Identification of sulfhydryl-containing proteins and further evaluation of the selenium-tagged redox homeostasis-regulating proteins.

Chemoproteomic profiling of sulfhydryl-containing proteins has consistently been an attractive research hotspot. However, there remains a dearth of probes that are specifically designed for sulfhydryl-containing proteins, possessing sufficient reactivity, specificity, distinctive isotopic signature, as well as efficient labeling and evaluation capabilities for proteins implicated in the regulation of redox homeostasis. Here, the specific selenium-containing probes (Se-probes) in this work displayed high specificity and reactivity toward cysteine thiols on small molecules, peptides and purified proteins and showed very good competitive effect of proteins labeling in gel-ABPP. We identified more than 6000 candidate proteins. In TOP-ABPP, we investigated the peptide labeled by Se-probes, which revealed a distinct isotopic envelope pattern of selenium in both the primary and secondary mass spectra. This unique pattern can provide compelling evidence for identifying redox regulatory proteins and other target peptides. Furthermore, our examiation of post-translational modification (PTMs) of the cysteine site residues showed that oxidation PTMs was predominantly observed. We anticipate that Se-probes will enable broader and deeper proteome-wide profiling of sulfhydryl-containing proteins, provide an ideal tool for focusing on proteins that regulate redox homeostasis and advance the development of innovative selenium-based pharmaceuticals.

Regulation of the stem­like properties of estrogen receptor­positive breast cancer cells through NR2E3/NR2C2 signaling.

Cancer stem cells (CSCs) are major drivers of metastasis, drug resistance and recurrence in numerous cancers. However, critical factors that can modulate CSC stemness have not been clearly identified. Nuclear receptor subfamily 2 group E member 3 (nr2e3) expression has been previously reported to be positively associated with drug sensitivity and favorable clinical outcomes in patients with estrogen receptor (ER)+ breast cancer. This suggests that nr2e3 expression may be inversely associated with CSC stemness in this type of tumor cells. The present study aimed to investigate the regulatory roles of NR2E3 in the stem-like properties of ER+ breast cancer cells and to identify the underlying mechanisms. Bioinformatics analysis was performed using the data derived from the Cancer Genome Atlas database. Nr2e3-specific shRNA and nuclear receptor subfamily 2 group C member 2 (nr2c2) overexpressed plasmids were constructed to silence and enhance the expression of nr2e3 and nr2c2, respectively. Transwell and wound healing experiments were conducted to evaluate the migration and invasion ability of MCF7 cells, while colony formation tests were used to evaluate the clonality. Flow cytometry was used to detect the percentage of CD44+CD24-/low cells. Reverse transcription-quantitative PCR and western blotting were performed to detect expression at the mRNA and protein levels. The results showed that compared with normal breast tissues and MCF10A cells, the expression of nr2e3 was increased in ER+ breast tumor tissues and cell lines. Nr2e3 silencing promoted the migration, invasion and colony-forming ability of the ER+ MCF7 cells. It also increased the expression of epithelial-mesenchymal transition markers and stem cell-related transcription factors, in addition to the percentage of CD44+CD24-/low cells. The expression of nr2e3 and nr2c2 was found to be positively correlated. Nr2e3 knockdown decreased the mRNA and protein expression levels of nr2c2, whereas nr2c2 overexpression reversed the elevated CD44+CD24-/low cell ratio and the increased migratory activity caused by nr2e3 silencing. The results of the present study suggest that NR2E3 may serve an important role in modulating the stem-like properties of ER+ breast cancer cells, where NR2E3/NR2C2 signaling may be a therapeutic target in ER+ breast cancer.

Detection of Cysteine Sulfenic Acid on E. coli Proteins with a Biotin-Benzoboroxole Probe.

S-sulfenylation of cysteine residues on proteins can effectively change protein structures and accordingly regulate their functions in vivo. Investigation of S-sulfenylation in different biological environments is thus vital for a systematic understanding of cellular redox regulation. In this work, a functional probe, biotin-benzoboroxole (Bio-ben), was designed for the detection of cysteine sulfenic acid (Cys-SOH). The performance of Bio-ben was characterized by small-molecule sulfenic acid, protein models, and proteome tests via mass spectra and western blotting. The results showed that Bio-ben was validated for cysteine sulfenic acid on proteins with good capture efficiency even at low concentrations. Compared with commonly used probes such as dimedone, the current probe has significantly shortened labeling time and exhibited comparable sensitivity. The proposed method provides a new approach for exploring S-sulfenylation in the oxidative modification of proteins and is helpful for related biological and clinical applications.

Quantitative Chemoproteomic Profiling of Targets of Au(I) Complexes by Competitive Activity-Based Protein Profiling.

Owing to the encouraging pharmacological action and acceptable toxicity profile, Au(I) complexes have attracted growing interest in the application of disease treatment. In order to investigate their potential target proteins and related bioinformation, herein, we screened four Au(I) complexes and explored the binding proteins utilizing a competitive activity-based protein profiling (ABPP) strategy, including identification experiments and reactivity classification experiments, which offers a simple and robust method to identify the target proteins of Au(I) complexes. We quantified the target proteins of the four Au(I) complexes and found that most of proteins were associated with cancer. In addition, the newly Au(I)-binding proteins and biological gold-protein interaction pathways were exhibited. Furthermore, we estimated the correlation between target proteins of Au(I) complexes and various cancers, which will promote the development of the gold anticancer drugs.

Fluorescence switch of gold nanoclusters stabilized with bovine serum albumin for efficient and sensitive detection of cysteine and copper ion in mice with Alzheimer's disease.

The near-infrared fluorescence of gold nanoclusters stabilized with bovine serum albumin (BSA -AuNCs) centered at 675 nm could be enhanced by cysteine and then effectively quenched by copper ion (Cu2+), therefore, cysteine and copper ion could be detected in sequence. At "on" state, fluorescence enhancement of BSA-AuNCs is generated due to the reaction between cysteine and BSA-AuNCs, via filling the surface defect of gold nanoclusters, while Cu2+ can further oxidize the reductive sulfydryl of cysteine and interact with amino acids presented in the BSA chain, inducing gold nanoclusters to aggregate, thus causing "off" state with fluorescence quenching. Fluorescence switch of BSA-AuNCs can be used for cysteine and Cu2+ detection in mice brain with Alzheimer's disease (AD) in vitro, with fast response, high chemical stability and sensitivity. Besides, it was able to image the endogenous Cu2+ in liver and heart of AD mice in situ. The results are promising, especially in the framework of early diagnosis of Alzheimer's disease.

Fibrisoma montanum sp. nov., isolated from soil of Mountain Danxia, China.

A Gram-stain-negative, non-motile, rod-shaped, aerobic bacterium, designated HYT19T, was isolated from soil of Mountain Danxia in southern China. It showed the highest similarity of 16S rRNA gene sequence (97.0%) and formed a monophyletic clade with Fibrisoma limi BUZ 3T. Strain HYT19T grew at 16-37 °C (optimum 28-30 °C) and at pH 6-7. The draft genome size of strain HYT19T was 7.8 Mb with a DNA G+C content of 54.0 mol%. The digital DDH and average nucleotide identity values between strain HYT19T and F. limi BUZ 3T were 28.8% and 85.1%, respectively. MK-7 was the sole respiratory quinone. The major polar lipids were phosphatidylethanolamine, unidentified aminophospholipid, two unidentified aminolipids, unidentified phospholipid and unidentified lipid. The strain contained C16:1ω5c, iso-C15:0, summed feature 3 (C16:1ω6c and/or C16:1ω7c), C16:0, iso-C17:0 3-OH and anteiso-C15:0 as the major fatty acids. On the basis of phylogenetic, genomic, phenotypic and chemotaxonomic analysis, we propose a new species Fibrisoma montanum sp. nov. of genus Fibrisoma. The type strain is HYT19T (= CCTCC AB 2018342T = JCM 33105T).

Sphingomonas gilva sp. nov., isolated from mountain soil.

A Gram-stain-negative, strictly aerobic, motile, yellow, rod-shaped bacterium, designated ZDH117T, was isolated from soilsampled atthe Danxialandformin Guangdong Province, PR China. The 16S rRNA gene sequence of strain ZDH117T had highest similarityvalues to Sphingomonas adhaesivaDSM 7418T (97.5 %), SphingomonasdesiccabilisCP1DT (97.3 %) and Sphingomonas ginsenosidimutans KACC 14949T (97.2 %). However, phylogenetic analyses based on 16S rRNA gene sequences demonstrated that strain ZDH117T clustered with Sphingomonas zeicaulis 541T (96.17 %) and Sphingomonas sanxanigenens DSM 19645T (95.95 %). The genomic average nucleotide identity values of ZDH117T with S. adhaesiva DSM 7418T, S. desiccabilis CP1DTand S. ginsenosidimutans KACC TT were 75.1, 75.2 and 75.0 %, respectively. The G+C content of the genomic DNA was 67.6 mol%. Strain ZDH117T was characterized to have ubiquinone-10 as the predominant respiratory quinone, sym-homospermidine as the major polyamine and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c), C14 : 0-2OH, C16 : 0 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) as the major cellular fatty acids (>5 % of total). The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid, an unidentified phospholipid and three unidentified lipids. On the basis of its phenotypic, chemotaxonomic and phylogenetic characteristics, strain ZDH117T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas gilva sp. nov. is proposed. The type strain is ZDH117T (=KCTC 62894T=CCTCCAB 2018262T).