Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β‐catenin by the ubiquitin‐proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)‐associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP‐dependent manner. Ubiquitination of Evi involves the E2‐conjugating enzyme UBE2J2 and the E3‐ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD‐dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.
Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future.
Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading.
Background: Oxidized substrates such as Tim13 acquire two disulfide bonds simultaneously, but Mia40 has one active redox center that accepts two electrons. Results: Mia40 can acquire up to six electrons when oxidizing substrates. Conclusion: Mia40 has the flexibility to accept several electrons. Significance: Mechanistic properties of the MIA pathway are unique compared with redox pathways in the endoplasmic reticulum and bacterial periplasm.
e18001 Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis, thereby enhancing the efficacy of RT and CRT. Xevinapant 200 mg/d (d1-14, Q3W) + CRT showed clinical benefit vs placebo + CRT in a phase (ph) 2 study of patients (pts) with LA SCCHN (NCT02022098). Exposure-response (E-R) relationships for efficacy and the composite safety endpoint of mucositis and/or dysphagia (AE_MD, a known RT-associated toxicity) were also identified. Two ph 3 studies of 200 mg/d xevinapant (d1-14, Q3W) + RT (XRay Vision; NCT05386550) or CRT (TrilynX; NCT04459715) followed by xevinapant monotherapy (MT) in pts with LA SCCHN are ongoing. We leverage the MOA of xevinapant to confirm RP3D strategy. Methods: Results from clinical and preclinical studies and quantitative pharmacology modeling were integrated. Results: Updated analyses of NCT02022098 data show an association between complete response (CR) and AE_MD in both treatment arms; AE_MD was the only AE with identified E-R relationship out of 13 AEs tested. In the xevinapant arm, the CR+AE_MD+ group had the highest median AUC but it overlapped with other groups (CR-AE_MD+, CR+AE_MD-, CR-AE_MD-) which all had similar AUCs. These data suggest that AE_MD is related to the RT-enhancing MOA of xevinapant and that decoupling efficacy from AE_MD incidence by exposure modulation is not feasible. Intermittent dosing for xevinapant was designed for recovery from epithelial toxicities considering the turnover of mucosal cells. In a human breast cancer mouse model, cIAP1 degradation with the intermittent regimen (100 mg/kg Q3D) at the end of the dosing interval was ~50% compared with the continuous one (30 mg/kg QD) expected to maintain maximal (max) cIAP1 degradation, but showed similar efficacy. This suggests continuous max cIAP1 degradation may not be required for efficacy, consistent with RT-enhancing MOA. Based on population PKPD modeling, at the RP3D for xevinapant max cIAP1 degradation was achieved during the dosing period; 91% of pts had at least 50% suppression at the end of the cycle. Xevinapant MT cycles are supported by enhanced anti-tumor activity with extended MT dosing following xevinapant/ RT cycles in MC38 syngeneic mouse model. Food-agnostic dosing is supported by similar AUCs observed in fed and fasted conditions. A human ADME study showed that urinary excretion is a minor elimination pathway, supporting no dose adjustment for pts with moderate renal impairment. Conclusions: Integrated assessment of all available clinical and nonclinical data supports the proposed RP3D strategy for xevinapant combined with RT or CRT, selected to maximize efficacy while minimizing potential adverse events and cumulative toxicity. Clinical trial information: NCT02022098 .
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