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Abstract The Legionella pneumophila effector MavC is a transglutaminase that carries out atypical ubiquitination of the host ubiquitin (Ub)‐conjugation enzyme UBE2N by catalyzing the formation of an isopeptide bond between Gln40Ub and Lys92UBE2N, which leads to inhibition of signaling in the NF‐κB pathway. In the absence of UBE2N, MavC deamidates Ub at Gln40 or catalyzes self‐ubiquitination. However, the mechanisms underlying these enzymatic activities of MavC are poorly understood at the molecular level. This study reports the structure of the MavC–UBE2N–Ub ternary complex representing a snapshot of MavC‐catalyzed crosslinking of UBE2N and Ub, which reveals the way by which UBE2N–Ub binds to the Insertion and Tail domains of MavC. Based on the structural and experimental data, the catalytic mechanism for the deamidase and transglutaminase activities of MavC is proposed. Finally, by comparing the structures of MavC and MvcA, the homologous protein that reverses MavC‐induced UBE2N ubiquitination, several essential regions and two key residues (Trp255MavC and Phe268MvcA) responsible for their respective enzymatic activities are identified. The results provide insights into the mechanisms for substrate recognition and ubiquitination mediated by MavC as well as explanations for the opposite activity of MavC and MvcA in terms of regulation of UBE2N ubiquitination.

Abstract Anti‐angiogenic therapies targeting inhibition of vascular endothelial growth factor (VEGF) pathway show clinical benefit in hypervascular hepatocellular carcinoma (HCC) tumors. However, HCC expresses massive pro‐angiogenic factors in the tumor microenvironment (TME) in response to anti‐angiogenic therapy, recruiting tumor‐associated macrophages (TAMs), leading to revascularization and tumor progression. To regulate cell types in TME and promote the therapeutic efficiency of anti‐angiogenic therapy, a supramolecular hydrogel drug delivery system (PLDX‐PMI) co‐assembled by anti‐angiogenic nanomedicines (PCN‐Len nanoparticles (NPs)) and oxidized dextran (DX), and loaded with TAMs‐reprogramming polyTLR7/8a nanoregulators (p(Man‐IMDQ) NRs) is developed for orthotopic liver cancer therapy. PCN‐Len NPs target tyrosine kinases of vascular endothelial cells and blocked VEGFR signaling pathway. p(Man‐IMDQ) NRs repolarize pro‐angiogenic M2‐type TAMs into anti‐angiogenic M1‐type TAMs via mannose‐binding receptors, reducing the secretion of VEGF, which further compromised the migration and proliferation of vascular endothelial cells. On highly malignant orthotopic liver cancer Hepa1‐6 model, it is found that a single administration of the hydrogel formulation significantly decreases tumor microvessel density, promotes tumor vascular network maturation, and reduces M2‐subtype TAMs, thereby effectively inhibiting tumor progression. Collectively, findings in this work highlight the great significance of TAMs reprogramming in enhancing anti‐angiogenesis treatment for orthotopic HCC, and provides an advanced hydrogel delivery system‐based synergistic approach for tumor therapy.