Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies / 生物工程学报

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.

The 40-91 aa sequence of porcine epidemic diarrhea virus ORF3 protein is the key structural domain controlling its location in cytoplasm / 生物工程学报

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.

Expression of CMTM6 in breast cancer tissues and its correlation with patients’ clinicopathological characteristics and prognosis / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression of chemokine-like factor-like MARVEL transmembrane domain-containing family member 6 (CMTM6) in breast cancer tissues and its correlation with clinicopathological features and prognosis of patients. Methods:Atotal of 136 breast cancer tissue chips (purchased from Superchip Company), including 42 pairs of matched cancer and paracancerous tissues, were used for this study. The expression level of CMTM6 in cancer and paracancerous tissues was detected by immunohistochemistry. The comparison of CMTM6 expression between breast cancer and paracancerous tissues was conducted by paired χ2 test. The relationship between CMTM6 expression in breast cancer tissues and the clinicopathological characteristics of patients was analyzed by χ2 test. Kaplan-Meier and Log rank test analyses were used to analyze the relationship between CMTM6 expression and the survival of patients, and Cox model was used to evaluate the effect of different indicators on the prognosis of patients. Results: The expression of CMTM6 in breast cancer tissues was significantly higher than that in paracancerous tissues (P<0.01). The expression of CMTM6 was correlated with pathological type of breast cancer and HER2 positivity (P<0.05). The survival time of patients in CMTM6 high expression group was significantly shorter than that of patients in CMTM6 low expression group (P<0.05). Pathological type (HR=10.374, 95%CI: 3.529-30.497, P<0.01), TNM stage (HR=4.599, 95%CI: 1.784-11.856, P<0.01), triple-negative breast cancer (HR=3.370, 95%CI: 1.055-10.761, P<0.05) and high expression of CMTM6 (HR=0.195, 95%CI: 0.073-0.518, P<0.01) were independent risk factors for prognosis of breast cancer patients. Conclusion: CMTM6 is highly expressed in breast cancer tissues, which can be used as a risk factor for prognosis evaluation of breast cancer patients.

Association between CMTM5 gene rs723840 single nucleotide polymorphism and coronary artery disease / 解放军医学杂志

Objective To elucidate the correlation between the single nucleotide polymorphism ofCKLF-like MARVEL transmembrane domain containing member 5 (CMTM5) gene rs723840 and the occurrence of coronary artery disease (CAD). Methods The present study is a case-control study. A total of 1110 hospitalized patients in Shijitan Hospital were enrolled in this study. Patients were divided into CAD group (n=560) and control group (n=550). CAD were diagnosed by coronary angiography, which was defined as at least one blood vessel diameter stenosis ≥50% according to the result of coronary angiography. Genotypes were determined by polymerase chain reaction (PCR) and using sequencing analysis to detect rs723840 of CMTM5 gene. The proportions of genotype and allele of CMTM5 gene were analyzed. Results The genotype frequencies in rs723840 C>T of CMTM5 gene conformed well to the Hardy-Weinberg equilibrium in both CAD group and control group. Between the two groups, the genotypes frequency (CC, CT and TT) in CAD and control groups were 53.9%, 40.9%, 5.2% and 69.3%, 28.7%, 2.0%, respectively (P<0.001). T allele frequency was significantly higher than that in C allele frequency (25.6% vs. 16.4%, OR=1.566, 95%CI 1.325-1.850, P<0.001). After adjusted for the risk factors of age, gender, BMI, smoking, hypertension, diabetes and hyperlipidemia, logistic regression analysis results indicated that CMTM5 was the susceptibility factors of CAD, which showed significant correlation with CAD. Conclusions A significant correlation was found between CMTM5 gene rs723840 polymorphism and the occurrence of CAD, T allele carriers are closely related to the occurrence of CAD.

Transmembrane domain dependent inhibitory function of FcγRIIB

FcγRIIB, the only inhibitory IgG Fc receptor, functions to suppress the hyper-activation of immune cells. Numerous studies have illustrated its inhibitory function through the ITIM motif in the cytoplasmic tail of FcγRIIB. However, later studies revealed that in addition to the ITIM, the transmembrane (TM) domain of FcγRIIB is also indispensable for its inhibitory function. Indeed, recent epidemiological studies revealed that a non-synonymous single nucleotide polymorphism (rs1050501) within the TM domain of FcγRIIB, responsible for the I232T substitution, is associated with the susceptibility to systemic lupus erythematosus (SLE). In this review, we will summarize these epidemiological and functional studies of FcγRIIB-I232T in the past few years, and will further discuss the mechanisms accounting for the functional loss of FcγRIIB-I232T. Our review will help the reader gain a deeper understanding of the importance of the TM domain in mediating the inhibitory function of FcγRIIB and may provide insights to a new therapeutic target for the associated diseases.

Identification and Characterization of Dopamine D4 Receptor (DRD4) Gene Sequence within and Among Non Mammalian Species.

A number of polymorphic tandem repeats in human dopamine D4 receptor (DRD4) have been identified in the exons, including a 12-bp repeat in the first exon and a 48-bp repeat in exon III located in the third cytoplasmic loop. However, to determine whether the tandem repeats is specific to humans or not, we have identified and characterized dopamine receptor D4 (DRD4) Exon III tandem repeats in public available nucleotide sequences from 13 different non mammalian species. We found that the tandem repeat was composed of 21-bp modules in sequences from the Mycobacterium smegmatis str. MC2 155, Salinibacter ruber DSM 13855, Danio rerio, Parus major, Corvus macrorhynchos, and Coturnix japonica. A tandem repeat consisting of 30-bp modules was identified in sequence from Melopsittacus undulates while in the Phalacrocorax capillatus and Numida meleagris we identified tandem repeats composed of 3-bp modules. Tandem repeats could not be identified in sequences from Carassius auratus, Phasianus colchicus and Gallus gallus. To understand the evolutionary history of the Exon I region of DRD4—which in humans contains a polymorphic 12bp tandem duplication, a polymorphic 13bp deletion, and other rare variants—we examined the homologous exon in these different species. There was a low degree of similarity between the sequences of bacterial species and those from members of the piscean and avian and with human sequence. We identified transmembrane domain of DRD4 gene and signature of G-protein coupled receptors in the amino acid sequences. The number of transmembrane segments varied pronouncedly between species from 0 to 7 and signature of G-protein coupled receptors was found only in piscean species and was also identified in one avian species (parus major). These findings suggest that an association between Drd4 gene polymorphisms and animal personality variation predates the divergence of the non mammalian and mammalian lineages. Furthermore, the analysis of Drd4 polymorphisms within and among populations may provide information for elucidating the phylogenetic relationship and such data may also provide a clue toward understanding the relation between the genetic variation and behavioral variation in animals.

Expression of CMTM3 and CMTM5 in clear cell renal cell carcinoma and its significance / 中华泌尿外科杂志

Objective To investigate the expression of CKLF-like MARVEL transmembrane (CMTM)domain-containing family in clear cell renal cell carcinoma(ccRCC)and its significance.Methods Seventy-five samples of ccRCC were collected,including 50 males and 25 females,mean age (59 ± 10)years.There were 34 cases in clinical stage Ⅰ,23 cases in stage Ⅱ,14 cases in stage Ⅲ and 4 cases in stage Ⅳ.The pathological differentiation was 3 cases of grade Ⅰ,1 case of grade Ⅰ-Ⅱ,35 cases of grade Ⅱ,10 cases of grade Ⅱ-Ⅲ,18 cases of grade Ⅲ,3 cases of grade Ⅲ-Ⅳ and 5 cases of grade Ⅳ.The expression of CMTM3 and CMTM5 proteins in 75 cases of ccRCC and corresponding adjacent normal kidney tissues was detected by tissue microarray and immunohistochemistry.Results The positive expression rate of CMTM3 and CMTM5 was 98.7％,97.3％ in the adjacent normal kidney tissues,and 44.0％,68.0％ in ccRCC tissues(P ＜ 0.05).The expression of CMTM3 and CMTM5 had no correlation with the gender,age,clinical staging and pathological differentiation(P ＞ 0.05).Conclusion CMTM3 and CMTM5 could be ccRCC suppressor genes,and their mutation or methylation might be an early event in the carcinogenesis of ccRCC,thus promise them to be potential biomarkers in early diagnosis.

Effect of CMTM5 on prostate cancer cell proliferation, migration and invasion / 中华泌尿外科杂志

Objective To study the effect and mechanism of CKLF-like Marvel transmembrane domain containing 5 (CMTMS) on prostate cancer cell proliferation,migration and invasion.Methods The inhibitory effects of CMTM5 on the migration of DU145 cells were studied in vitro by wound healing assay.The expression of the cell signal pathway PI3K-AKT protein was detected by Western blot.The inhibition of tumor growth was also studied in transplanted prostate cancer nude mice model treated with CMTM5 adenovirus.The expression of CMTM5 and ki-67 in transplanted prostate cancer tissue of the nude mice model was analyzed immunohisochemistically.Prostate tumor volume in the nude mice model and the proliferation were measured two weeksafter.injection..Results Wound healing assay showed that over-expression of CMTM5 can inhibit the migration of DU145 cells.The expression of pAKT and NF-kB was significantly decreased after the overexpression of CMTM5.The tumor volume (573.39 ± 175.24) mm3,weight (0.55 ± 0.11) g and proliferation index of prostate in CMTM5 orthotopic injection nude mice model were significantly smaller and decreased than those in the control group (1482.50 ± 327.86) mm3 and (1.31 ± 0.29) g (P ＜ 0.05).Conclusions Both in vitro and in vivo experiments demonstrate that overexpression of CMTM5 could suppress prostate cancer cell proliferation,migration and invasion.The effect may be conducted by PI3K-AKT signaling pathway.