Biomedical subjects

Yinsheng Wang

Publications and source records attributed to Yinsheng Wang.

At least 19 recordsLinked to original sources

PubMed2024

Mass Spectrometry-Based Proteomics for Assessing Epitranscriptomic Regulations.

Epitranscriptomics is a rapidly evolving field that explores chemical modifications in RNA and how they contribute to dynamic and reversible regulations of gene expression. These modifications, for example, N6-methyladenosine (m6A), are crucial in various RNA metabolic processes, including splicing, stability, subcellular localization, and translation efficiency of mRNAs. Mass spectrometry-based proteomics has become an indispensable tool in unraveling the complexities of epitranscriptomics, offering high-throughput, precise protein identification, and accurate quantification of differential protein expression. Over the past two decades, advances in mass spectrometry, including the improvement of high-resolution mass spectrometers and innovative sample preparation methods, have allowed researchers to perform in-depth analyses of epitranscriptomic regulations. This review focuses on the applications of bottom-up proteomics in the field of epitranscriptomics, particularly in identifying and quantifying epitranscriptomic reader, writer, and eraser (RWE) proteins and in characterizing their functions, posttranslational modifications, and interactions with other proteins. Together, by leveraging modern proteomics, researchers can gain deep insights into the intricate regulatory networks of RNA modifications, advancing fundamental biology, and fostering potential therapeutic applications.

Proteomics↗

PubMed2007

Derivatization with Girard reagent T combined with LC-MS/MS for the sensitive detection of 5-formyl-2'-deoxyuridine in cellular DNA.

Nucleoside 5-formyl-2'-deoxyuridine (FodU) is a major thymidine lesion generated by reactive oxygen species. In vitro and in vivo replication studies revealed that FodU can be mutagenic. A reliable and sensitive quantification method is, therefore, important for assessing the biological implications of this lesion. However, the detection limit of FodU by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was relatively poor compared with those of other oxidative DNA base damages. In this paper we described a new approach for the highly sensitive detection of FodU. We derivatized FodU with Girard reagent T to form a hydrazone conjugate harboring a precharged quaternary ammonium moiety, which enabled the facile detection of the resulting conjugate by positive-ion electrospray ionization MS. We also showed that the combination of derivatization with LC-MS/MS on a linear-ion-trap mass spectrometer could allow for the quantification of FodU at a detection limit of 3-4 fmol, which is approximately 20-fold better than that for the direct analysis of the underivatized compound. By using isotope-labeled FodU as the internal standard and this derivatization method, we further quantified, by using LC-MS/MS, the yield of FodU formed in cellular DNA.

Betaine↗

PubMed2007

The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro.

Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.

Alcohol Dehydrogenase↗

PubMed2006

Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal.

Methylglyoxal (MG) is an important glycating agent produced under physiological conditions. MG could react with DNA and proteins to generate advanced glycation end products. Human hemoglobin, the most abundant protein in blood cells, has not been systematically investigated as the target protein for methylglyoxal modification. Here we examined carefully, by using HPLC coupled with tandem mass spectrometry (LC-MS/MS), the covalent modifications of human hemoglobin induced by methylglyoxal. Our results revealed that hemoglobin could be modified by methylglyoxal, and the major form of modification was found to be the hydroimidazolone derivative of arginine residues. In addition, Arg-92 and Arg-141 in the alpha chain as well as Arg-40 and Arg-104 in the beta chain were modified, whereas two other arginine residues, that is, Arg-31 in the alpha chain and Arg-30 in the beta chain, were not modified. Semiquantitative measurement for adduct formation, together with the analysis of the X-ray structure of hemoglobin, showed that the extents of arginine modification were highly correlated with the solvent accessibilities of these residues. The facile formation of hydroimidazolone derivatives of arginine residues in hemoglobin by methylglyoxal at physiologically relevant concentrations suggested that this type of modification might occur in vivo. The unambiguous determination of the sites and extents of methylglyoxal modifications of arginines in hemoglobin provided a basis for understanding the implications of these modifications and for employing this type of hemoglobin modification as molecular biomarkers for clinical applications.

Amino Acid Sequence↗

PubMed2006

Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine.

The replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a BrdU or its closely related 5-bromo-2'-deoxycytidine (BrdC). Our results showed that two types of crosslink products could be induced from d(BrCG), d(BrUG), d(GBrU), or d(ABrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5' flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5' neighboring nucleoside was a dG than while it was a dA, and BrdC induced the formation of crosslink products much more efficiently than BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.

Base Sequence↗

PubMed2006

Statins activate AMP-activated protein kinase in vitro and in vivo.

BACKGROUND: Statins exert pleiotropic effects on the cardiovascular system, in part through an increase in nitric oxide (NO) bioavailability. AMP-activated protein kinase (AMPK) plays a central role in controlling energy and metabolism homeostasis in various organs. We therefore studied whether statins can activate AMPK, and if so, whether the activated AMPK regulates nitric oxide (NO) production and angiogenesis mediated by endothelial NO synthase, a substrate of AMPK in vascular endothelial cells. METHODS AND RESULTS: Western blotting of protein extracts from human umbilical vein endothelial cells treated with atorvastatin revealed increased phosphorylation of AMPK at Thr-172 in a time- and dose-dependent manner. The AMPK activity, assessed by SAMS assay, was also increased accordingly. The phosphorylation of acetyl-CoA carboxylase at Ser-79 and of endothelial NO synthase at Ser-1177, 2 putative downstream targets of AMPK, was inhibited by an adenovirus that expressed a dominant-negative mutant of AMPK (Ad-AMPK-DN) and compound C, an AMPK antagonist. The positive effects of atorvastatin, including NO production, cGMP accumulation, and in vitro angiogenesis in Matrigel, were all blocked by Ad-AMPK-DN. Mice given atorvastatin through gastric gavage showed increased AMPK, acetyl-CoA carboxylase, and endothelial NO synthase phosphorylation in mouse aorta and myocardium. CONCLUSIONS: Statins can rapidly activate AMPK via increased Thr-172 phosphorylation in vitro and in vivo. Such phosphorylation results in endothelial NO synthase activation, which provides a novel explanation for the pleiotropic effects of statins that benefit the cardiovascular system.

AMP-Activated Protein Kinases↗

PubMed2006

Recognition and incision of oxidative intrastrand cross-link lesions by UvrABC nuclease.

Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.

Base Sequence↗

PubMed2006

Collisionally activated dissociation of protonated 2'-deoxycytidine, 2'-deoxyuridine, and their oxidatively damaged derivatives.

We examined the collisionally activated dissociation (CAD) pathways of protonated 2'-deoxycytidine (dC), 5-formyl-2'-deoxycytidine (5-FmdC), 5-hydroxy-2'-deoxycytidine (5-OHdC), 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), and their corresponding stable isotope-labeled compounds to gain insights into the effects of modifications on the fragmentation pathways of the pyrimidine bases. Multi-stage MS (MSn) results showed that protonated cytosine, its 5-hydroxyl- and 5-hydroxymethyl-substituted derivatives, but not its 5-formyl-substituted analog, could undergo Dimroth-like rearrangement in the gas-phase. The elimination of HNCO was one of the major fragmentation pathways observed for the protonated ions of all dC derivatives except for 5-hydroxymethylcytosine, which underwent this loss only after a H2O molecule had been eliminated. In addition, the protonated cytosine and 5-hydroxycytosine can undergo a facile elimination of NH3 molecule. This loss, however, was not observed for protonated 5-hydroxymethylcytosine, 5-formylcytosine, and their uracil analogs. Taken together, our study demonstrated that modifications could alter markedly the CAD patterns of the protonated pyrimidine bases. The results from this study provided a basis for the identifications of other modified pyrimidine bases/nucleosides by tandem mass spectrometry.

Chromatography, High Pressure Liquid↗

PubMed2006

Acetylation and phosphorylation of high-mobility group A1 proteins in PC-3 human tumor cells.

In this paper, we examined the posttranslational modifications (PTMs) of high-mobility group A1 (HMGA1) proteins in PC-3 human prostate cancer cells that are either treated or not treated with a histone deacetylase inhibitor, sodium butyrate. We found that, from a reversed-phase C4 column, the HMGA1a protein eluted in two different fractions with distinct forms of PTMs: Ser98, Ser101, and Ser102 were phosphorylated and Arg25 was methylated for both fractions; only the minor fraction, however, is hyperphosphorylated where Ser35, Thr52, and Thr77 were also phosphorylated. In addition, Lys14 was acetylated in the major but not the minor HMGA1a fraction isolated from the PC-3 cells that were not treated with butyrate. Likewise, HMGA1b, which is a splicing variant of HMGA1a, was acetylated on Lys14 and phosphorylated on the corresponding residues, i.e., Thr41, Thr66, Ser87, Ser90, and Ser91. The acetylation and phosphorylation of the HMGA1a and HMGA1b proteins may affect their interactions with other protein factors, which in turn may modulate the binding of HMGA1 proteins to DNA and regulate gene expression. In addition, the specifically posttranslationally modified HMGA1 proteins may serve as molecular biomarkers for cancer diagnosis and prognosis.

Acetylation↗

PubMed2006

Facile photocyclization chemistry of 5-phenylthio-2'-deoxyuridine in duplex DNA.

We report here the synthesis of 5-phenylthio-2'-deoxyuridine (d(PhS)U), its incorporation into oligodeoxynucleotides (ODNs), and its photocyclization chemistry. Irradiation of dinucleoside monophosphate d((PhS)UG) and d(PhS)U-bearing duplex ODNs with 254 nm light results in the facile formation of a cyclic product where the C6 of uracil is covalently bonded to the C2 of the phenyl ring. The chemistry reported here may serve as the basis for the efficient preparation of a new class of duplex DNA with an extended pi system. [reaction: see text]

Cyclization↗

PubMed2006

Differentiation of 2'-O- and 3'-O-methylated ribonucleosides by tandem mass spectrometry.

Recent studies revealed that the 3'-terminal nucleotides in plant microRNAs were methylated on the ribose at the 2' or 3' hydroxyl groups. Here we examined the fragmentation of the electrospray-produced [M + H]+ and [M - H]- ions of 2'- and 3'-O-methylated ribonucleosides. It turned out that the predominant fragmentation pathway for the [M + H]+ ions of ribose-methylated nucleosides was the neutral loss of the methylated ribose, which made it impossible to distinguish 2'-O-methylation from 3'-O-methylation by positive-ion MS/MS. However, characteristic fragment ions, resulting from the cleavage through the ribose rings, were produced for the [M - H]- ions of each pair of ribose-methylated nucleosides. In this respect, the neutral loss of a 90-Da fragment (C3H6O3) was observed for 2'-O-methylated cytidine, guanosine and adenosine, but not for their 3'-O-methylated counterparts. On the other hand, the neutral loss of a 60-Da fragment (C2H4O2) was found for 3'-O-methyluridine, but not for 2'-O-methyluridine.

Methylation↗

PubMed2006

Phosphorylation of human high mobility group N1 protein by protein kinase CK2.

High mobility group (HMG) N1 protein, formerly known as HMG 14, is a member of the chromosomal HMG protein family. Protein kinase CK2 was previously reported to be able to phosphorylate bovine HMGN1 in vitro; Ser89 and Ser99, corresponding to Ser88 and Ser98 in human HMGN1, were shown to be major and minor recognition sites, respectively. In this report, we employed mass spectrometry and examined both the extent and the sites of phosphorylation in HMGN1 protein catalyzed by recombinant human protein kinase CK2. We found that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in HMGN1 can be phosphorylated by the kinase in vitro. All five sites were previously shown to be phosphorylated in MCF-7 human breast cancer cells in vivo. Among these five sites, Ser6, Ser7, and Ser85 were new sites of phosphorylation induced by protein kinase CK2 in vitro.

Amino Acid Sequence↗

PubMed2006

Fragmentation of the deprotonated ions of peptides containing cysteine, cysteine sulfinic acid, cysteine sulfonic acid, aspartic acid, and glutamic acid.

We examined the fragmentation of the electrospray-produced [M-H]- and [M-2H]2- ions of a number of peptides containing two acidic amino acid residues, one being aspartic acid (Asp) or glutamic acid (Glu), and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)], on an ion-trap mass spectrometer. We observed facile neutral losses of H2S and H2SO2 from the side chains of cysteine and C(SO2H), respectively, whereas the corresponding elimination of H2SO3 from the side chain of C(SO3H) was undetectable for most peptides that we investigated. In addition, the collisional activation of the [M-H]- ions of the C(SO2H)-containing peptides resulted in the cleavage of the amide bond on the C-terminal side of the C(SO2H) residue. Moreover, collisional activation of the [M-2H]2- ions of the above Asp-containing peptides led to the cleavage of the backbone N-Calpha bond of the Asp residue to give cn and/or its complementary [zn-H2O] ions. Similar cleavage also occurred for the singly deprotonated ions of the otherwise identical peptides with a C-terminal amide functionality, but not for the [M-H]- ions of same peptides with a free C-terminal carboxylic acid. Furthermore, ab initio calculation results for model cleavage reactions are consistent with the selective cleavage of the backbone N-Calpha bond in the Asp residue.

Amino Acid Sequence↗

PubMed2006

Identification and quantification of a guanine-thymine intrastrand cross-link lesion induced by Cu(II)/H2O2/ascorbate.

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. It was shown that X- or gamma-ray irradiation of aqueous solutions of DNA, during which *OH is one of the major ROS, can lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32P-postlabeling studies suggested that the intrastrand cross-link lesions may arise from Fenton reaction, which also induces the formation of *OH; the structures of the proposed intrastrand cross-link lesions, however, have not been determined. Here, we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H2O2/ascorbate could lead to the formation of an intrastrand cross-link lesion, i.e., G wedge T, where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G wedge T. In addition, the yield of the intrastrand cross-link was approximately 3 orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-formyl-2'-deoxyuridine. The induction of intrastrand cross-link lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.

Ascorbic Acid↗

PubMed2006

Synthesis and thermodynamic studies of oligodeoxyribonucleotides containing tandem lesions of thymidine glycol and 8-oxo-2'-deoxyguanosine.

Thymidine glycol (Tg), which is also known as 5,6-dihydroxy-5,6-dihydrothymidine, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) are two major types of DNA damage products induced by reactive oxygen species (ROS). Here, we report the synthesis of oligodeoxyribonucleotides (ODNs) containing both Tg and 8-oxodG. The dual incorporation of the two single-base lesions was achieved by using a phosphoramidite building block of 8-oxodG with ultramild base protecting group and a building block of Tg whose nucleobase hydroxyl groups were protected with acetyl functionality. The availability of ODNs carrying neighboring 8-oxodG and Tg provided authentic substrates for assessing the formation and examining the replication and repair of this kind of tandem lesions. In addition, thermodynamic parameters derived from melting temperature data revealed that tandem lesions destabilized the double helix to a greater extent than either of the two single-base lesions alone. The thermodynamic results could offer a basis for understanding the repair of the tandem base lesions.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2006

The photochemistry of 5-BrdCpdC, dCp5-BrdCpdA, and dCp5-BrdCdT in aqueous solution.

We isolated an intrastrand crosslink product from the Pyrex-filtered UV light irradiation of 5-BrdCpdC in aqueous solution. ESI-MS, MS/MS, and 2-D nuclear Overhauser effect spectroscopy (NOESY) results showed that the C5 carbon atoms of the two cytosines are covalently bonded. The same cross-link product can also be induced from the similar UV irradiation of dCp5-BrdCpdA and dCp5-BrdCpdT.

Chromatography, High Pressure Liquid↗

PubMed2005

Formation of intrastrand cross-link products between cytosine and adenine from UV irradiation of d((Br)CA) and duplex DNA containing a 5-bromocytosine.

Here, we showed that Pyrex-filtered UV light irradiation of d((Br)CA) gave rise to three types of intrastrand cross-link products, that is, d(C[5-N6]A), d(C[5-2]A), and d(C[5-8]A), where the C5 carbon atom of cytosine is covalently bonded to the N6 nitrogen atom, C2, and C8 carbon atoms of adenine, respectively. Furthermore, we demonstrated by LC-MS/MS that the UV irradiation of a 5-bromocytosine-containing duplex oligodeoxynucleotide (ODN) led to the formation of five cross-link products, that is, C[5-N6]A, C[5-2]A, C[5-8]A, A[2-5]C, and A[8-5]C, under both aerobic and anaerobic conditions. LC-MS/MS quantification results showed that the yields for the formation of these cross-link products are different. The presence of molecular oxygen reduces the yields for the formation of all cross-link products except A[2-5]C. To our knowledge, this is the first report about the formation of intrastrand cross-link products between cytosine and adenine in duplex DNA. The chemistry discovered here may facilitate the future preparations of oxidative cross-link lesion-bearing substrates for biochemical and biophysical studies.

Adenine↗

PubMed2005

Thermodynamic and in vitro replication studies of an intrastrand G[8-5]C cross-link lesion.

We recently identified, from the gamma-irradiation mixture of duplex DNA, a new intrastrand G[8-5]C cross-link lesion, in which the C8 atom of guanine and the C5 atom of its 3' neighboring cytosine are covalently bonded, and carried out in vitro replication studies for the lesion-bearing substrate with a translesion synthesis polymerase, yeast polymerase eta. Here we extended the in vitro replication studies to two replicative polymerases, exonuclease-deficient bacteriophage T7 DNA polymerase (T7(-)) and HIV reverse transcriptase (HIV-RT). Primer extension assays showed that both polymerases stopped synthesis after incorporating a nucleotide opposite the 3'-cytosine in the G[8-5]C lesion. Steady-state kinetic measurements for nucleotide incorporation opposite the 3'-cytosine of the lesion showed that both T7(-) and HIV-RT preferentially incorporated the correct nucleotide, dGMP. We also examined the thermal stabilities and base pairing properties of G[8-5]C in d(ATGGCG[8-5]CGCTAT). The G[8-5]C lesion destabilizes the duplex form by approximately 4 kcal/mol in free energy at 25 degrees C relative to the undamaged parent duplex, and the thermally most stable duplex has natural bases opposite the lesion.

Base Sequence↗