Protein nitration is an important post-translational modification regulating protein structure and function, especially for heme proteins. Myoglobin (Mb) is an ideal protein model for investigating the structure and function relationship of heme proteins. With limited structural information available for nitrated heme proteins from experiments, we herein performed a molecular dynamics study of human Mb with successive nitration of Tyr103, Tyr146, Trp7 and Trp14. We made a detailed comparison of protein motions, intramolecular contacts and internal cavities of nitrated Mbs with that of native Mb. It showed that although nitration of both Tyr103 and Tyr146 slightly alters the local conformation of heme active site, further nitration of both Trp7 and Trp14 shifts helix A apart from the rest of protein, which results in altered internal cavities and forms a water channel, representing an initial stage of Mb unfolding. The computational study provides an insight into the nitration of heme proteins at an atomic level, which is valuable for understanding the structure and function relationship of heme proteins in non-native states by nitration.
The intermediates and byproducts formed during the ozonation of microcystin-LR (MC-LR, m/z = 995.5) and the probable degradation pathway were investigated at different initial molar ratios of ozone to MC-LR ([O3]0/[MC-LR]0). Seven reaction intermediates with m/z ? 795.4 were observed by LC/MS, and four of them (m/z = 815.4, 827.3, 853.3 and 855.3) have not been previously reported. Meanwhile, six aldehyde-based byproducts with molecular weights of 30-160 were detected for the first time. Intermediates structures demonstrated that ozone reacted with two sites of MC-LR: the diene bonds in the Adda side chain and the Mdha amino acid in the cyclic structure. The fragment from the Adda side chain oxidative cleavage could be further oxidized to an aldehyde with a molecular weight of 160 at low [O3]0/[MC-LR]0. Meanwhile, the polypeptide structure of MC-LR was difficult to be further oxidized, unless [O3]0/[MC-LR]0 > 10. After further oxidation of the intermediates, five other aldehyde-based byproducts were detected by GC/MS: formaldehyde, acetaldehyde, isovaleraldehyde, glyoxal and methylglyoxal. Formaldehyde, isovaleraldehyde and methylglyoxal were the dominant species. The yields of the aldehydes varied greatly, depending on the value of [O3]0/[MC-LR]0.
A method based on HPLC with UV detection was developed for the quantitative determination of chloramphenicol (CAP) residues in aquatic products. The samples were extracted with ethyl acetate-ammonium hydroxide (98 + 2, v/v), followed by a cleanup step using an immunoaffinity column. The analytes were determined by HPLC-UV. Optimal conditions for the extraction and cleanup procedures are described. The linear regression equation was y = 91.47x - 8.60 with R = 0.9998 (y = peak area and x = CAP concentration) and showed a good reproducibility. The LOQ was 0.25 microg/kg for determining CAP spiked in the aquatic products. The mean recoveries of CAP from fish and shrimp samples fortified at 0.25-1.0 microg/kg were 88.7-93.1 and 92.0-97.3%, respectively; the repeatability RSDs were less than 8.1%. It was concluded that the method is simple, highly sensitive, and low cost for quantitatively measuring CAP residues in aquatic products. Analyte identification was confirmed by HPLC/MSIMS analysis.
The associations between cyclooxygenase-2 (COX-2) polymorphisms (-765G>C, -1195G>A, and -587G>A) and risk of gastric cancer have been investigated, but the results were inconsistent. The aim of this study was to explore the associations between COX-2 polymorphisms and risk of gastric cancer using a meta-analytic method. We searched the databases of PubMed, Embase, and Wanfang (Chinese database) to identify the eligible studies. Odds ratio and 95 % confidence interval (OR and 95 % CI) were used as effect size, and combined analyses were conducted using fixed- or random-effects model. Overall, ten studies for COX-2-765G>C, six studies for -1195G>A, and three studies for -587G>A were included in this study. The results for combined analysis for COX-2-765G>C indicated that C allele was significantly associated with increased risk of gastric cancer compared with G allele, especially for Asians (OR and 95 % CI: 1.58 (1.06-2.35), P z-test?=?0.03, and P heterogeneity <0.01 for CC+GC vs. GG). In addition, the A allele of COX-2-1195G>A was also significantly associated with risk of gastric cancer compared with G allele (OR and 95 % CI: 1.20 (1.09-1.32), Pz-test <0.001, and Pheterogeneity?=?0.82 for A carriers vs. G carriers). In contrast, the COX-2-587G>A polymorphism was not associated with risks of gastric cancer. In summary, this meta-analysis indicated that the COX-2-765G>C and -1195G>A polymorphisms were significantly associated with risk of gastric cancer development.
Uranium is harmful to human health due to its radiation damage and the ability of uranyl ion (UO2(2+)) to interact with various proteins and disturb their biological functions. Cytochrome b5 (cyt b5) is a highly negatively charged heme protein and plays a key role in mediating cytochrome c (cyt c) signaling in apoptosis by forming a dynamic cyt b5-cyt c complex. In previous molecular modeling study in combination with UV-Vis studies, we found that UO2(2+) is capable of binding to cyt b5 at surface residues, Glu37 and Glu43. In this study, we further investigated the structural consequences of cyt b5 and cyt c, as well as cyt b5-cyt c complex, upon uranyl binding, by fluorescence spectroscopic and circular dichroism techniques. Moreover, we proposed a uranyl binding site for cyt c at surface residues, Glu66 and Glu69, by performing a molecular modeling study. It was shown that uranyl binds to cyt b5 (KD=10 ?M), cyt c (KD=87 ?M), and cyt b5-cyt c complex (KD=30 ?M) with a different affinity, which slightly alters the protein conformation and disturbs the interaction of cyt b5-cyt c complex. Additionally, we investigated the functional consequences of uranyl binding to the protein surface, which decreases the inherent peroxidase activity of cyt c. The information of uranyl-cyt b5/cyt c interactions gained in this study likely provides a clue for the mechanism of uranyl toxicity.
Deletion of 3p is one of the most frequent genetic alterations in esophageal squamous cell carcinoma (ESCC), suggesting the existence of one or more tumor suppressor genes (TSGs) within these regions. In this study, one TSG, CACNA2D3 at 3p21.1, was characterized.
Deletions on chromosome 3p occur often in many solid tumors, including esophageal squamous cell carcinoma (ESCC), suggesting the existence at this location of one or more tumor suppressor genes (TSG). In this study, we characterized RBMS3 gene encoding an RNA-binding protein as a candidate TSG located at 3p24. Downregulation of RBMS3 mRNA and protein levels was documented in approximately 50% of the primary ESCCs examined. Clinical association studies determined that RBMS3 downregulation was associated with poor clinical outcomes. RBMS3 expression effectively suppressed the tumorigenicity of ESCC cells in vitro and in vivo, including by inhibition of cell growth rate, foci formation, soft agar colony formation, and tumor formation in nude mice. Molecular analyses revealed that RBMS3 downregulated c-Myc and CDK4, leading to subsequent inhibition of Rb phosphorylation. Together, our findings suggest a tumor suppression function for the human RBMS3 gene in ESCC, acting through c-Myc downregulation, with genetic loss of this gene in ESCC contributing to poor outcomes in this deadly disease.
Bovine liver cytochrome b (5) (cyt b (5)), with heme bound noncovalently, has been converted into a cyt c-like protein (cyt b (5) N57C) by constructing a thioether linkage between the heme and the engineered cysteine residue. With no X-ray or NMR structure available, we herein performed a molecular modeling study of cyt b (5) N57C. On the other hand, using amino acid sequence information for a newly discovered member of the cyt b (5) family, domestic silkworm cyt b (5) (DS cyt b (5)), we predicted the protein structure by homology modeling in combination with MD simulation. The modeling structure shows that both Cys57 in cyt b (5) N57C, and Cys56, a naturally occurring cysteine in DS cyt b (5), have suitable orientations to form a thioether bond with the heme 4-vinyl group, as the heme is in orientation A. In addition to providing structural information that was not previously obtained experimentally, these modeling studies provide insight into the formation of cyt c-like thioether linkages in cytochromes, and suggest that c-type cyt b (5) maturation involves a b-type intermediate.
Amplification of 19q is a frequent genetic alteration in many solid tumors, and SEI1 is a candidate oncogene within the amplified region. Our previous study found that the oncogenic function of SEI1 was associated with chromosome instability. In this study, we report a novel mechanism of genomic instability involving the SEI1-SET-NM23H1 pathway. Overexpression of SEI1 was observed in 57 of 100 of esophageal squamous cell carcinoma cases. Functional study showed that SEI1 had strong tumorigenic ability, and overexpression of SEI1 could induce the genomic instability by increasing micronuclei formation and reducing the number of chromosomes. Further study found that SEI1 was able to upregulate SET expression and subsequently promote the translocation of a small amount of NM23H1 from the cytoplasm to the nucleus. Nuclear NM23H1 can induce DNA damage through its DNA nick activity. Unlike CTL attack, only a small amount of NM23H1 translocated into the nucleus (<10%) induced by the overexpression of SEI1. Further study found that the small amount of NM23H1 only induced minor DNA damage and subsequently increased genomic instability, rather than inducing irreparable DNA damage and initiating apoptosis by CTL attack. Sister chromatid exchange experiment found that the translocation of small amount of NM23H1 into the nucleus induced by the overexpressions of SEI1/SET could increase the frequency of sister chromatid exchange. In addition, overexpression of SEI1 was associated with poor prognosis of esophageal squamous cell carcinoma. Taken together, these findings define a novel mechanism of genomic instability and malignant progression in esophageal cancers, a deadly disease of increasing incidence in developed countries.
Myoglobin (Mb) is an ideal scaffold protein for rational protein design mimicking native enzymes. We recently designed a nitrite reductase (NiR) based on sperm whale Mb by introducing an additional distal histidine (Leu29 to His29 mutation) and generating a distal tyrosine (Phe43 to Tyr43 mutation) in the heme pocket, namely L29H/F43Y Mb, to mimic the active site of cytochrome cd (1) NiR from Ps. aeruginosa that contains two distal histidines and one distal tyrosine. The molecular modeling and dynamics simulation study herein revealed that L29H/F43Y Mb has the necessary structural features of native cytochrome cd (1) NiR and can provide comparable interactions with nitrite as in native NiRs, which provides rationality for the protein design and guides the protein engineering. Additionally, the present study provides an insight into the relatively low NiR activity of Mb in biological systems.
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