Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.

In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory cis-element DNA and a cryo-EM structure of GlnR-TAC which comprises Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved ß flap, σAR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.

Structural and Functional Analyses of the Transcription Repressor DgoR From Escherichia coli Reveal a Divalent Metal-Containing D-Galactonate Binding Pocket.

The transcription repressor of D-galactonate metabolism, DgoR, from Escherichia coli belongs to the FadR family of the GntR superfamily. In the presence of D-galactonate, DgoR binds to two inverted repeats overlapping the dgo cis-acting promoter repressing the expression of genes involved in D-galactonate metabolism. To further understand the structural and molecular details of ligand and effector interactions between D-galactonate and this FadR family member, herein we solved the crystal structure of C-terminal domain of DgoR (DgoR_C), which revealed a unique divalent metal-containing substrate binding pocket. The metal ion is required for D-galactonate binding, as evidenced by the dramatically decreased affinity between D-galactonate and DgoR in the presence of EDTA, which can be reverted by the addition of Zn2+, Mg2+, and Ca2+. The key amino acid residues involved in the interactions between D-galactonate and DgoR were revealed by molecular docking studies and further validated with biochemical studies by site-directed mutagenesis. It was found that changes to alanine in residues R102, W181, T191, and R224 resulted in significantly decreased binding affinities for D-galactonate, as determined by EMSA and MST assays. These results suggest that the molecular modifications induced by a D-galactonate and a metal binding in the DgoR are required for DNA binding activity and consequently, transcriptional inhibition.

AMPK decreases ERK1/2 activity and cancer cell sensitivity to nutrition deprivation by mediating a positive feedback loop involving eEF2K.

Nutrition deprivation (ND) is a common feature of the tumor microenvironment. Tumor cells, therefore, frequently develop resistance mechanisms against ND. One of these mechanisms is the activation of the AMP-activated protein kinase (AMPK), which promotes cell survival under ND. AMPK activation promotes the activity of eukaryotic elongation factor 2 kinase (eEF2K), thereby blocking protein synthesis. The results of the present study indicated the inhibiting effect of AMPK activation on mitogen-activated protein kinase (ERK1/2) activity, which in turn downregulates G1/S transition and promotes cell survival by mediating eEF2K under ND. The knockdown of ERK1/2 enhances cancer cell survival under ND. In the presence of nutrients, eEF2k interacts with dual-specificity mitogen-activated protein kinase kinase (MEK)1/2, conferring a positive feedback loop via MEK1/2-ERK1/2-ribosomal protein S6 kinase α-1-eEF2K signaling, leading to the constitutive activation of ERK1/2. By contrast, under acute ND, AMPK activation blocked the interaction between eEF2K and MEK1/2, contributing to the increased resistance of cancer cells to ND. The present findings reveal a previously undiscovered mechanism that uses AMPK activation to mediate ERK1/2-regulated protein synthesis and cell survival by inhibiting eEF2K-MEK1/2 interaction under ND conditions.

Expression of NUAK2 in gastric cancer tissue and its effects on the proliferation of gastric cancer cells.

The present study aimed to analyze the expression and effects of NUAK2 in gastric cancer and adjacent normal gastric tissues. The protein expression levels of NUAK2 were detected by western blot analysis. The effects of NUAK2 expression on the proliferation of gastric cancer cells was detected using an MTT and BrdU incorporation assay. Furthermore, the effects of NUAK2 on proliferation and cancer stem cell markers, both protein and microRNA (miRNA), were investigated by western blot analysis and miRNA microarrays, respectively. The results demonstrated that NUAK2 was able to significantly promote the proliferation of SGC-7901 gastric cancer cells. In addition, NUAK2 overexpression decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S phase. Western blot analysis and miRNA microarrays revealed that overexpression of NUAK2 resulted in increased expression levels of proliferation markers, including c-myc, proliferating cell nuclear antigen, cyclin-dependent kinase 2, miRNA 21, and gastric cancer stem cell markers, including aldehyde dehydrogenase 1, CD44 and CD133. In conclusion, NUAK2 expression differed between the tumor and normal gastric tissues. NUAK2 was able to promote the proliferation of gastric cancer cells and regulate their cell cycle. Proliferation and cancer stem cell markers were upregulated by NUAK2 expression. Therefore, the results from the present study suggest that NUAK2 may be a promising target for gastric cancer therapy in the future.

TGF-α gene variations and increased susceptibility of gastric cancer in an Eastern Chinese Han population.

Transforming growth factor-alpha (TGF-α) correlates with deep invasion, advanced stage and poor prognosis in gastric cancer. Genetic variants in the 3' untranslated region (UTR) of TGF-α gene may influence the stability and post-transcriptional regulation of mRNA and contribute to gastric cancer predisposition. To test this hypothesis, we genotyped five polymorphisms in 3'UTR (rs3771527, rs503314, rs473698, rs3732253 and rs538118) and one in 3' near region (rs11466306) of the TGF-α gene by polymerase chain reaction-ligation detection reaction methods (PCR-LDR). We found that GA/AA genotype of rs11466306 in the 3' near gene could increase the risk of overall gastric cancer (adjusted OR = 1.499, 95%CI: 1.101-2.041), compared to the wild homozygous GG genotype. Meanwhile, the risk effect was more obvious in the intestinal gastric cancer and gastric noncardia cancer (adjusted OR = 1.682, 95%CI: 1.188-2.380; adjusted OR = 1.495, 95%CI: 1.072-2.086, respectively), but not for the diffuse type and gastric cardia cancer (p > 0.05). CT/TT genotype for rs3732253 in the 3' UTR was associated with increased risk of intestinal gastric cancer (adjusted OR = 1.464, 95%CI: 1.036-2.069), compared to their wild homozygous genotypes. These findings indicate that potentially functional TGF-α gene variant may contribute to the risk of intestinal gastric cancer and/or gastric noncardia cancer and could be used as molecular markers for detecting intestinal gastric cancer and/or gastric noncardia cancer in Chinese Han population.

Association among polymorphisms in EGFR gene exons, lifestyle and risk of gastric cancer with gender differences in Chinese Han subjects.

BACKGROUND: The epidermal growth factor receptor (EGFR) gene plays a key role in tumor survival, invasion, angiogenesis, and metastatic spread. Recent studies showed that gastric cancer (GC) was associated with polymorphisms of the EGFR gene and environmental influences, such as lifestyle factors. In this study, seven known SNPs in EGFR exons were investigated in a high-risk Chinese population in Jiangsu province to test whether genetic variants of EGFR exons and lifestyle are associated with an increased risk of GC. METHODOLOGY/PRINCIPAL FINDINGS: A hospital-based case-control study was performed in Jiangsu province. The results showed that smoking, drinking and preference for salty food were significantly associated with the risk of GC. The differences of lifestyle between males and females might be as the reason of higher incidence rates in males than those in females. Seven exon SNPs were genotyped rs2227983,rs2072454,rs17337023,rs1050171,rs1140475, rs2293347, and rs28384375. It was noted that the variant rs2072454 T allele and TT genotype were significantly associated with an increased risk of GC. Interestingly, our result suggested the ACAGCA haplotype might be associated with decreased risk of GC. However, no significant association was examined between the other six SNPs and the risk of GC both in the total population and the age-matching population even with gender differences. CONCLUSIONS: Smoking, drinking and preference for salty food were significantly associated with the risk of GC in Jiangsu province with gender differences. Although only one SNP (rs2072454) was significantly associated with an increased risk of GC, combined the six EGFR exon SNPs together may be useful for predicting the risk of GC.

Functional epidermal growth factor gene polymorphisms and risk of gastric cancer.

Epidermal growth factor (EGF) is critical in cancer progression and various genotypes of the EGF gene have been reported to be correlated with susceptibility to gastric cancer; however, the results are unclear. The aim of this study was to explore the associations of functional EGF gene polymorphisms and risk of gastric cancer in a Chinese population. We genotyped seven functional single-nucleotide polymorphisms (SNPs) of the EGF gene [rs3756261, rs11568835 and rs4444903 in the promoter region; rs11568943, rs2237051 and rs11569017 in the non-synonymous exon region and rs3733625 in the 3' untranslated region (UTR)] in a hospital-based case-control study, including 387 gastric cancer cases and 392 healthy controls by polymerase chain reaction-ligation detection reaction (PCR-LDR) methods. We found that individuals carrying the AG/GG genotype of rs2237051 (Ile to Met) in the exon region had an increased risk of gastric cancer (adjusted OR=1.577; 95% CI=1.163-2.138), compared with the wild-type homozygous AA genotype. The heterozygous AG/GG genotype of rs3733625 in the 3'UTR demonstrated a protective effect (adjusted OR=0.690; 95% CI=0.501-0.951), compared with the homozygous AA genotype. In addition, the effects of the two SNPs were more evident in intestinal gastric cancer (P<0.05); however, this was not case for the diffuse type (P>0.05). These findings indicate that a change in amino acids from isoleucine to methionine of rs2237051 and rs3733625 in the 3'UTR may contribute to the etiology of intestinal gastric cancer in the Chinese population.

Relationship between EGF, TGFA, and EGFR Gene Polymorphisms and Traditional Chinese Medicine ZHENG in Gastric Cancer.

In traditional Chinese medicine (TCM), correct syndrome differentiation is the most important principle guiding the prescription of Chinese herbal formulae for the treatment of gastric cancer (GC). We aimed to reveal the genetic mechanisms underlying GC syndrome differentiation (ZHENG) in a population of 387 GC patients. Twenty-nine single nucleotide polymorphisms (SNPs) in EGF, TGFA, and EGFR were investigated. Two SNPs, rs11466285 in TGFA and rs884225 in EGFR, were significantly associated with the distribution of ZHENG (P < 0.05). The rs11466285 TT genotype increased the risk of damp heat with toxin (DHT) and deficiency of both Qi and yin (DQY) compared with obstruction of blood stasis (OBS). The rs884225 AA genotype could increase the risk of DQY and deficiency of both Qi and blood (DQB) compared with yin deficiency due to stomach heat (YDSH). Parallel comparison among the SNPs and syndrome types revealed that DQB was distinct from YDSH, disharmony between the liver and stomach, stagnation of phlegm muddiness (SPM), OBS, and other syndromes at several SNP loci (P < 0.05). The rs11466285 TT and rs884225 AA genotypes exhibit increased risk of DQB compared with OBS and SPM (P < 0.05), respectively. In conclusion, the formation of GC ZHENG was related to EGF, TGFA, and EGFR gene polymorphisms.

Association among lifestyle, clinical examination, polymorphisms in CDH1 gene and Traditional Chinese Medicine syndrome differentiation of gastric cancer.

OBJECTIVE: To explore the association among life-style, clinical examination, polymorphisms in CDH1 gene and Traditional Chinese Medicine (TCM) syndrome differentiation of gastric cancer (GC). METHODS: A hospital-based population of 387 GC patients was investigated in Jiangsu province. Relevant information regarding lifestyle and clinical examination were collected by a standard questionnaire. Four known single nucleotide polymorphisms (SNPs) in CDH1 were investigated by polymerase chain reaction-ligation detection reaction methods. Statistical analysis was conducted by SPSS 16.0 software. RESULTS: The results showed that meal duration and the status of glutamic pyruvic transaminase were significantly associated with TCM syndrome differentiation of GC (both P < 0.05). None of the four SNPs in the E-cadherin (CDH1) gene achieved significant differences in their distributions among the nine syndrome types of GC (both P > 0.05). However, significant differences were observed in rs13689 genotype distributions between several pairs of syndrome types of GC, suggesting that rs13689 is correlated with the syndrome differentiation of GC. CONCLUSION: Integrated analysis of lifestyle, clinical examination and CDH1 gene polymorphisms can contribute to a better understanding of the GC syndrome types and may improve the efficacy of interventions by stratifying disease according to TCM criteria.

CDH1 gene polymorphisms, plasma CDH1 levels and risk of gastric cancer in a Chinese population.

The genetic polymorphisms in E-cadherin gene (CDH1) may affect invasive/metastatic development of gastric cancer by altering gene transcriptional activity of epithelial cell. Our study aims to explore the associations among CDH1 gene polymorphisms, and predisposition of gastric cancer. We genotyped four potentially functional polymorphisms (rs13689, rs1801552, rs16260 and rs17690554) of the CDH1 gene in a case-control study of 387 incident gastric cancer cases and 392 healthy controls by polymerase chain reaction-ligation detection reaction methods (PCR-LDR) and measured the plasma CDH1 levels using enzyme immunoassay among the subjects. The median and inter-quartile range were adopted for representing the mean level of non-normally distributed data, and we found the level of plasma CDH1 in gastric cancer patients (median: 171.00 pg/ml; inter-quartile range: 257.10 pg/ml) were significantly higher than that of controls (median: 137.40 pg/ml; inter-quartile range: 83.90 pg/ml, P = 0.003). However, none of the four polymorphisms or their haplotypes achieved significant differences in their distributions between gastric cancer cases and controls, and interestingly, in the subgroup analysis of gastric cancer, we found that CA genotype of rs26160 and CG genotype of rs17690554 were associated with the risk of diffuse gastric cancer, compared with their wild genotypes (OR = 2.98, 95 % CI: 1.60-5.53; OR = 2.10, 95 % CI: 1.14-3.85, respectively, P < 0.05). In conclusion, our results indicated that plasma CDH1 levels may serve as a risk marker against gastric cancer and variant genotypes of rs26160 and rs17690554 may contribute to the etiology of diffuse gastric cancer in this study. Further studies are warranted to verify these findings.