Dynamic monitoring of MALT1 paracaspase activity by fluorescently quenched activity-based probes

点击翻译按钮获取中文摘要

Deregulated protease activity of MALT1 paracaspase is associated with various human diseases. For example, in highly aggressive subforms of diffuse large B cell lymphomas (DLBCL), MALT1 is constitutively active. Clinical trials with MALT1 directed inhibitors have started, but it is essential here to stratify patients to see if they can benefit this treatment. In addition, MALT1 has recently also been implicated in inflammatory bowel diseases. In this study, I will develop fluorescently quenched activity-based probes (qABPs) as chemical tools to profile protease activity of MALT1 by flow cytometry and real-time imaging. The design of MALT1-qABPs will be based on the acyloxymethyl ketone (AOMK) reactive group, which is selective for cysteine proteases. It will be attached to a tetrapeptide with a sequence crucial for selectivity and cell permeability. In the mechanism-based reaction of AOMKs with cysteine proteases, the acyloxy group is expelled, which allows for introduction of a fluorescent quenching group, leading to a ‘smart’ probe that is non-fluorescent itself, but only becomes fluorescent after reaction with its target. The covalent bond formed between the probe and the MALT1 then prevents diffusion of the resulting fluorescence signal and also allows downstream biochemical analysis of target engagement. For optimization in flow cytometry and real-time imaging, I will implement various different fluorophore-quencher pairs. After initial biochemical validation, MALT1-qABPs will be implemented in studies towards clinical applications to unambiguously classify lymphoma subtypes. To this end, flow cytometry application of these qABPs will be optimized using cell culture models and eventually cells from resected lymphnodes of patients suffering from lymphoma. This material will be used to (1) quantify the activity state of MALT1, and (2) monitor the efficacy of MALT1 inhibitors. In a separate work package, real-time imaging with MALT1-qABPs will be demonstrated. Here, the reported upregulation of MALT1 in the intestine during colitis will be analyzed. Specifically, a mouse model chemically induced to develop colitis will be used for ex vivo and in vivo fluorescent microscopy. On this basis, I will investigate fundamental research questions to shed light on the unknown roles of MALT1 within inflammatory bowel disease.