A novel base editor SpRY-ABE8eF148A mediates efficient A-to-G base editing with a reduced off-target effect.

Adenine base editors (ABEs) can mediate two transition mutations, A-to-G and T-to-C, which are suitable for repairing G·C-to-T·A pathogenic variants, the most significant human pathogenic variant. By combining the protospacer adjacent motif (PAM)less SpRY nuclease with F148A-mutated TadA∗8e deaminase, we developed a new editor, SpRY-ABE8eF148A, in this study, which has narrowed the editing range and enhanced A-to-G editing efficiency in most sites with NR/YN PAMs. Furthermore, compared with SpRY-ABE8e, SpRY-ABE8eF148A significantly decreased the RNA off-target effect. Therefore, this engineered base editor, SpRY-ABE8eF148A, expanded the editing scope and improved the editing precision for G·C-to-T·A pathogenic variants. Besides, we established a bioinformatics tool, adenine base-repairing sgRNA database of pathogenic variant (ARDPM), to facilitate the development of precise editors.

O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.

SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.

O-GlcNAcylation regulates the methionine cycle to promote pluripotency of stem cells.

Methionine metabolism is critical for the maintenance of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) pluripotency. However, little is known about the regulation of the methionine cycle to sustain ESC pluripotency. Here, we show that adenosylhomocysteinase (AHCY), an important enzyme in the methionine cycle, is critical for the maintenance and differentiation of mouse embryonic stem cells (mESCs). We show that mESCs exhibit high levels of methionine metabolism, whereas decreasing methionine metabolism via depletion of AHCY promotes mESCs to differentiate into the three germ layers. AHCY is posttranslationally modified with an O-linked ß-N-acetylglucosamine sugar (O-GlcNAcylation), which is rapidly removed upon differentiation. O-GlcNAcylation of threonine 136 on AHCY increases its activity and is important for the maintenance of trimethylation of histone H3 lysine 4 (H3K4me3) to sustain mESC pluripotency. Blocking glycosylation of AHCY decreases the ratio of S-adenosylmethionine versus S-adenosylhomocysteine (SAM/SAH), reduces the level of H3K4me3, and poises mESC for differentiation. In addition, blocking glycosylation of AHCY reduces somatic cell reprogramming. Thus, our findings reveal a critical role of AHCY and a mechanistic understanding of O-glycosylation in regulating ESC pluripotency and differentiation.

O-GlcNAcylation of PGK1 coordinates glycolysis and TCA cycle to promote tumor growth.

Many cancer cells display enhanced glycolysis and suppressed mitochondrial metabolism. This phenomenon, known as the Warburg effect, is critical for tumor development. However, how cancer cells coordinate glucose metabolism through glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle is largely unknown. We demonstrate here that phosphoglycerate kinase 1 (PGK1), the first ATP-producing enzyme in glycolysis, is reversibly and dynamically modified with O-linked N-acetylglucosamine (O-GlcNAc) at threonine 255 (T255). O-GlcNAcylation activates PGK1 activity to enhance lactate production, and simultaneously induces PGK1 translocation into mitochondria. Inside mitochondria, PGK1 acts as a kinase to inhibit pyruvate dehydrogenase (PDH) complex to reduce oxidative phosphorylation. Blocking T255 O-GlcNAcylation of PGK1 decreases colon cancer cell proliferation, suppresses glycolysis, enhances the TCA cycle, and inhibits tumor growth in xenograft models. Furthermore, PGK1 O-GlcNAcylation levels are elevated in human colon cancers. This study highlights O-GlcNAcylation as an important signal for coordinating glycolysis and the TCA cycle to promote tumorigenesis.

O-GlcNAcylation of core components of the translation initiation machinery regulates protein synthesis.

Protein synthesis is essential for cell growth, proliferation, and survival. Protein synthesis is a tightly regulated process that involves multiple mechanisms. Deregulation of protein synthesis is considered as a key factor in the development and progression of a number of diseases, such as cancer. Here we show that the dynamic modification of proteins by O-linked ß-N-acetyl-glucosamine (O-GlcNAcylation) regulates translation initiation by modifying core initiation factors eIF4A and eIF4G, respectively. Mechanistically, site-specific O-GlcNAcylation of eIF4A on Ser322/323 disrupts the formation of the translation initiation complex by perturbing its interaction with eIF4G. In addition, O-GlcNAcylation inhibits the duplex unwinding activity of eIF4A, leading to impaired protein synthesis, and decreased cell proliferation. In contrast, site-specific O-GlcNAcylation of eIF4G on Ser61 promotes its interaction with poly(A)-binding protein (PABP) and poly(A) mRNA. Depletion of eIF4G O-GlcNAcylation results in inhibition of protein synthesis, cell proliferation, and soft agar colony formation. The differential glycosylation of eIF4A and eIF4G appears to be regulated in the initiation complex to fine-tune protein synthesis. Our study thus expands the current understanding of protein synthesis, and adds another dimension of complexity to translational control of cellular proteins.