He Zhu

Background and Aims: HCC is a highly heterogeneous disease that is caused largely by genomic copy number variations. Herein, the mechanistic and therapeutically targeted role of vacuolar protein sorting 72 homologue (VPS72), a novel copy number variation cis-driven gained gene identified by genome-wide copy number variation and transcriptome analyses in HCC, is not well understood. Approach and Results: First, overexpression of VPS72 enhanced the initiation and progression of HCC in vitro and in vivo. Mechanistically, VPS72 interacted with the oncoproteins MYC and actin-like 6A (ACTL6A) and promoted the formation of the ACTL6A/MYC complex. Furthermore, ACTL6A regulated VPS72 protein stability by weakening the interaction between tripartite motif containing 21 (TRIM21) and VPS72. Thus, the interaction between VPS72 and ACTL6A enhanced the affinity of MYC for its target gene promoters and promoted their transcription, thereby contributing to HCC progression, which was inhibited by adeno-associated virus serotype 8 (AAV8)-mediated short hairpin RNA (shRNA) against VPS72. Conclusions: This study reveals the molecular mechanism of ACTL6A/VPS72/MYC in HCC, providing a theoretical basis and therapeutic target for this malignancy.

TurboID, a proximity labeling method based on mutant biotin ligase, is an efficient new technique for recognizing protein–protein interactions and has been successfully applied to living cells. Sialic acid is typically the terminal monosaccharide attached to many glycoproteins and plays many important roles in many biological processes. However, the lack of enrichment methods for the terminal sialic acid glycosylation in vivo hinders the identification and analysis of this glycosylation. Here, we introduced TurboID to identify the terminal sialic acid glycosylation in living cells. SpCBM, the carbohydrate-binding domain of sialidase from Streptococcus pneumoniae, was fused with TurboID and overexpressed in HeLa cells. After streptavidin-based purification and detection by mass spectrometry (MS), we identified 31 terminal sialic acid N-glycosylated sites and 1359 putative terminal sialic acid glycosylated proteins, many of which were located in the cytoplasm and nucleus.