公开(公告)号：US20210208141A1

公开(公告)日：2021-07-08

申请号：US17021260

申请日：2020-09-15

申请人： The Scripps Research Institute

发明人： Benjamin Cravatt ,  Chu Wang ,  Keriann Backus

IPC分类号： G01N33/573 ,  G01N33/68 ,  C12N9/12

摘要： Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods. Here, we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify a select set of proteins that constitute “hot spots” for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxynonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by oxidative stress.

公开(公告)号：US10782295B2

公开(公告)日：2020-09-22

申请号：US14911316

申请日：2014-08-13

申请人： THE SCRIPPS RESEARCH INSTITUTE

发明人： Benjamin Cravatt ,  Chu Wang ,  Keriann Backus

IPC分类号： G01N33/573 ,  C12N9/12 ,  C40B30/00 ,  G01N33/68

摘要： Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods. Here, we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify a select set of proteins that constitute hot spots for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxnonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by oxidative stress.

公开(公告)号：US20160252509A1

公开(公告)日：2016-09-01

申请号：US14911316

申请日：2014-08-13

申请人： THE SCRIPPS RESEARCH INSTITUTE

发明人： Benjamin Cravatt ,  Chu Wang ,  Keriann Backus

IPC分类号： G01N33/573 ,  C12N9/12

摘要： Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods. Here, we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify a select set of proteins that constitute hot spots for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxnonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by oxidative stress.

摘要翻译： 细胞产生具有修饰和影响蛋白质功能的潜力的亲电子产物。 化学方法提供了定性鉴定内源性亲电试剂靶向的蛋白质的方法; 然而，确定这些反应的能力和特异性以鉴定蛋白质组中最敏感的部位进行亲电改性需要更多的定量方法。 在这里，我们描述了一种基于竞争活动的分析方法，用于在人类蛋白质组中平行定量亲电子化合物对1000+半胱氨酸的反应性。 使用这种方法，我们确定了一组蛋白质，构成了各种脂质衍生的亲电试剂（包括氧化应激产物4-羟基壬烯醛（HNE））修饰的热点。 我们显示这些蛋白质之一的ZAK激酶被保守的活性位点近端半胱氨酸上的HNE标记，导致酶的抑制，从而产生可以通过氧化应激抑制JNK通路激活的负反馈机制。