The C-terminal selenenylsulfide of extracellular/non-reduced thioredoxin reductase endows this protein with selectivity to small-molecule electrophilic reagents under oxidative conditions.

Mammalian cytosolic thioredoxin reductase (TrxR1) serves as an antioxidant protein by transferring electrons from NADPH to various substrates. The action of TrxR1 is achieved via reversible changes between NADPH-reduced and non-reduced forms, which involves C-terminal selenolthiol/selenenylsulfide exchanges. TrxR1 may be released into extracellular environment, where TrxR1 is present mainly in the non-reduced form with active-site disulfide and selenenylsulfide bonds. The relationships between extracellular TrxR1 and tumor metastasis or cellular signaling have been discovered, but there are few reports on small-molecule compounds in targeted the non-reduced form of TrxR1. Using eight types of small-molecule thiol-reactive reagents as electrophilic models, we report that the selenenylsulfide bond in the non-reduced form of TrxR1 functions as a selector for the thiol-reactive reagents at pH 7.5. The non-reduced form of TrxR1 is resistant to hydrogen peroxide/oxidized glutathione, but is sensitive to certain electrophilic reagents in different ways. With 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and S-nitrosoglutathione (GSNO), the polarized selenenylsulfide bond breaks, and selenolate anion donates electron to the dynamic covalent bond in DTNB or GSNO, forming TNB-S-Se-TrxR1 complex or ON-Se-TrxR1 complex. The both complexes lose the ability to transfer electrons from NADPH to substrate. For diamide, the non-reduced TrxR1 actually prevents irreversible damage by this oxidant. This is consistent with the regained activity of TrxR1 through removal of diamide via dialysis. Diamide shows effective in the presence of human cytosolic thioredoxin (hTrx1), Cys residue(s) of which is/are preferentially affected by diamide to yield disulfide, hTrx1 dimer and the mixed disulfide between TrxR1-Cys497/Sec498 and hTrx1-Cys73. In human serum samples, the non-reduced form of TrxR1 exists as dithiothreitol-reducible polymer/complexes, which might protect the non-reduced TrxR1 from inactivation by certain electrophilic reagents under oxidative conditions, because cleavage of these disulfides can lead to regain the activity of TrxR1. The details of the selective response of the selenenylsulfide bond to electrophilic reagents may provide new information for designing novel small-molecule inhibitors (drugs) in targeted extracellular/non-reduced TrxR1.

"DSN-mismatched CRISPR″sensor for highly selective and sensitive detection of under-expressed miR-let-7a.

Several microRNAs (miRNAs) are expressed at lower levels in specific tumors, e.g., miR-let-7a in non-small cell lung cancer (NSCLC). This makes it challenging to analyze their lower abundance versus specifically elevated miRNAs. Here, we describe a novel fluorescent biosensor for the highly selective and sensitive detection of miR-let-7a constructed by combining miRNA screening assisted by a duplex-specific nuclease (DSN) with CRISPR-Cas12a system signal amplification. We meticulously designed a mismatch in the first three to four bases at the 5'-end of the capture DNA to improve the signal-to-noise ratio of the CRISPR-Cas12a system. Within this "DSN-mismatched CRISPR" fluorescence strategy, miR-let-7a was accurately screened by DSN-assisted cleavage, and the mismatched capture DNA unbound to target miRNA could trigger the CRISPR-Cas12a system to produce a mass of trans-cleave fluorescence signals. This "turn-off" approach was suitable for detecting decreased levels of miRNAs. This approach can not only discriminate the single-base mismatched let-7 family but also reach a limit of detection at 64.17 fM as well as be quantified from 100 fM to 500 pM. The miR-let-7a levels were then measured in clinical serum samples from healthy volunteers and patients with NSCLC. This study holds promise for the development of a universal under-expressed miRNA assay for early diagnosis and treatment of cancers.

Mechanistic insights into the inhibitory effects of palmitoylation on cytosolic thioredoxin reductase and thioredoxin.

Overnutrition can lead to oxidative stress, but its underlying mechanism remains unclear. In this study, we report that human liver-derived HepG2 cells utilize cytosolic thioredoxin reductase (TrxR1) and thioredoxin (hTrx1) to defend against the high glucose/palmitate-mediated increase in reactive oxygen species. However, enhanced TrxR1/hTrx1 palmitoylation occurs in parallel with a decrease in their activities under the conditions studied here. An autoacylation process appears to be the major mechanism for generating palmitoylated TrxR1/Trx1 in HepG2 cells. A novel feature of this post-translational modification is the covalent inhibition of TrxR1/hTrx1 by palmitoyl-CoA, an activated form of palmitate. The palmitoyl-CoA/TrxR1 reaction is NADPH-dependent and produces palmitoylated TrxR1 at an active site selenocysteine residue. Conversely, S-palmitoylation occurs at the structural Cys62/Cys69/Cys72 residues but not the active site Cys32/Cys35 residues of hTrx1. Palmitoyl-CoA concentration and the period of incubation with TrxR1/hTrx1 are important factors that influence the inhibitory efficacy of palmitoyl-CoA on TrxR1/hTrx1. Thus, an increase in TrxR1/hTrx1 palmitoylation could be a potential consequence of high glucose/palmitate. The time-dependent inactivation of the NADPH-TrxR1-Trx1 system by palmitoyl-CoA occurs in a biphasic manner - a fast phase followed by a slow phase. Kinetic analysis suggests that the fast phase is consistent with a fast and reversible association between TrxR1/hTrx1 and palmitoyl-CoA. The slow phase is correlated with a slow and irreversible inactivation, in which selenolate/thiolate groups nucleophilically attack the α-carbon of bound palmitoyl-CoA, leading to the formation of thioester/selenoester bonds. hTrx1 can enhance rate of fast phase but limits the rate of slow phase when it is present in a preincubation mixture containing NADPH, TrxR1 and palmitoyl-CoA. Therefore, hTrx1 may provide palmitoylation sites or partially protect the TrxR1 active site selenol/thiol group(s) from palmitoylation. Our data suggest that Se/S-palmitoylation acts as an important modulator of TrxR1/hTrx1 activities, representing a novel potential mechanism that underlies overnutrition-induced events.