Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. selleck chemicals llc Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.The coupling of solid-state electrolytes with a Li-metal anode and state-of-the-art (SOA) cathode materials is a promising path to develop inherently safe batteries with high energy density (>1000 Wh L-1). However, integrating metallic Li with solid-electrolytes using scalable processes is not only challenging, but also adds extraneous volume since SOA cathodes are fully lithiated. Here we show the potential for “Li-free” battery manufacturing using the Li7La3Zr2O12 (LLZO) electrolyte. We demonstrate that Li-metal anodes >20 μm can be electroplated onto a current collector in situ without LLZO degradation and we propose a model to relate electrochemical and nucleation behavior. A full cell consisting of in situ formed Li, LLZO, and NCA is demonstrated, which exhibits stable cycling over 50 cycles with high Coulombic efficiencies. These findings demonstrate the viability of “Li-free” configurations using LLZO which may guide the design and manufacturing of high energy density solid-state batteries.Metastasis is the main cause of laryngeal cancer-related death; its molecular mechanism remains unknown. Here we identify protein arginine methyltransferase 5 (PRMT5) as a new metastasis-promoting factor in laryngeal carcinoma, and explore its underlying mechanism of action in regulating laryngeal cancer progression. We illustrated that PRMT5 expression was positively correlated with tumor stages, lymphatic metastasis, and unfavorable outcome. Functional assays revealed that PRMT5 promoted laryngeal carcinoma cell proliferation, migration, and invasive capacity in vitro, as well as lymph-node metastasis in vivo. The ectopic expression of PRMT5 induced EMT with downregulation of E-cadherin and upregulation of N-cadherin, snail, and MMP9. Mechanistic results revealed that the metastatic effects could be attributed to PRMT5-mediated activation of Wnt signaling, and Wnt4 is an important driver of Wnt/β-catenin signaling pathway. Wnt4 silencing could reverse PRMT5-induced cell proliferation, migration, and invasion capacities. Furthermore, inhibition of the Wnt/β-catenin signaling pathway abolished the effect of PRMT5-induced proliferation, whereas activation of the pathway enhanced the effect of PRMT5 overexpression on cell proliferation. These results demonstrated that the oncogenic role of PRMT5 could be attributed to PRMT5/Wnt4 axis-mediated activation of the Wnt/β-catenin signaling pathway. PRMT5 may serve as a novel prognostic marker and a therapeutic target for lymphatic metastasis of laryngeal carcinoma.As a key enzyme in de novo pyrimidine biosynthesis, the expression level of dihydroorotate dehydrogenase (DHODH) has been reported to be elevated in various types of malignant tumors and its tumor-promoting effect was considered to relate to its pyrimidine synthesis function. Here, we revealed one intriguing potential mechanism that DHODH modulated β-catenin signaling in esophageal squamous cell carcinoma (ESCC). We demonstrated that DHODH directly bound to the NH2 terminal of β-catenin, thereby, interrupting the interaction of GSK3β with β-catenin and leading to the abrogation of β-catenin degradation and accumulation of β-catenin in the nucleus, which in turn, resulted in the activation of β-catenin downstream genes, including CCND1, E2F3, Nanog, and OCT4. We further demonstrated that the regulation of β-catenin by DHODH was independent of DHODH catalyzing activity. Univariate and multivariate analyses suggested that DHODH expression might be an independent prognostic factor for ESCC patients. Collectively, our study highlights the pivotal role of DHODH mediated β-catenin signaling and indicates that DHODH may act as a multi-functional switcher from catalyzing pyrimidine metabolism to regulating tumor-related signaling pathways in ESCC.HOPS/Tmub1 is a ubiquitously expressed transmembrane ubiquitin-like protein that shuttles between nucleus and cytoplasm during cell cycle progression. HOPS causes cell cycle arrest in G0/G1 phase, an event associated to stabilization of p19Arf, an important tumor suppressor protein. Moreover, HOPS plays an important role in driving centrosomal assembly and maintenance, mitotic spindle proper organization, and ultimately a correct cell division. Recently, HOPS has been described as an important regulator of p53, which acts as modifier, stabilizing p53 half-life and playing a key role in p53 mediating apoptosis after DNA damage. NF-κB is a transcription factor with a central role in many cellular events, including inflammation and apoptosis. Our experiments demonstrate that the transcriptional activity of the p65/RelA NF-κB subunit is regulated by HOPS. Importantly, Hops-/- cells have remarkable alterations of pro-inflammatory responses. Specifically, we found that HOPS enhances NF-κB activation leading to increase transcription of inflammatory mediators, through the reduction of IκBα stability. Notably, this effect is mediated by a direct HOPS binding to the E3 ubiquitin ligase TRAF6, which lessens TRAF6 stability ultimately leading increased IKK complex activation. These findings uncover a previously unidentified function of HOPS/Tmub1 as a novel modulator of TRAF6, regulating inflammatory responses driven by activation of the NF-κB signaling pathway. The comprehension on how HOPS/Tmub1 takes part to the inflammatory processes in vivo and whether this function is important in the control of proliferation and tumorigenesis could establish the basis for the development of novel pharmacological strategies.