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Here we report the design of N 2 -carboxy-4-aryl-1,2,3-triazole-lysines (CATKs) and their site-specific incorporation into proteins via genetic code expansion. When introduced into the protein dimer interface, CATKs permitted spontaneous, proximity-driven, site-selective crosslinking to generate covalent protein dimers in living cells, with phenyl-bearing CATK-1 exhibiting high reactivity toward the proximal Lys and Tyr. Furthermore, when introduced into the N-terminal β-strand of either a single-chain VHH antibody or a supercharged monobody, CATK-1 enabled site-specific, inter-strand, orthogonal crosslinking with a proximal Tyr located on the opposing β-strand. Compared with a non-crosslinked monobody, the orthogonally crosslinked monobody displayed improved cellular uptake and enhanced proteolytic stability against an endosomal enzyme. The robust crosslinking reactivity of CATKs should facilitate the design of novel protein topologies with improved physicochemical properties., (© 2022 Wiley-VCH GmbH.)

Cancer cells are often hypersensitive to the targeting of transcriptional regulators, which may reflect the deregulated gene expression programs that underlie malignant transformation. One of the most prominent transcriptional vulnerabilities in human cancer to emerge in recent years is the bromodomain and extraterminal (BET) family of proteins, which are coactivators that link acetylated transcription factors and histones to the activation of RNA polymerase II. Despite unclear mechanisms underlying the gene specificity of BET protein function, small molecules targeting these regulators preferentially suppress the transcription of cancer-promoting genes. As a consequence, BET inhibitors elicit anticancer activity in numerous malignant contexts at doses that can be tolerated by normal tissues, a finding supported by animal studies and by phase I clinical trials in human cancer patients. In this review, we will discuss the remarkable, and often perplexing, therapeutic effects of BET bromodomain inhibition in cancer., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.)

Plasma selenium and NRF2 promoter variants (e.g., rs6721961) are associated with cardiovascular disease risk in the general population. However, epidemiological evidence on the interaction between plasma selenium and NRF2 genetic susceptibility in relation to incident coronary heart disease (CHD) risk remains scarce, especially among individuals with type 2 diabetes (T2D). Thus, we examined whether rs6721961 in the NRF2 gene might modify the association between plasma selenium levels and incident CHD risk among people with T2D. During a mean (SD) follow-up period of 6.90 (2.96) years, 798 incident CHD cases were identified among 2,251 T2D cases. Risk-allele carriers of rs6721961 had a higher risk of incident CHD among people with T2D (adjusted hazard ratio [HR] 1.17; 95% CI 1.02-1.35) versus nonrisk-allele carriers. Each 22.8-μg/L increase in plasma selenium levels was associated with a reduced risk of incident CHD among risk-allele carriers with T2D (HR 0.80; 95% CI 0.71-0.89), whereas no association was found in those without risk alleles (P for interaction = 0.004), indicating that the NRF2 promoter polymorphism might modify the association between plasma selenium levels and incident CHD risk among people with T2D. Our study findings suggest redox-related genetic variants should be considered to identify populations that might benefit most from selenium supplementation. More mechanistic studies are warranted. [ABSTRACT FROM AUTHOR]

Here we report the design of N2‐carboxy‐4‐aryl‐1,2,3‐triazole‐lysines (CATKs) and their site‐specific incorporation into proteins via genetic code expansion. When introduced into the protein dimer interface, CATKs permitted spontaneous, proximity‐driven, site‐selective crosslinking to generate covalent protein dimers in living cells, with phenyl‐bearing CATK‐1 exhibiting high reactivity toward the proximal Lys and Tyr. Furthermore, when introduced into the N‐terminal β‐strand of either a single‐chain VHH antibody or a supercharged monobody, CATK‐1 enabled site‐specific, inter‐strand, orthogonal crosslinking with a proximal Tyr located on the opposing β‐strand. Compared with a non‐crosslinked monobody, the orthogonally crosslinked monobody displayed improved cellular uptake and enhanced proteolytic stability against an endosomal enzyme. The robust crosslinking reactivity of CATKs should facilitate the design of novel protein topologies with improved physicochemical properties. [ABSTRACT FROM AUTHOR]

Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development. [ABSTRACT FROM AUTHOR]

Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli was explored. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC but not cytoplasmic ones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. To our knowledge, this is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable. [ABSTRACT FROM AUTHOR]