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

MALT1副半乳糖酶的蛋白酶活性失调与多种人类疾病有关。例如，在弥漫性大B细胞淋巴瘤（DLBCL）的高侵袭性亚型中，MALT1具有组成性活性。MALT1定向抑制剂的临床试验已经开始，但有必要对患者进行分层，看看他们是否能从这种治疗中获益。此外，MALT1最近也被认为与炎症性肠病有关。在这项研究中，我将开发荧光淬灭活性探针（qABPs）作为化学工具，通过流式细胞术和实时成像来分析MALT1的蛋白酶活性。MALT1-qABPs的设计将基于对半胱氨酸蛋白酶具有选择性的酰基氧甲基酮（AOMK）反应基团。它将附着在一个四肽上，其序列对选择性和细胞渗透性至关重要。在AOMKs与半胱氨酸蛋白酶的基于机理的反应中，乙酰基被排出，从而允许引入荧光猝灭基团，从而导致“智能”探针本身不具有荧光，但只有在与目标反应后才具有荧光。探针和MALT1之间形成的共价键阻止了所产生的荧光信号的扩散，也允许对目标接合进行下游生化分析。为了优化流式细胞术和实时成像，我将实现各种不同的荧光团猝灭器对。在初步生化验证后，MALT1-qABPs将在临床应用研究中实施，以明确区分淋巴瘤亚型。为此，这些qABPs的流式细胞术应用将通过细胞培养模型和淋巴瘤患者切除淋巴结的细胞来优化。该材料将用于(1)量化MALT1的活性状态，(2)监测MALT1抑制剂的功效。在一个单独的工作包中，将演示使用MALT1-qABPs进行实时成像。在这里，我们将分析报道的结肠炎期间肠道中MALT1的上调。具体来说，化学诱导结肠炎的小鼠模型将用于离体和体内荧光显微镜。在此基础上，我将探讨基础研究问题，以阐明MALT1在炎症性肠病中的未知作用。

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.