPCs pulsed with peptide pools) – (response to aAPCs)].The CMV pp65-specific CD4eviously . Brieflyt-test (with a two-tailed test of significance). Values of P < 0.05 were considered significant. GraphPad Prism 7, NumPy /5×105 CD4+ T cells) > HLA-DP (75 SFCs/5×105) > HLA-DQ (54 SFCs/5×105), and that by HLA-DR was significantly higher than that by HLA-DQ (p = 0.01). However, there was no significant difference between the response by HLA-DQ and the response by HLA-DP. Among positive CD4+ T cell responses above 10 SFCs/5×105 CD4+ T cells, the magnitude of response above 100 SFCs/5×105 CD4+ T cells was considered strong. The magnitude under the strong response was considered as a weak response. The proportion of donors with strong response was 40% in HLA-DR, 11% in HLA-DQ, and 20% in HLA-DP.Among 20 HLA-DR alleles, the magnitudes of response by DRB1*04:06 were significantly higher than those of DRB1*13:02, DRB1*09:01, DRB1*08:03, DRB1*07:01, DRB1*14:05, DRB1*04:05 . These results suggest that the CMV infection contributes differently to HLA class I and class II diversity.The diversity of MHC molecules at the population level could be driven by the magnitude of response to a given pathogen , 37, 38.+ T cell response to CMV pp65 within individuals. In 49% of donors, only one allotype showed a strong response (p < 0.0001). These results are the first to directly prove allele dominance in HLA class II molecules within an individual.Since up to 6 HLA class II alleles are expressed in one individual, we analyzed the number of allotypes used in CD4We speculated that the dominant alleles, which showed a strong response at least once, are not always dominant within individuals, but there is a penetrance between dominant alleles. As expected, the probabilities of the highest responses varied among the dominant alleles   (p=0.5551). This expression pattern was similar to the magnitude of CD4+ T cell responses that were also higher in HLA-DR locus than in HLA-DQ and HLA-DP locus, as shown in + T cell responses according to the allotype . The membrane expression of HLA-DR and HLA-DP were not correlated with the magnitude of CD4+ T cell response of HLA-DR and HLA-DP .Similar to HLA-DR, -DQ, -DP loci in PBMCs , the mem(i) In the case of promiscuous epitopes restricted by HLA loci, T cell clones compete for a restriction between HLA alleles in an individual. (ii) In the case of selective epitopes restricted by specific HLA locus, T cell clones respond independently. However, since the aAPCs expressing a single HLA allotype were used, there is a limitation that the possible interaction or competition of multiple HLAs on true APC could not be observed. It is considered that cross-reactivity or competition for multiple HLA allotypes should be systematically investigated using T cell lines or TCR of dominant clones that responds to a single HLA allele . The participants provided their written informed consent to participate in this study.Y-SH and T-GK conceived and designed the study. Y-SH, H-AJ, and Y-HL performed experiments. S-MK, I-CB, H-JS, and H-IC contributed reagents/materials/analysis tools. Y-SH and T-GK analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.This study was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C3417).H-JS and H-IC were employed by the company ViGenCell Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rhizopus chinensis, we identified lipase (Lipr27RCL) mutagenesis sites that were associated with enhanced flexibility based upon B-factor analysis and multiple sequence alignment. We found that two mutated isoforms (Lipr27RCL-K64N and Lipr27RCL-K68T) exhibited enhanced thermostability and improved residual activity, with respective thermal activity retention values of 37.88% and 48.20% following a 2 h treatment at 50°C relative to wild type Lipr27RCL. In addition, these Lipr27RCL-K64N and Lipr27RCL-K68T isoforms exhibited 2.4- and 3.0-fold increases in enzymatic half-life following a 90 min incubation at 60°C. Together these results indicate that novel mutant lipases with enhanced thermostability useful for industrial applications can be predicted based upon B-factor analysis and constructed via site-directed mutagenesis.In order to improve the thermostability of lipases derived from  Lipases  mediate the hydrolysis of triglycerides into monoglycerides, fatty acids, diglycerides, and glycerol at oil-water interfaces , and canRhizopus-derived lipases have been shown to exhibit superior stability in acidic conditions, making them ideal for industrial applications. Dozens of commercial Rhizopus lipases have been produced to date, with Rhizopus chinensis CCTCC M201021 being the most commonly used strain for lipase production  that can lead to the deactivation of lipases lacking heat resistance. As such, it is vital that heat-resistant lipases should be developed so that they may be utilized as superior biocatalysts . A site-directed mutagenesis kit and DMT chemically competent cells were from TransGen . The R. chinensis lipr27RCL gene cloned in the pPIC9K vector was deposited in our laboratory. We obtained 4-nitrophenol palmitate (4-NPP) from Sigma. Mutagenic primers were synthesized by Shuoqing . All other chemicals were commercially available analytical grade materials.r27RCL as a template to construct four single-mutant lipase constructs  via introduction of point mutations into lipr27RCL through site-directed mutagenesis with a Fast MultiSite Mutagenesis System based on provided directions. The primers for these mutations are compiled in P. pastoris, and DNA sequencing was used to confirm the identity of the resulting recombinant strains. PCR thermocycler settings were as follows: 5 min at 94°C, then 28 cycles of 30 s at 94°C, 2.5 min at 55°C, and 2 min at 72°C.For site-directed mutagenesis experiments, we utilized pPIC9K-SalI was used to linearize the WT pPIC9K/Lipr27RCL and mutant constructs, which were then individually transformed into P. pastoris GS115 via electroporation. Yeast extract peptone dextrose (YPD) medium supplemented with 200 μg/mL G-418 (Geneticin) was used to select for clones, which were then grown for 2 days at 30°C in a 50 mL volume of buffered glycerol-complex media. This media was then exchanged for 50 mL of buffered methanol-complex media to drive expression of the lipase proteins. Protein purification was conducted using an Amicon centrifugal filter device (cutoff 10.000). Proteins were then sequentially purified via Q SepharoseTM Fast Flow anion exchange column chromatography and Phenyl-Sepharose 4 FF hydrophobic chromatography column chromatography, after which SDS-PAGE was used to confirm purification results  as the quantity of enzyme necessary to mediate the release of 1 μmol p-nitrophenol (p-NP) per minute from 4-NPP . Thermosm, VKmax, and kcat), enzymes were measured in a Tris–HCl (pH 9) buffer solution at 37°C with 4-PNN as a substrate provided across a range of concentrations (0.078125–10 mM), with reactions monitored based on p-NP production. Lineweaver–Burk plots were then used to fit the results and to determine these kinetic parameter values  from Applied Photophysics Limited (United Kingdom). All spectra were recorded at 20°C. Conditions, including pathlength: 1 mm, time-per-point: 1 s (25us × 40000), step size: 1 nm, and bandwidth: 1 nm, were used for scanning the 200–250 nm spectra range. CD spectra were collected from the solution of 50 mM sodium phosphate buffer  with the lipase concentration of 0.1 mg/ml. After three scans, the final spectrum was corrected by removing the recorded baseline of the PBS control medium.The X-ray crystal structure of Lipr27RCL was taken from PDB 6A0W, while structures for the four recombinant lipases Lipr27RCL-K64N, Lipr27RCL-W65I, Lipr27RCL-D66T, and Lipr27RCL-K68T, were constructed using the SWISS-MODEL server , using tThere is a linear relationship between B-factors determined based upon X-ray diffraction data and the mean square displacement of atoms relative to their average positions. As such, B-factors derived from protein crystal structures offer invaluable insights into the flexibility, stability, and dynamics of individual proteins. In the present study, we extracted and normalized the B-factor values of Cα atoms for Lipr27RCL from its crystal structure . ResidueBased on the results from B-factor analysis and multiple sequence alignment, we used site-directed mutagenesis to produce mutant forms of Lipr27RCL in which the 64K, 65W, 66D, and 68K residues had been mutated to 64N, 65I, 66T, and 68T, respectively. The resultant single mutant lipases  were termed Lipr27RCL-K64N, Lipr27RCL-W65I, Lipr27RCL- D66T, and Lipr27RCL-K68T for the purposes of this study. These lipases had the same molecular mass (39.50 kDa) as did Lipr27RCL when analyzed via SDS-PAGE . FollowiWe next assessed the changes in lipase thermostability as a result of this mutagenic campaign by assessing residual activity of these four Lipr27RCL enzymes and the WT isoform following a 90 min incubation at a range of temperatures. We found that all five of these enzymes functioned best at 40°C . Both Lip-NP method was used to monitor these reactions, with purified proteins being combined with a range of 4-PNN concentrations (0.078125–10 mM). Kinetic measurements revealed that the apparent Michaelis constant (Km) values for Lipr27RCL, Lipr27RCL-K64N, Lipr27RCL-W65I, Lipr27RCL-D66T, and Lipr27RCL-K68T were 0.36, 0.29, 0.45, 0.38, and 0.29 mM, respectively  of Lipr27RCL-K64N and Lipr27RCL-K68T was also increased . The Km and kcat/Km values for Lipr27RCL-D66T were similar to those for WT Lipr27RCL. However, the Km of Lipr27RCL-W65I was 1.25-fold that of Lipr27RCL, with a clear decline in kcat/Km  . The CD The CD spectra showed that compared with Lipr27RCL, the ellipticities of the α-helix and β-sheet of the Liper27RCL-K64N isoform changed obviously, while the ellipticity of α-helix of Lipr27RCL-K68T isoform had minor changes with almost no changes of β-sheet . These sTo fully understand the basis of enhanced thermostability in these recombinant lipases, MD simulations for Lipr27RCL and two single mutants (Lipr27RCL-K64N and Lipr27RCL-K68T) with improved thermostability were conducted at 60°C. To investigate the improved thermostability of mutants, novel interactions formed with the mutational sites were monitored during the simulations. It is discovered hydrogen bonding interactions between N64⋯S58 in Lipr27RCL-K64N was stronger than that in K64⋯S58 Lipr27RCL. Specifically, the hydrogen bond forming probability between K/N64⋯S58 were 70.6% and 62.5% in Lipr27RCL-K64N and Lipr27RCL, respectively . AdditioSimilarly, RMSF value of the mutated residue T68 in Lipr27RCL-K68T was smaller than that of K68 in Lipr27RCL, and the smaller RMSF value of T68 indicate the mutated site was relative stable during simulation . T68 in The development of thermostable lipases has clear and practical potential to meet with industrial demands. Furthermore, generating such stable lipases may simultaneously improve their kinetic efficiency . In the Normalized B-factor analysis of Lipr27RCL was the key step in the design of thermostable recombinant isoforms of this enzyme, given that B-factors offer insight into protein fluctuations, which are indicative of atom rigidity relative to their corresponding positions , and preBased on sequence and structural analyses, we found further evidence suggesting that Lipr27RCL-K64N and Lipr27RCL-K68T may exhibit greater thermostability relative to other untested lipase isoforms, leading us to focus our in depth characterization efforts on these isoforms. We had also constructed a quadruple mutant lipase  for this study, but found its thermostability to be 40% lower than the WT Lipr27RCL in a matter of minutes. This is likely at least in part due to the loss of hydrogen bond network interactions introduced by the I65 mutation, thereby adversely impacting the formation of hydrogen bond networks and the α-helix around N64 and T68. This loss of hydrogen bond networks in turn reduced the regional thermostability, markedly decreasing the thermostability of this quadruple mutant lipase. As such, other lipase isoforms containing the I65 mutation will similarly not display improved thermostability owing to this loss of hydrogen bond network interactions. Similarly, lipase isoforms bearing T66 mutations are likely to exhibit limited thermostability due to the lack of stable interactions between this residue and other surrounding residues. As such, the characterization of lipase isoforms containing mutated I65 or T66 residues was not tested in this work. However, the double mutant lipase (Lipr27RCL-K64N/K68T) exhibited cumulative thermostability of Lipr27RCL-K64N and Lipr27RCL-K68T , indicatIn summary, we found that we were able to produce novel mutant lipases (Lipr27RCL-K64N and Lipr27RCL-K68T) with improved thermostability through a combination of B factor analysis and site-directed mutagenesis, and these resultant enzymes represent attractive candidates for use in industrial applications.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.CZ performed the major experiments containing enzyme production, purification, and CD analysis. ZJ and MT carried out the experiments containing site-directed mutagenesis and characterization of lipases. LW, WQ, JH, and ZH helped with protein purification. NH analyzed the B-factor and MD simulation. QW and YM prepared experimental materials. BX, JD, and RZ dealt with the problems encountered in the experiment and coordinated the study. CZ wrote the manuscript. ZH and NH revised this manuscript. All authors read and approved the final manuscript.LW, WQ, JH, and ZZ were employed by Yunnan Walvax Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pharmacological inhibition or knockdown of ATGL alone prevents cAMP-dependent TXNIP degradation and thus significantly decreases glucose uptake and lactate secretion. Conversely, overexpression of ATGL amplifies the cAMP response, yielding increased glucose uptake and lactate production. Similarly, knockdown of TXNIP elicits enhanced basal glucose uptake and lactate secretion, and increased cAMP further amplifies this phenotype. Overexpression of TXNIP reduces basal and cAMP-stimulated glucose uptake and lactate secretion. As a proof of concept, we replicated these findings in human primary adipocytes and observed TXNIP degradation and increased glucose uptake and lactate secretion upon elevated cAMP signaling. Taken together, our results suggest a crosstalk between ATGL-mediated lipolysis and glucose uptake.Traditionally, lipolysis has been regarded as an enzymatic activity that liberates fatty acids as metabolic fuel. However, recent work has shown that novel substrates, including a variety of lipid compounds such as fatty acids and their derivatives, release lipolysis products that act as signaling molecules and transcriptional modulators. While these studies have expanded the role of lipolysis, the mechanisms underpinning lipolysis signaling are not fully defined. Here, we uncover a new mechanism regulating glucose uptake, whereby activation of lipolysis, in response to elevated cAMP, leads to the stimulation of thioredoxin-interacting protein (TXNIP) degradation. This, in turn, selectively induces glucose transporter 1 surface localization and glucose uptake in 3T3-L1 adipocytes and increases lactate production. Interestingly, cAMP-induced glucose uptake  Adipose tissue metabolism plays a critical role in maintaining healthy homeostasis for glucose and lipid metabolism . InsulinGlucose uptake is fundamental for cell metabolism and is mediated through a set of GLUTs and their associated machinery. Specialized cells, such as adipocytes, express GLUT4, which is largely dependent on insulin signaling for activation and surface translocation , 14. AnoHere, we show that elevated levels of cAMP rapidly degrade TXNIP in a dose-dependent manner and thereby release GLUT1 for surface localization. GLUT1 surface localization stimulates glucose uptake, which to a large extent is further metabolized to lactate. We uncover a mechanism by which ATGL activity regulates cAMP-dependent TXNIP degradation that in turn regulates GLUT1-, but not GLUT4-, mediated glucose uptake in adipocytes.A). We also exposed 3T3-L1 adipocytes to 8-Br-cAMP in a time-dependent manner up to 2 h and found that degradation of TXNIP initiated from 60 min onward . To determine whether other standard stimuli that increase the level of cAMP also cause degradation of TXNIP, we used multiple standard stimuli, such as forskolin, isoproterenol, and epinephrine. We found that, in parallel with 8-Br-cAMP, forskolin, isoproterenol, and epinephrine also significantly degrade TXNIP levels (C1–C3). Interestingly, cAMP-stimulated TXNIP protein expression changes are not reflected in mRNA levels, suggesting that cAMP-mediated TXNIP degradation is a post-translational event (3H-2-deoxy-d-glucose (DOG) uptake. We found that 8-Br-cAMP, as expected, increased glucose uptake both in a concentration- and time-dependent manner, which is paralleled with TXNIP degradation (D and E). Similar to that observed with 8-Br-cAMP, administration of other cAMP agonists, such as forskolin, isoproterenol, and epinephrine, also showed increased glucose uptake in 3T3-L1 adipocytes .Besides providing glycerol and fatty acids, lipolysis also generates several lipid derivatives and substrate molecules that regulate metabolism through multiple mechanisms, including gene transcription and post-translational modifications , 5. A goFig. 1al event . TXNIP, al event . In a neG and H). Both protein kinase B (AKT) and AMP-activated protein kinase (AMPK) have previously been shown to regulate glucose uptake via TXNIP degradation . These data suggest that cAMP acts via another pathway. Furthermore, we also tested insulin signaling activation, which was found to be unchanged (I). We observed that the PKA inhibitor, H89, significantly reduces cAMP-mediated glucose uptake in 3T3-L1 adipocytes and TXNIP degradation . Interestingly, these effects were present in neither 3T3-L1 preadipocytes  nor C2C12 myoblasts, two cell types lacking significant intracellular triglyceride stores and associated signaling pathways . These data support the role of TXNIP as a potent regulator of glucose uptake. Since cells not responding to cAMP with increased glucose uptake (preadipocytes and C2C12 cells) lack lipid droplets and associated pathways, which appear pivotal for TXNIP degradation, it is possible that they therefore could not upregulate glucose uptake in response to cAMP, despite the presence of the GLUT1 transporter (A and B).We took an aliquot of supernatant after 2 h of cAMP incubation and measured the lactate level. We found that, similar to that with glucose uptake, lactate production is concomitantly increased with increasing concentrations of cAMP, and this was also found for other cAMP agonists , G and Hradation , 16. We nchanged , and furnchanged I. We obspathways . Howevernsporter . Moreovensporter , A and BG) by providing internal glucose substrate for lactate secretion, we used the GSK3-specific inhibitor, SB216763. We found that GSK inhibition does not cause any broad alteration in the cAMP response, which makes any direct involvement of GSK3 kinase unlikely (A and B). Finally, to rule out any off-target effects of 8-Br-cAMP because of the chemical 8-Br moiety, or any similar overlapping pathway activated by cGMP, we used the similar analog, 8-Br-cGMP. We observed that 8-Br-cGMP did not show any alteration at the dose that is effective for 8-Br-cAMP  to initiate glycogenolysis. To investigate whether GSK3 phosphorylation contributes to the lactate production observed G by provunlikely , A and B-Br-cAMP .A and B). As hypothesized, and linked to TXNIP degradation, we found that atglistatin also completely abolished the cAMP stimulatory effect on glucose uptake and lactate production (C and D). We further investigated the potency of atglistatin in inhibiting lipolysis by measuring the lipolytic end product, glycerol. We measured atglistatin-mediated ATGL suppression by both varying cAMP concentration and using several doses of atglistatin (A and B). We observed that atglistatin robustly inhibited the cAMP-stimulated glycerol secretion without affecting HSL phosphorylation in the same setting, suggesting that atglistatin specifically blocks ATGL activity as previously reported (C).We next used atglistatin, a specific inhibitor of ATGL, which is the first lipase activated in response to elevated cAMP—primary signal for lipolysis , 19. Intoduction , C and Dlistatin , A and Breported , 20 . In agreement with our previous results, we found that cAMP-mediated glucose uptake is largely regulated through ATGL activity as knockdown of ATGL almost completely abrogated the cAMP-mediated effects. This is very similar to the atglistatin effect, which is a specific inhibitor of ATGL (C and D). Unlike ATGL, HSL and MGL knockdown still maintain a significant response to cAMP in terms of glucose uptake and lactate production (B and C). In line with these findings, we could demonstrate that knockdown of only ATGL led to significant inhibition of TXNIP degradation (D). We further validated our results with stable knockdown for ATGL though a specific shRNA approach. We observed that, similar to atglistatin and siRNA knockdown, shRNA-mediated knockdown of ATGL completely abrogates the cAMP-mediated glucose uptake and inhibits TXNIP degradation (A and B).Cellular lipolysis is essentially carried out by three lipases: ATGL, HSL, and MGL, which work in concert to break down TAG to fatty acids and glycerol. Our data suggest that lipolysis leads to degradation of TXNIP, and linked to this, increased glucose uptake and lactate secretion. To study the relative contribution of these three lipases to the observed phenotype of cAMP-mediated glucose uptake, lactate secretion, and TXNIP degradation, we knocked down ATGL, HSL, or MGL in 3T3-L1 adipocytes , A1–A3.  of ATGL , C and Doduction , B and Cradation D. We furradation , A and BE–G). This further supports the notion that ATGL-mediated lipolysis is necessary for cAMP-induced glucose uptake and lactate secretion.Overexpression (OE) of ATGL in 3T3-L1 adipocytes elicits a significant increase in glucose uptake and lactate secretion , E–G. ThA). We found that GLUT1 knockdown completely abolished cAMP-mediated glucose uptake, whereas GLUT4 knockdown had essentially no effect on cAMP-mediated glucose uptake in 3T3-L1 adipocytes as compared with a control siRNA . Similar trends were found for lactate release . This suggests that cAMP acts via ATGL and that this triggers a TXNIP degradation that specifically stimulates GLUT1 but not GLUT4 surface localization. We also adapted a complementary approach using stable knockdown of GLUT1 through shRNA in adipocytes and observed, as expected, a blunting of cAMP-mediated glucose uptake , we observed a dose-dependent increase in 8-Br-cAMP–linked increase in surface localization of GLUT1 (D) but not of GLUT4 (E). HA–GLUT4 constructs remain unresponsive to cAMP stimulation, however, as expected, insulin rapidly caused GLUT4 translocation in a dose-dependent manner, which was abolished by the AKT inhibitor, MK2206 (F). Furthermore, we tested whether cAMP-mediated GLUT1 surface localization was affected by pharmacological inhibition or knockdown of ATGL. We also found that ATGL inhibition by atglistatin or ATGL knockdown significantly reduces cAMP-mediated GLUT1 surface localization (G and H). Moreover, we found that TXNIP knockdown significantly upregulated GLUT1 surface localization and that was further amplified by 8-Br-cAMP (I). In conclusion, it appears that the increase in glucose uptake in 3T3-L1 adipocytes mediated by cAMP stimulation and linked to TXNIP degradation is mediated by GLUT1 but not GLUT4.As surrogate markers for surface localization of GLUTs, we used GLUT1 and GLUT4 tagged with FLAG and human influenza hemagglutinin (HA), respectively. In 3T3-L1 adipocytes, transfected to stably express these constructs, we used an indirect ELISA method to quantitate the surface to total ratio of GLUT1 and GLUT4. Mimicking previous glucose uptake experiments D, we obsof GLUT1 D but notof GLUT4 E. HA–GLU, MK2206 F. Furthelization , G and H-Br-cAMP I. In conA) and observed that TXNIP knockdown significantly upregulated glucose uptake in the basal state and also significantly amplified the cAMP response (B). There was also a modest effect on lactate secretion (C). In a next step, we used retrovirus to stably overexpress mouse TXNIP with a FLAG tag (D). TXNIP OE reduced glucose uptake in both the basal and cAMP-stimulated state (E), which was in stark contrast to the TXNIP knockdown phenotype (B). Similarly, lactate secretion was significantly downregulated (F). Enhanced lactate secretion, a sign of stimulated glycolysis, might be a way to rapidly replete the total cellular ATP pool. Thus, we observed that cAMP, in a time-dependent manner, reduced the total ATP pool (A), which is in concurrence with TXNIP degradation (B), glucose uptake (E), and lactate secretion (G) data. Moreover, coincubation with an ATGL inhibitor restricted the decrease in the ATP pool (B). Overall, these evidences suggest that adipocytes, in response to acute elevated cAMP levels, upregulate glycolysis, which in turn acts to replenish the ATP pool.We used siRNA to knockdown TXNIP expression in 3T3-L1 adipocytes A and obsresponse B. There ecretion C. In a nFLAG tag D. TXNIP ed state E, which henotype B. Similaegulated F. EnhancATP pool A, which radation B, glucose uptake E, and laecretion G data. MATP pool B. OveralA). We also observed increased glucose uptake and lactate production (B and C). In conclusion, these results indicate that the cAMP-mediated effects observed in 3T3-L1 adipocytes also are present in primary human adipocytes.To extend the physiological relevance of these observations, we studied how human primary adipocytes respond to increased cAMP levels. Similar to that we have observed in 3T3-L1 adipocytes, human primary adipocytes degrade TXNIP in response to increased cAMP stimulation A. We alsoduction , B and Cvia the circulation. Glucose will be metabolized to release ATP during glycolysis and in the tricarboxylic acid cycle. Moreover, instead of going directly into the mitochondria for ATP production, pyruvate can be reduced to form lactate, which in turn can be released to the circulation and serve as an important energy source for other cells . This is supported by a previous report studying glucose uptake in muscle cells in response to adrenergic stimulation (B). A selective knockdown of ATGL, but not that of HSL or MGL, will decrease TXNIP degradation and GLUT1-mediated glucose uptake (D and 4D). Moreover, the specific inhibitor of ATGL activity, atglistatin, also decreases TXNIP degradation and GLUT1-mediated glucose uptake (A–C and 4H). These data support the view that during induced cellular levels of cAMP, and when insulin levels are low, a cAMP-induced and TXNIP degradation–dependent mechanism will ensure the uptake of glucose that is necessary for cellular biosynthetic purposes. To ensure that this mechanism is active when fatty acid is the major energy substrate, the GLUT1-mediated glucose uptake is dependent on ATGL activity . Although many aspects of lipolysis have been studied in detail and are well characterized, recently, new functions of lipolytic products as signaling and transcriptional modulators have been identified  (B7880), atglistatin (SML1075), dorsomorphin, polybrene (hexadimethrine bromide), 2-deoxyglucose, SB 216763 (GSK inhibitor) (S3442-5MG), and H89 (B-1427) were purchased from Sigma–Aldrich. Antibodies against TXNIP (#14715), ATGL (#2138) Akt, phospho-Akt, phospho-AS160 (Thr642), and actin-horseradish peroxidase (#12262) were obtained from Cell Signaling Technologies. GLUT1 and GLUT4 antibody Akt inhibitor MK-2206 was purchased from Cayman. Super Signal West Pico and Super Signal West Dura chemiluminescent substrate were obtained from Pierce (Thermo Fisher Scientific). RNeasy mini kit was purchased from Qiagen. Radiochemicals and 3T3-L1 fibroblasts (American Type Culture Collection) were maintained in culture and differentiated into adipocytes as previously described. Lipofectamin 2000 and RNAiMAX (Life Technology) were used for transfections of mammalian cells according to the manufacturer's protocol. Retroviral particles were produced in human embryonic kidney 293T cells after transfection with a standard combination of expression and packaging vectors (Primary human preadipocytes were purchased from Promo cell (C-12730) and cultured according to manufacturer's instructions. Primary human preadipocytes were differentiated using differentiation media (811D-250) according to their instruction. Experiments were performed after full differentiation protocol and 95% adipocyte differentiation.Full-length human ATGL (MR226260) and TXNIP (MR206196) with C-terminal triple FLAG tag (3XFLAG) in pcDNA3.1 vectors were purchased from Origene and subcloned into retroviral pBabe-puro (ATGL) or pRetroXTRE3G (TXNIP) vectors for OE. FLAG-rGLUT1 vector was ordered from Addgene (#89571) and HA-GLUT4-GFP construct was kindly gifted from Prof. McGraw Lab, Weill Cornell Medicine, NY, USA. Both GLUT1 and GLUT4, along with their tagged versions (FLAG and HA), were subcloned into pBabe-puro vector for stable cell generation. ATGL (SASI_Mm01_00033377), HSL (SASI_Mm01_00090382), MGL (SASI_Mm01_00106711), TXNIP siRNA (SASI_Mm01_00138870), GLUT1 (SASI_Mm01_00068163), and GLUT4 (SASI_Mm01_00032134) together with scramble siRNA (SIC001) as a control were purchased from Sigma.pLKO.1 vector was purchased from Addgene (#10878), and the following set of shRNAs were cloned using standard procedures. After confirming shRNA clone presence, constructs were used for virus particle production, and subsequent stable cell generation was performed as mentioned in the previous section. Empty vector was used as a control throughout the experiments.Forward: CCGGCATCTCCCTGACTCGTGTTTCCTCGAGGAAACACGAGTCAGGGAGATGTTTTTGReverse: AATTCAAAAACATCTCCCTGACTCGTGTTTCCTCGAGGAAACACGAGTCAGGGAGATGForward: CCGGTGAGGAGTTCTACAATCAAACCCTCGAGGGTTTGATTGTAGAACTCCTCATTTTTGReverse: AATTCAAAAATGAGGAGTTCTACAATCAAACCCTCGAGGGTTTGATTGTAGAACTCCTCA3H(N)]-DOG taken up by cells, essentially as described earlier (3H(N)] (0.125 μCi/well) and 50 μM cold DOG and incubated for 10 min. Cells were then immediately washed 3 times with chilled PBS and lysed in 0.1% Triton-100. A small aliquot from the lysate was taken for protein measurement, and the remainder transferred to scintillation fluid for radioactive counting. The CPM values were normalized to the total cellular protein level for each well.Glucose uptake in differentiated 3T3-L1 adipocytes was assessed by analysis of 2-[1,2- earlier . Assays Differentiated adipocytes stably expressing tagged GLUT1 or GLUT4 were serum starved for 2 h in Dulbecco's modified Eagle's medium serum-free media. During this starvation, 8-Br-cAMP was incubated throughout starvation to assess GLUT1/GLUT4 surface localization. As a positive control for GLUT4, insulin was added during the last 20 min to trigger GLUT4 translocation. To see the effect of atglistatin on GLUT1 surface levels, cells were preincubated with atglistatin before adding 8-Br-cAMP. After completion of serum starvation, cells were immediately washed with chilled PBS and fixed with 3.7% formaldehyde for 7 min. Cells were washed 3 times with PBS and incubated with FLAG or HA antibody for 1 h at 37 °C. Cells were washed 3 times with PBS and incubated with secondary horseradish peroxidase–linked antibody for 1 h. Cells were washed 2 to 3 times, and 3,3′,5,5′-tetramethylbenzidine substrate was added. After 10 to 20 min of development, the reaction was stopped with 0.1 N HCl, and absorbance was measured at 450 nm. Total FLAG/HA  was determined using permeabilization with Triton X-100 (0.01%) and subtracted from surface values to obtain surface-to-total ratio. Control cells were taken as a blank control and subtracted from all values.For Western blots, cells were lysed in CST buffer , supplemented with protease and phosphatase inhibitor cocktails according to the manufacturer's recommendations. Protein concentrations were measured using the BCA protein assay kit (ThermoFisher). Proteins were separated by SDS-PAGE on NuPAGE Novex 4 to 12% Bis–Tris protein gels and transferred to polyvinylidene fluoride membranes. Specific proteins were detected with the indicated primary antibodies. Horseradish peroxidase–coupled secondary antibody was visualized with SuperSignal West Pico or SuperSignal West Dura Chemiluminescent Substrates on a LAS-4000 Luminescent Image analyzer (FujiFilm). Density of the bands was quantified using ImageJ software , and results were expressed as fold changes compared with the signal observed in the control cells (infected with empty vector) after normalization to actin level.Total RNA from adipocytes was isolated using RNeasy mini kit according to the manufacturer's instructions. Reverse transcription of 1 μg of total RNA was carried out using the First-Strand cDNA synthesis kit. Expression levels of specific mRNAs were quantified by real-time PCR using Power SYBR green PCR Master Mix on ABI Prism 7900 Sequence Detection System  and normalized to 36B4. All samples were analyzed in triplicate, and mean values were calculated.Lactate was measured in cAMP-treated supernatant samples using EnzyChrom L-lactate Assay Kit (ECL-1000) as per manufacturer's protocol. Total cellular ATP content was determined using EnzyLight ATP Assay Kit (EATP-100) according to manufacturer's protocol. Glycerol measurements were performed with supernatant samples taken after various treatments and secreted glycerol measured using the Glycerol Assay Kit (MAK117) according to manufacturer's protocol.p-Values were calculated by ANOVA and Student's t test. p-Values of <0.05 were defined as statistically significant.Data are expressed as mean ± SD. All the data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


Branched-chain α-keto acid dehydrogenase kinase (BCKDK), the key enzyme of branched-chain amino acids (BCAAs) metabolism, has been reported to promote colorectal cancer (CRC) tumorigenesis by upregulating the MEK-ERK signaling pathway. However, the profile of BCKDK in metastatic colorectal cancer (mCRC) remains unknown. Here, we report a novel role of BCKDK in mCRC. BCKDK is upregulated in CRC tissues. Increased BCKDK expression was associated with metastasis and poor clinical prognosis in CRC patients. Knockdown of BCKDK decreased CRC cell migration and invasion ex vivo, and lung metastasis in vivo. BCKDK promoted the epithelial mesenchymal transition (EMT) program, by decreasing the expression of E-cadherin, epithelial marker, and increasing the expression of N-cadherin and Vimentin, which are mesenchymal markers. Moreover, BCKDK-knockdown experiments in combination with phosphoproteomics analysis revealed the potent role of BCKDK in modulating multiple signal transduction pathways, including EMT and metastasis. Src phosphorylated BCKDK at the tyrosine 246 (Y246) site in vitro and ex vivo. Knockdown and knockout of Src downregulated the phosphorylation of BCKDK. Importantly, phosphorylation of BCKDK by Src enhanced the activity and stability of BCKDK, thereby promoting the migration, invasion, and EMT of CRC cells. In summary, the identification of BCKDK as a novel prometastatic factor in human CRC will be beneficial for further diagnostic biomarker studies and suggests novel targeting opportunities. Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies and accounted for 1.8 million new cases and 540 000 deaths worldwide in 2018 . In addiOne of the ten characteristics of tumors is the alteration of cell metabolism, which is both a cause and an effect of tumor progression. In the past decade, many enzymes involved in glucose and lipid metabolisms have become the focus of research, including pyruvate kinase M2 (PKM2) , hexokinBCAAs can be employed for protein synthesis or oxidized for energy by tumors . Many caThe branched-chain α-keto acid dehydrogenase (BCKDH) complex catalyzes the second rate-limiting irreversible step of BCAAs catabolism , and theHowever, the relevance of BCKDK to human cancer remains poorly understood. Therefore, we investigated the role of BCKDK in human cancer and found that elevated BCKDK promoted CRC tumorigenesis by upregulating the MEK-ERK signaling pathway . MEK wasThe non-receptor protein tyrosine kinase Src, was the first tyrosine kinase found to be significantly more active in the progression of most solid tumors . Src kinIn the present study, we found that BCKDK expression was elevated in mCRC tissues and positively associated with poor survival of CRC patients. However, at the cellular level, direct supplementation with BCAAs failed to facilitate the migration and invasion of CRC cells, indicating that BCKDK exerted its prometastatic function through a BCAAs-independent manner in CRC cells. Using in vitro kinase assays in combination with a publicly available phosphorylation prediction software program, we identified a novel upstream of BCKDK, Src, which phosphorylated BCKDK at the tyrosine 246 (Y246) site in vitro and ex vivo. Importantly, phosphorylation of BCKDK at Y246 by Src enhanced the activity and stability of BCKDK, leading to enhanced migration, invasion, and EMT of CRC cells. Furthermore, elevated p-BCKDK (Y246) expression was also proved to be associated with CRC metastasis and poor prognosis in CRC patients. As an upstream regulator of BCKDK, inhibition of Src downregulated the phosphorylation of BCKDK, suggesting the necessary role of Src in maintaining the prometastatic function of BCKDK. Taken together, these results demonstrate the significance of the Src/BCKDK axis in human CRC and provide a promising novel target for mCRC targeted therapy.n = 70, ***p < 0.001) and rectal adenocarcinoma  than in corresponding normal tissues.To understand the role of BCKDK in CRC progression, the mRNA expression level of BCKDK was first analyzed by using the Oncomine database. The online analysis showed that the mRNA level of BCKDK was significantly higher in colorectal carcinoma  compared with nonmetastatic patient tissues (n = 74) . Moreover, the BCKDK protein expression profile and the clinical information of CRC patients were combined to further determine whether BCKDK protein expression was associated with clinical prognosis, and this analysis was achieved by applying the Kaplan-Meier analysis. The outcomes illustrated that CRC patients with high BCKDK protein levels had significantly worse overall survival (OS) than those with low levels . Based on the above results, we conclude that BCKDK plays an important role in mCRC and might be a valuable indicator for poor prognosis in CRC.Next, the protein level of BCKDK was evaluated in the tissue microarrays (TMAs) of CRC patients by immunohistochemical (IHC) staining. The results indicated that BCKDK protein levels were upregulated in metastatic CRC tissues . As BCKDK is a negative regulator of BCAAs catabolism, knockdown of BCKDK accelerates BCAAs breakdown. Although previous results have demonstrated that high BCAAs stimulation could not accelerate the migration and invasion of CRC cells  antibody indicated that Src could phosphorylate BCKDK-WT, whereas a mutation in BCKDK at Y246 eliminated the phosphorylation Fig. .Despite the fact that BCKDK is known to reside in the mitochondrial subcellular compartment, a recent study showed that BCKDK was preferentially localized in the cytosolic fraction, and was also detected in the mitochondrial fraction . Src is 4-Src-His were transiently transfected into HEK293T cells, and phosphorylation of BCKDK was determined by western blot with the p-BCKDK (Y246) antibody. The results indicated that Src phosphorylated BCKDK in a dose-dependent manner under EGF treatment. Moreover, as shown in Fig. 4-Src-His and pCMV-BCKDK-WT-Flag were individually transfected or co-transfected into HEK293T cells, the phosphorylation of BCKDK at Y246 was dramatically elevated upon Src transfection. In the previous work, we found that the Y246 mutant of BCKDK abrogated the phosphorylation effect of Src on BCKDK in vitro, and we wondered the BCKDK mutant affects the phosphorylation effect of Src on BCKDK ex vivo. Therefore, Flag-BCKDK-WT and Flag-BCKDK-Y246F were co-transfected with His-Src, respectively, into HEK293T cells. Western blot using the p-BCKDK (Y246) antibody was performed to evaluate the phosphorylation effect of Src on BCKDK ex vivo. The results suggested that Src could phosphorylate BCKDK-WT, whereas a mutation in BCKDK at Y246 significantly downregulated the phosphorylation effect  and knockout (Src−/−) mouse embryonic fibroblasts (MEFs) cells were used to detect p-BCKDK (Y246) expression by western blot. Indeed, as shown in Fig. +/+ cells, we observed absolute inhibition of p-BCKDK (Y246) expression and prominent inhibition of p-MEK1/2 (S221) and p-BCKDHA (S293) expression in Src−/− cells. Altogether, these results indicate that intracellular Src inhibition leads to decreased phosphorylation of BCKDK and its downstream targets. In other words, phosphorylation of BCKDK at Y246 by Src enhances the activity of BCKDK by promoting the phosphorylation of its downstream molecules.To further identify whether it is possible that loss of Src induces completely inhibits the phosphorylation of BCKDK, Src WT , Src+/+ and Src−/− cells were treated with cycloheximide (CHX) to prevent de novo protein synthesis, and the remaining BCKDK protein level was investigated. Indeed, Src+/+ significantly elevated the half-life of BCKDK compared with Src−/− .Interestingly, we observed that the expression of total BCKDK was also sharply decreased in Srclls Fig. , suggest−/− Fig. . In addi−/− Fig. . These r4-Src-His, WT, and Y246F forms of BCKDK were transfected into HEK293T cells. After 48 h, the cell lysates were harvested and immunoprecipitated with BCKDK antibody, and the ubiquitination signal was measured by western blot. We found that the presence of Src markedly reduced the ubiquitination of BCKDK-WT, whereas the Y246 mutation resulted in increased ubiquitination of BCKDK . Then, transwell cell migration and invasion assays were conducted to elevate the metastatic phenotypes of cells. As shown in Fig. p < 0.05, **p < 0.01, ***p < 0.001). Consistently, in the HT29 cell line, the migration and invasion rates were remarkably upregulated in BCKDK-WT cells compared with control and BCKDK-Y246F cells .As phosphorylation is one of the most extensively studied posttranslational modifications that orchestrates a variety of cellular functions , it is nTo better understand the effects of BCKDK phosphorylation on CRC cell EMT, we performed western blot analysis. As shown in Fig. n = 38) compared with non-mCRC tissues (n = 40) (***p < 0.001). Moreover, Kaplan-Meier survival analysis illustrated that elevated p-BCKDK (Y246) expression was associated with bad prognosis in CRC patients . These demonstrate that p-BCKDK (Y246), as well as BCKDK, is upregulated in mCRC tissues and linked to worse prognosis of CRC patients.In the previous work, we demonstrated that BCKDK expression was elevated in mCRC tissues and associated with poor OS in CRC patients Fig. . TherefoTo gain comprehensive insight into the BCKDK signaling network, especially signaling involved in cancer cell metastasis, we combined BCKDK knockdown with phosphoproteomics analysis . ControlThese phosphoproteins were divided into four groups according to their experiment/control ratio, named Q1 to Q4 : Q1 (0 < ratio ≤ 1/2), Q2 (1/2 < ratio ≤ 1/1.5), Q3 (1.5 < ratio ≤ 2), and Q4 (ratio > 2). With a threshold of fold-change >1, Q1–Q2 and Q3–Q4 were the down- and upregulated groups, respectively. Enrichment-based clustering analyses were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to profile cellular pathways of Q1–Q4 groups regulated by BCKDK Fig. . SubsequBased on the above KEGG analysis, a selected list of BCKDK-downregulated phosphoproteins is shown in Fig. n = 10 per group), were adopted to validate the significance of BCKDK in CRC metastasis in vivo. As shown in Fig. p < 0.001). In addition, hematoxylin-eosin (H&E) staining was used to detect the location of metastatic nodules, and the results indicated that BCKDK knockdown resulted in a lower incidence of lung metastasis in vivo  metastatic HCT116 cells, into BALB/C nude mice  and were cultured in Dulbecco’s modified Eagles’ medium  with 10% FBS. All cells applied in this study were cultured at 37 °C in a humidified 5% CO2 atmosphere. The cell transfection assays were performed using Simple-fect reagent  following the manufacturer’s instructions. Corresponding blank vectors were added for adjustment to ensure that an equal amount of total DNA was transfected for each individual group. Puromycin and G418  were applied for knockdown and overexpression stable cell lines screening, respectively.The HCT116, SW620, DLD1, HCT15, SW480, HT29, and HEK293T cell lines were purchased from American Type Culture Collection . The CRC cell lines HCT116, SW620, HCT15, HT29, SW480, and DLD1 were grown in RPMI 1640 Medium  with 10% fetal bovine serum , and HEK293T cells were cultured in Dulbecco’s modified Eagles’ medium  supplemented with 10% FBS. Colorectal adenocarcinoma LoVo cell line was obtained from the Cell Bank of the Chinese Academy of Sciences  and were cultured with F-12K medium  containing 10% FBS. The SrcThe β-actin  and BCKDK (E-12)  antibodies were purchased from Santa Cruz Technology, Inc. . Flag antibody  was purchased from Sigma-Aldrich . His Mouse monoclonal antibody  was obtained from Pregene, Inc . All the following antibodies were purchased from Cell Signaling Technology : p-BCKDHA (S293) , BCKDHA , MEK1/2 , p-MEK1/2 (S221) , p-ERK1/2 (T202/Y204) , ERK1/2 , p-RPS6 (S235/236) , p-RPS6 (S240/244) , RPS6, E-cadherin , N-cadherin , and Vimentin . p-BCKDK (Y246) antibody was prepared by Abiocode, Inc . Rabbit  and mouse  antibodies were obtained from EarthOx Life Sciences . We purchased BT2 (Cat No. ZC-26488) from ZZBIO. CO.LTD . Dasatinib (Cat No. S1021) was purchased from Selleckchem, Inc .The plasmids of pCMV-BCKDK-WT-Flag, pCMV-BCKDK-Y246F-Flag, pLKO.1-shSrc (#1-#7) and pLKO.1-shBCKDK (#1-#2) were constructed by our laboratory. All PCR products were confirmed by sequencing analysis . The sequences for shRNAs against human BCKDK are: 1. 5′-CCGGTCAGGACCCATGCACGGCTTTCTCGAGAAAGCCGTGCATGGGTCCTGATTTTTG-3′; 2. 5′-CCGGACGCTGACTTCGAGGCTTGGACTCGAGTCCAAGCCTCGAAGTCAGCGTTTTTTG-3′. The sequences for shRNAs against Src are: 1. 5′-CCGGGCTCGGCTCATTGAAGACAATCTCGAGATTGTCTTCAATGAGCCGAGCTTTTTG-3′; 2. 5′-CCGGGACAGACCTGTCCTTCAAGAACTCGAGTTCTTGAAGGACAGGTCTGTCTTTTTG-3′; 3. 5′-CCGGGTCATGAAGAAGCTGAGGCATCTCGAGATGCCTCAGCTTCTTCATGACTTTTTG-3′; 4. 5′-CCGGTCAGAGCGGTTACTGCTCAATCTCGAGATTGAGCAGTAACCGCTCTGATTTTTG-3′; 5. 5′-CCGGGCTGACAGTTTGTGGCATCTTCTCGAGAAGATGCCACAAACTGTCAGCTTTTTTG-3′; 6. 5′-CCGGCATCCTCAGGAACCAACAATTCTCGAGAATTGTTGGTTCCTGAGGATGTTTTTTG-3′; 7. 5′-CCGGCATCCTCAGGAACCAACAATTCTCGAGAATTGTTGGTTCCTGAGGATGTTTTTG-3′. A scrambled siRNA with a sequence lacking significant homology to the human genome database was used as the control siRNA (shMOCK).3VO4, 1% NP-40, 0.1% SDS, 1 mmol/L aprotinin, and 1 mmol/L PMSF), sonicated three times and centrifuged to obtain the supernatant. For co-IP assays, IP buffer  was used to acquire IP samples. After sonicated and centrifuged, the supernatant fractions containing equal amounts of protein were subjected to incubation with corresponding antibody and protein A/G plus-agarose beads . For western blot, protein samples were boiled and separated on 7.5–15% SDS-PAGE gels followed by transferring to PVDF membranes . After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 30 min, the membranes were incubated with specific primary antibodies overnight at 4 °C. Finally, antibody-bound proteins were detected by chemiluminescence .Cells cultured for western blot were harvested in RIPA buffer  and shBCKDK#2 cells were labeled with “light isotopic amino acids” (L-Lysine/L-Arginine). To achieve more than 98% labeling efficiency, the cells was cultured for more than six generations before being harvested. First, the labeling efficiency was detected. Next, a total of 2 mg of mixed cell lysates  were digested with trypsin. The tryptic peptides were fractionated by high pH reverse-phase HPLC using Agilent 300Extend C18 column . Then each fraction was subjected to phosphopeptide enrichment. The specific peptides were selected for in-solution digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. The resulting MS data were processed using Maxquant search engine (v.1.5.2.8). Finally, bioinformatics methods analysis were performed to analysis the MS data.For Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based phosphoproteome experiments, HCT116 cells were infected with lentiviruses encoding control hairpin (shMOCK) or shRNA specific for BCKDK (shBCKDK#2), then BCKDK knockdown efficiency was tested by Western blot before proceeding to phosphoproteomics experiments. Subsequently, cells were metabolically labeled with the SILAC protein quantitation kit : the control cells were labeled with “heavy isotopic amino acids”  to evaluate the migration of cells.The wound healing assays were applied to determine the migration ability of cells. 2 × 105 cells suspended in 150 mL serum-free medium were seeded onto the upper chamber of 24-well plates, and 700 mL of medium with 10% FBS was added to the lower chamber. 48 h later, the medium was removed from the upper chamber. The non-invading cells on the upper side of the chamber were removed thoroughly with a clean cotton swab. Then cells on the bottom side of the membrane were fixed with 4% paraformaldehyde for 30 min, the migrated cells were stained with 0.1% of crystal violet (Sangon Biotech) for 15 min Finally, the stained cells were counted by microscopy. Results represent the average number of cells in three fields per membrane in triplicate inserts. As for cell invasion assays, the matrigel was diluted according to the manufacturer’s recommendations and added onto the chambers before seeding cells, then performed in the same manner as cell migration assays.Chambers  without or with matrigel  were used to investigate migration and invasion ability of cells, respectively. Systems without Matrigel were used to measure the migration ability of cells. 1 × 10All the experiments above were performed in triplicate, and results were presented as mean value ± SD.4/well) were seeded in 96-well plates for 24 h, then treated with BT2  for 24 and 48 h, respectively. Subsequently, 20 μL 0.5% MTT was added per well and incubated for 4 h. The culture media was discarded and 150 μL of dimethyl sulfoxide (DMSO) was added. After 15 min, when the crystal was fully dissolved, the absorbance value was measured at OD490 nm on an enzyme immunoassay analyzer (Bio-Rad).The cell viability was analyzed by MTT assay. HCT116 and SW620 cells (1 × 1032P] ATP) at 37 °C for 2 h . The reactions were stopped by adding 5× SDS loading buffer. Finally, the phosphorylation signal was detected by autoradiography .The inactive human BCKDK recombinant protein (# TP303601) was obtained from Beijing OriGene Technology. Src active kinase was purchased from Millipore Corp . The inactive substrate (BCKDK) and the active kinase (Src) were incubated in a 30 μL reaction system  were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 30 min, and blocked in 10% FBS for 30 min Then the cells were incubated overnight with primary antibodies  at 4 °C. On the second day, the cells were incubated for 2 h at room temperature with the Alexa Fluor 488 (green for BCKDK) or Alexa Fluor 546 (red for Src) secondary antibodies while keeping in the dark. The chromatin was stained by DAPI. Images were captured by LSM700 confocal microscopy .TMAs of human CRC were obtained from Urology, Xijing hospital of the Fourth Military Medical University. This study was performed with the permission of the ethical committee of the Fourth Military Medical University. The IHC staining analysis was performed using BCKDK (1:50) and p-BCKDK (Y246) (1:50) antibodies. The immunostaining intensity were graded following the Remmele scoring method. The positive group was defined by score > 3. Representative images were obtained by magnified  with an Olympus Imaging System Microscope.+/+ and Src−/−) and transiently transfected HEK293T cells were treated with 20 and 80 ng/mL EGF, respectively. Then 100 μg/mL of cycloheximide  was supplemented to the medium to block the new protein synthesis. Cells were harvested at a range of time points and the levels of aim proteins were analyzed by western blot.The MEFs cells (Srcn = 10 per group) were injected in their tail veins with 1 × 106 control (shMOCK) cells or BCKDK knockdown (shBCKDK#2) cells. The mice were observed daily and sacrificed after 8 weeks. The number of tumor nodules on the surface of lungs was counted and statistically evaluated. The collected lungs were fixed with formalin and submitted for hematoxylin-eosin staining (H&E). Medical Ethics Committee of the Tongji Medical College, Huazhong University of Science and Technology approved the protocols for animal experiments.The BALB/C-nu/nu nude mice were obtained from Beijing Vital River Laboratory Animal Technology Corp. For in vivo tumor metastasis assays, 5–6-week-old BALB/C-nu/nu nude mice . All data were collected from three independent experiments and the results were expressed as mean ± SD. 1-way ANOVA or a two-tailed Student’s t test was performed to analyze the statistically significance. p values < 0.05 was considered as significant. *p < 0.05; **p < 0 01; ***p < 0.001.Statistical analysis in this study were performed using Supplementary figure and table legendsFigure S1Figure S2Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4


TM and TRIKAFTATM are approved for people who have the F508del mutation on at least one allele. In this study we examine the effects of potentiator and corrector combinations on the rare mutation c.3700A>G. This mutation produces a cryptic splice site that deletes six amino acids in NBD2 (I1234-R1239del). Like F508del it causes protein misprocessing and reduced chloride channel function. We show that a novel cystic fibrosis transmembrane conductance regulator CFTR modulator triple combination , rescued I1234-R1239del-CFTR activity to WT-CFTR level in HEK293 cells. Moreover, we show that although the response to ORKAMBI was modest in nasal epithelial cells from two individuals homozygous for I1234-R1239del-CFTR, a substantial functional rescue was achieved with the novel triple combination. Interestingly, while both the novel CFTR triple combination and TRIKAFTATM treatment showed functional rescue in gene-edited I1234-R1239del-CFTR-expressing HBE cells and in nasal cells from two CF patients heterozygous for I1234-R1239del/W1282X, nasal cells homozygous for I1234-R1239del-CFTR showed no significant response to the TRIKAFTATM combination. These data suggest a potential benefit of CFTR modulators on the functional rescue of I1234-R1239del -CFTR, which arises from the rare CF-causing mutation c.3700A>G, and highlight that patient tissues are crucial to our full understanding of functional rescue in rare CFTR mutations.The combination therapies ORKAMBI CFTR gene which result in defects in protein quantity, biosynthetic processing, stability, regulation and/or channel gating. While >2000 mutations have been identified in the CFTR gene, caused mainly by missense, frameshift, splicing or nonsense mutations [TM) [TM; ivacaftor) [The Cystic Fibrosis transmembrane conductance regulator, CFTR, mediates PKA-regulated, ATP-dependent chloride and bicarbonate flux across apical membranes of polarized epithelial cells of organs, such as the lungs, pancreas and intestine ,2,3. Cysutations , only abutations . A highlons [TM) , and foracaftor) ,7,8,9,10TM-approved mutations on both alleles and remain without CFTR modulator therapy. While many of these mutations may be considered rare, regional or founder population effects may cause certain mutations to be more prevalent within groups or areas. Examples include the W1282X mutation in Jewish populations, M1101K among Hutterite communities, and c.3700G>A, a mutation common in individuals with ancestry from Bedouin tribes and the second most common CF mutation in the Middle East [A significant number of CF patients worldwide lack F508del or KALYDECOdle East .CFTR variant c.3700A>G has been predicted to create a missense mutation . As we demonstrated, I1234V-CFTR caused no change in the CFTR protein folding, processing and function when expressed in an overexpression system [CFTR gene and CFTR mRNA obtained from nasal cultures of a CF patient homozygous for c.3700A>G, showed that this mutation activates a cryptic donor splice site (with no wild-type (WT) transcript), resulting in deletion of six amino acids (I1234-R1239del) in NBD2, which like the F508del mutation, was shown to cause a primary defect in folding and processing [TM (lumacaftor + ivacaftor), failed to rescue functional expression of this mutant to a level anticipated to be therapeutically relevant. In a subsequent study, it was shown that “amplifier” therapy meant to stabilize CFTR mRNA, in combination with the components of ORKAMBITM in in vitro studies of patient nasal epithelial cultures increased the levels of immature CFTR protein and significantly increased the ORKAMBITM response [TM in I1234-R1239del-CFTR expressing Fisher Rat Thyroid (FRT) cells, gene-edited Human Bronchial Epithelial (HBE) cells expressing I1234-R1239del-CFTR and in primary nasal cultures from sibling CF donors bearing I1234-R1239del/W1282X mutations. Interestingly, they found that nasal cultures from a donor homozygous for c.3700A>G did not show CFTR functional rescue by TRIKAFTATM. They also evaluated the effects of TRIKAFTATM treatment on two individuals homozygous for the c.3700A>G mutation. This study showed some improvements in both patients, with one showing a decrease in sweat chloride and symptomatic improvement, and the other showing a small improvement in lung function. Results from further follow up studies on these and additional individuals are awaited.The n system . Detaileocessing . Howeverresponse . This suresponse , showed CFTR mutations outside of their current indications [Small molecule CFTR therapies are in development from a variety of companies, including Vertex, Abbvie, Proteostasis and others. While it would be prohibitively expensive and difficult to develop a separate modulator therapy for every mutation, theratyping of different mutations in overexpression systems and follow-up with patient tissues may prove an effective pipeline to allow repurposing of modulators for all CF mutations where they may be effective. We and other groups have examined modulators, approved or in various stages of clinical trials, in vitro on patient tissues possessing various rare ications ,18,19,20ications ,21. MoreTM on the rare c.3700A>G mutation in Human Embryonic Kidney (HEK)-293 cells and 16HBE cells. We also tested the small molecule combinations in patient derived nasal epithelial cultures from two patients homozygous for I1234-R1239del and two heterozygous patients I1234-R1239del/W1282X. We found that the specific modulator combination (AC1 + AC2-2 + AP2) rescued functional expression of I1234-R1239del-CFTR to normal function in patient-derived nasal epithelial cells.In this study we examine the effects of the novel Abbvie modulators and TRIKAFTAHuman embryonic kidney (HEK) 293 GripTite™ cells (HEK293)  were maintained as previously described ,23.TM ALI, StemCell Tech., Vancouver, BC, Canada).I1234_R1239del/W1282X nasal epithelial cells were obtained through Dr. Carlos Milla at the CF Center at Stanford University, and I1234_R1239del homozygous nasal cells were obtained from family members at The Hospital for Sick Children (after obtaining informed consent). The subsequent nasal epithelial cell culture was performed as previously described ,25,26. CThe small molecule modulators of CFTR used in this study were: VX-770, VX-661 and VX-809 , VX-445 , CFTR-sSome of the Abbvie compounds used in this study are structurally related to clinical candidates currently in clinical trials for subjects who are homozygous for F508del and were obtained by Abbvie Inc., North Chicago, IL, USA. Corrector AC1 X281602) is described as compound 72 in a recently published review by Kym et al. 1602 is d and is aHEK293 cells were seeded in 96-well plates  transfected with either WT- or I1234_R1239del-CFTR constructs as previously described ,33. The 16HBE cells, a gift from Dr. D.C. Gruenert (USF), were used for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing to introduce the I1234_1239del mutation as previously described . I1234_R3, 2.13 mM K2HPO4, 0.38 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2 and 10 mM glucose) was maintained at pH 7.4 and 37 °C and continuously gassed with a 5% CO2/95% O2 mixture. The transepithelial potential (Vte) was recorded in open-circuit mode and the baseline resistance (Rte) was measured following repeated, brief short-circuit current pulses (1 µA every 30 s). The results are presented as equivalent transepithelial current (Ieq), which was calculated using Ohm’s law. Following the epithelial sodium channel (ENaC) inhibition with 30 µM of Amiloride , CFTR was stimulated with 10 µM forskolin and 1 µM VX-770 for cells treated with VX-809 or VX-661 + VX-445, or 1.5 µM for AP2 for cells treated with AC1 + AC2-2. Then, CFTR was inhibited with 5 µM CFTRInh-172  [Primary nasal epithelial cultures were studied in a non-perfused Ussing chamber  after 48 h treatment with 0.1% DMSO, 3 µM VX-809, 0.5 µM AC1 + 3 µM AC2-1, 0.5 µM AC1 + 3 µM AC2-2 or 3 µM VX-661 + 3 µM VX-445. The buffer solution  ,24.p-values < 0.05 were considered significant. Data with multiple comparisons were assessed using Tukey’s multiple-comparison test with α = 0.05. Spearman correlation was used for correlation analysis.Data are represented as mean ± S.E.M. GraphPad Prism 7.0 software  was used for all statistical tests. One-way ANOVA were conducted as appropriate, and The CFTR mutation I1234_R1239del-CFTR was expressed in HEK293 cells and the effects of various modulators on its chloride channel function and protein processing were examined. Regulated CFTR chloride channel function was measured using the FLIPR assay as the membrane depolarization stimulated by forskolin addition in cells exposed to an outward chloride gradient. As expected for a CFTR mediated effect, the depolarization was reversed by the addition of CFTRInh-172. As previously demonstrated , we saw Next, we tested the effects of the modulators on the function of CFTR in nasal epithelial cultures from two patients homozygous for the I1234_R1239del-CFTR mutation. As for the HEK293 cells, preincubation with VX-809, the combination of AC1 and AC2-1 and the combination of AC1 and AC2-2 all resulted in improvements in CFTR channel activity. The greatest improvement was again seen for the treatment with the combination of AC1 and AC2-2, which rescued I1234_R1239del-CFTR up to ~130% of the mean forskolin response in non-CF cultures A,B. ThisTM, which contains two correctors (VX-445 and VX661) and a potentiator (VX770), has been approved by the Food and Drug Administration (FDA) for the treatment of CF patients with at least one copy of the F508del mutation. Therefore, we compared the effects of TRIKAFTATM with the combination of AC1, AC2-2 and AP2 on the functional rescue of I1234_R1239del-CFTR in nasal epithelial cultures obtained from individuals homozygous for the I1234_R1239del mutation. Interestingly, in agreement with a recent studies by Phuan and colleagues [TM did not rescue the I1234_R1239del-CFTR function in nasal epithelial cultures . Similaell line  where thLukacs and group have demonstrated that VX-661 and VX-809 are type I correctors that stabilize the MSD2-NBD1 interface, and other correctors stabilize NBD1 (type III) or NBD2 (type II) ,37. VX-4TM in the HBE cell line that was edited to harbor this mutation on both alleles. We hypothesized that this discrepancy could be due to the differential genetic factors (such as modifier genes) or environmental factors at play among the 4 participant samples and HBE cell line that could induce different residual CFTR protein levels. The participant disease status at the time of nasal brushing, or brush method differences between the two locations where the nasal samples were collected, could also be factors.Here we show that the combination of VX-770, VX-661 and VX-445 improved functional rescue of the I1234-R1239del mutant in nasal cultures from two patients heterozygous for the mutation (I1234_R1239del/W1282X) but not in nasal cultures from two patients homozygous for c.3700A>G, in agreement with the previous study by Phuan et al. . This fiTM led to modest symptomatic responses [Interestingly, despite the lack of robust responses in-vitro, clinical treatment of the two patients, homozygous for c.3700A>G with TRIKAFTAesponses . In factesponses . This stTM [TM [TM or TRIKAFTATM.The G551D-CFTR mutation was the first with a highly effective modulator therapy in KALYDECOTM . The F50TM [TM . MechaniTM [TM , inhibitTM [TM ,40, and In this work, we demonstrated the utility and value of comparing different combinations of CFTR modulators and drugs using nasal epithelial tissues from individuals bearing a rare CF-causing mutations ,21,24,25