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Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation. Synopsis: Polyglutamylation by the tubulin tyrosine ligase-like (TTLL) family enzymes predominantly regulates cytoskeletal proteins. This study shows that the glutamylase TTLL11 polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3) at its C-terminus, thus changing its biochemical and signaling properties. TTLL11-mediated polyglutamylation of DVL3 takes place at the alpha-carboxyl group of the terminal methionine and extends the protein with a chain of glutamates. Polyglutamylation of DVL3 regulates its phase separation and phosphorylation. Polyglutamylated DVL3 is more active in the Wnt/planar cell polarity (PCP) pathway. TTLL11 is required for morphogenetic events controlled by PCP in Xenopus. Glutamylase TTLL11 catalyzes a novel type of carboxy-terminal polyglutamylation to suppress DVL3 phase separation and enhance its activity in the planar cell polarity pathway. [ABSTRACT FROM AUTHOR]

Abstract Multiple myeloma (MM) is a cancer of plasma cells caused by abnormal gene expression and interactions within the bone marrow (BM) niche. The BM environment significantly influences the progression of MM. Celastrol, a natural compound derived from traditional Chinese medicine, exhibits significant anticancer effects. This study aimed to identify specific targets of celastrol and develop more effective and less toxic treatment options for MM. Celastrol is used as a probe to determine its specific target, pyridoxine‐5′‐phosphate oxidase (PNPO). Increased levels of PNPO are associated with poor outcomes in MM patients, and PNPO promotes MM cell proliferation and induces osteoclast differentiation through exosomes. Mechanistically, PNPO oxidizes disheveled 3M282 (DVL3), leading to abnormal activation of the Wnt/β‐catenin pathway. Based on the critical sites of PNPOR95/K117, Eltrombopag is identified as a potential therapeutic candidate for MM. In addition, the experiments showed its efficacy in mouse models. Eltrombopag inhibited the growth of MM cells and reduced bone lesions by disrupting the interaction between PNPO and DVL3, as supported by preliminary clinical trials. The study highlights the importance of PNPO as a high‐risk gene in the development of MM and suggests that Eltrombopag may be a promising treatment option.

The focus of this paper is the human Dishevelled 3 protein (hDvl3), an essential component of the Wnt signalling pathway that contributes to their regulation. Mass spectrometry-based analysis of hDvl3 phosphorylations induced by eight associated kinases was performed revealing several dozens of phosphorylation sites. The main outcome of this study was the description of Dvl phosphorylation “patterns” induced by individual kinases