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

MALT 1 paracaspase的蛋白酶活性失调与多种人类疾病相关。例如，在弥漫性大B细胞淋巴瘤（DLBCL）的高度侵袭性亚型中，MALT 1是组成型活性的。针对MALT 1的抑制剂的临床试验已经开始，但对患者进行分层以确定他们是否能受益于这种治疗是至关重要的。此外，MALT 1最近也被认为与炎症性肠病有关。在这项研究中，我将开发荧光猝灭的基于活性的探针（qABPs）作为化学工具，通过流式细胞术和实时成像来分析MALT 1的蛋白酶活性。MALT 1-qABP的设计将基于酰氧基甲基酮（AOMK）反应基团，其对半胱氨酸蛋白酶具有选择性。它将被连接到一个四肽序列的选择性和细胞渗透性至关重要。在AOMK与半胱氨酸蛋白酶的基于机制的反应中，酰氧基被排出，这允许引入荧光猝灭基团，导致本身不发荧光的“智能”探针，但仅在与其靶标反应后才发荧光。然后，探针和MALT 1之间形成的共价键防止所得荧光信号的扩散，并且还允许对靶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剂对。在初步生物化学验证后，MALT 1-qABP将在临床应用研究中实施，以明确分类淋巴瘤亚型。为此，将使用细胞培养模型和最终来自患有淋巴瘤的患者的切除淋巴结的细胞来优化这些qABP的流式细胞术应用。该材料将用于（1）定量MALT 1的活性状态，和（2）监测MALT 1抑制剂的疗效。在一个单独的工作包中，将演示使用MALT 1-qABP的实时成像。在这里，将分析结肠炎期间肠道中MALT 1的上调。具体而言，化学诱导发展结肠炎的小鼠模型将用于离体和体内荧光显微镜检查。在此基础上，我将研究基础研究问题，以阐明MALT 1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.