Histone posttranslational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that ...determines the chromatin state. In addition, histone “histidine” methylation was initially reported over 50 years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry. We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linker histones carried a Nτ-methylhistidine modification, and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Protein methylation is mainly observed in lysine, arginine, and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing ...Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18, and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the ...distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC–tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Histone post-translational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that ...determines the chromatin state. In addition, histone "histidine" methylation was initially reported over 50 years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry (MS). We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linker histones carried a Nτ-methylhistidine modification and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Background and aims
The recent advancement of next-generation sequencing (NGS) has enabled the identification of cancer-related somatic aberrations in advanced gastric cancer. However, these remain ...unclear in early gastric cancers, especially in intramucosal gastric cancers.
Patients and methods
Thirty-one well-differentiated (tub1) intramucosal gastric cancers obtained by endoscopic submucosal dissection (ESD) from 29 patients were analyzed. After precise collection of tumors and non-tumors from formalin-fixed paraffin-embedded tissues using laser-captured microdissection (LCM), target sequencing analysis of 50 cancer-related genes was performed using an Ion-Proton sequencer.
Results
The most frequent hotspot mutation was found in
TP53
(17 lesions, 54.8%) followed by the
Wnt
-signaling pathway genes,
APC
and
CTNNB1
(6 lesions, 19.4%). The mutations in
TP53
and the
Wnt
-signaling genes were mutually exclusive (
p
= 0.004). There was a tendency that
H. pylori
infection (
p
= 0.082) and macroscopic protrusion (
p
= 0.095) was associated with the presence of these mutations. Only 10 lesions (59%) among 17 lesions with proven
TP53
mutations were positive for p53 immunostaining demonstrating the superiority of the mutational analysis. In addition, focal gene amplification of
ERBB2
(16%) was found frequently in these early stage lesions.
Conclusions
Using LCM and NGS, mutations in
TP53
and the
Wnt-
signaling pathway were frequently found and were mutually exclusive in the earliest stage of gastric carcinogenesis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Cross-dehydrogenative coupling between 3-hydroxycarbazoles and 2-naphthols has been achieved by using a mesoporous silica-supported oxovanadium catalyst.
Cross-dehydrogenative coupling between ...3-hydroxycarbazoles and 2-naphthols has been achieved by using a mesoporous silica-supported oxovanadium catalyst.
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IJS, KILJ, NUK, UL, UM, UPUK
Large‐scale genomic sequencing of colorectal cancers has been reported mainly for Western populations. Differences by stage and ethnicity in the genomic landscape and their prognostic impact remain ...poorly understood. We investigated 534 Japanese stage III colorectal cancer samples from the Phase III trial, JCOG0910. Targeted‐capture sequencing of 171 potentially colorectal cancer‐associated genes was performed, and somatic single‐nucleotide variants and insertion–deletions were determined. Hypermutated tumors were defined as tumors with MSIsensor score >7 and ultra‐mutated tumors with POLE mutations. Genes with alterations associated with relapse‐free survival were analyzed using multivariable Cox regression models. In all patients (184 right‐sided, 350 left‐sided), mutation frequencies were TP53, 75.3%; APC, 75.1%; KRAS, 43.6%; PIK3CA, 19.7%; FBXW7, 18.5%; SOX9, 11.8%; COL6A3, 8.2%; NOTCH3, 4.5%; NRAS, 4.1%; and RNF43, 3.7%. Thirty‐one tumors were hypermutated (5.8%; 14.1% right‐sided, 1.4% left‐sided). Modest associations were observed: poorer relapse‐free survival was seen with mutant KRAS (hazard ratio 1.66; p = 0.011) and mutant RNF43 (2.17; p = 0.055), whereas better relapse‐free survival was seen with mutant COL6A3 (0.35; p = 0.040) and mutant NOTCH3 (0.18; p = 0.093). Relapse‐free survival tended to be better for hypermutated tumors (0.53; p = 0.229). In conclusion, the overall spectrum of mutations in our Japanese stage III colorectal cancer cohort was similar to that in Western populations, but the frequencies of mutation for TP53, SOX9, and FBXW7 were higher, and the proportion of hypermutated tumors was lower. Multiple gene mutations appeared to impact relapse‐free survival, suggesting that tumor genomic profiling can support precision medicine for colorectal cancer.
We demonstrated that the overall spectrum of mutations in our Japanese stage III colorectal cancer cohort (534 patients) was largely similar to that in Western populations, but the frequencies of mutation for TP53 (75.3%), SOX9 (11.8%), and FBXW7 (18.5%) were higher, and the proportion of hypermutated tumors (5.8%) was lower. We also revealed that poorer relapse‐free survival was seen with mutant KRAS (hazard ratio 1.66; p = 0.011) and mutant RNF43 (2.17; p = 0.055) as well as hypermutated tumors (0.53; p = 0.229), whereas better relapse‐free survival was seen with mutant COL6A3 (0.35; p = 0.040) and mutant NOTCH3 (0.18; p = 0.093), suggesting that tumor genomic profiling has the potential to support precision medicine for patients with colorectal cancer.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We performed in situ conductivity measurements by multiple microscopic probes of Ag nanofilms grown on a periodic array of indium atomic wires on Si(111). Within the investigated thickness range the ...transport properties differ markedly from the bulk case. The ratio between the in-plane conductivity along and perpendicular to the In wires is 1.4 for the 3 monolayer (0.7 nm) thick Ag film and approaches unity with increasing film thickness. The observed anisotropy at the initial stage of the film growth is far larger than expected from the quasi-one-dimensional quantum well electronic structure of the films and ascribed to the carrier scattering at the film/vacuum and film/substrate interfaces. Our findings show that an ordered monolayer inserted at the interface enables drastic changes of the transport behavior of the whole metal nanofilm.
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CMK, CTK, FMFMET, IJS, NUK, PNG, UM
Background and Aim
There are no globally approved, distinguishing criteria enabling the classification of gastric adenomas and intramucosal carcinomas for differential diagnosis of noninvasive ...neoplasia (NIN).
Methods
Next‐generation sequencing of 50 cancer‐related genes was undertaken on 68 pathologically diagnosed microdissected gastric neoplasms (25 adenomas, 27 intramucosal carcinomas, and 16 submucosal carcinomas) obtained during endoscopic submucosal dissection. Findings from magnifying endoscopy with narrow‐band imaging (M‐NBI) of 52 NINs (the 25 adenomas and 27 intramucosal carcinomas) were compared with these data.
Results
Among all 68 neoplasms, the most frequently mutated genes were APC (76% in adenoma, 11.1% in intramucosal carcinoma, and 0% in submucosal carcinoma; P < 0.001) and TP53 in intramucosal and submucosal carcinomas (8% in adenoma, 48.1% in intramucosal carcinoma, and 75% in submucosal carcinoma; P < 0.001). Dividing the NIN neoplasms into five groups according to their mutational status (A1: APC mutation, A2: APC + α mutation, B: APC + TP53 mutation, C: TP53 mutation, D: no mutation in either APC or TP53) resulted in almost identical diagnoses by pathology and M‐NBI for groups A1 (12/13, 92%), C (12/13, 92%), and D (16/17, 94%) but not for groups A2 (3/7, 43%) or B (0/2, 0%). This finding implies that NINs with the APC + α mutation have carcinoma‐like endoscopic features despite most being judged as adenomas by pathology.
Conclusion
A diagnosis of NINs by pathology or M‐NBI in the subset of gastric tumors classified by cancer‐related mutations is not completely identical, suggesting the possible additional role of M‐NBI in diagnosing NINs. Further studies are needed to confirm this.
Through a semiquantitative analysis of cancer‐related genes, it was found that noninvasive intraepithelial neoplasia (NIN) diagnosed as adenoma by both pathology and magnifying endoscopy with narrow‐band imaging (M‐NBI) had a profile of a single APC mutation, while NINs diagnosed as intramucosal carcinoma by both methods had a profile of TP53 mutation in most of the cases. On the other hand, some NINs were diagnosed as adenomas by pathology but were diagnosed as intramucosal carcinomas by M‐NBI, and such NINs with discrepant diagnoses had a characteristic profile of APC plus other mutation(s), suggesting that the M‐NBI finding reflects biological change and might be important for the diagnosis of NINs.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Next generation sequencing (NGS) has revealed a great deal about cancer-related somatic changes in esophageal squamous cell neoplasia; however, the changes in the very early stages remain unclear.
...(87%) and
(20%) hot spot mutations were frequently found in early lesions.
was the most common mutation (LGIN/HGIN, 86%; EP, 83%; LPM, 95%; MM/SM1, 80%), followed by
(29%, 28%, 16% and 10%, respectively); the frequency of other mutations increased as the disease advanced (
< 0.01). Copy number variation analysis revealed copy number aberrations in multiple genes, including
amplification (48%). NGS was superior to p53 immunostaining for detecting
mutations (74% vs. 87%); in combination, the two tests improved detectability to 94%. Clinically, smoking was associated with the occurrence of
mutations in these early lesions (
= 0.049).
Fifty-four early esophageal neoplasia lesions from 47 patients treated by endoscopic resection (low-grade intraepithelial neoplasia LGIN,
1; high-grade intraepithelial neoplasia HGIN
7; invasion limited to epithelium EP/M1,
18; lamina propria mucosae LPM/M2,
19; muscularis mucosae MM/M3,
8; and upper third of the SM SM1,
2) were isolated from formalin-fixed paraffin-embedded tissue specimens by laser-capture microdissection. Target sequencing of 50 cancer-related genes was performed with an Ion Proton sequencer; their association with the clinical characteristics was investigated.
Mutations of
and
, and
amplification were common in early esophageal squamous neoplasia, while other mutations accumulated with disease progression. An understanding of these molecular events might provide a molecular basis for early lesion treatment.