High-content imaging, immunoblot and immunofluorescence data related to: Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH

G3BP is the central node within stress-induced protein–RNA interaction
networks known as stress granules (SGs). The SG-associated proteins
Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1,
promoting and inhibiting SG formation, respectively. Herein, we present
the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived
short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62
within a third NTF2-binding site outside those covered by USP10, as
confirmed using biochemical and biophysical-binding assays.
Nano-differential scanning fluorimetry revealed reduced thermal stability
of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced
significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex
immunoprecipated from human U2OS cells was more resistant to acidic buffer
washes than G3BP1/Caprin-1. Acidification of cellular condensates by
approximately 0.5 units relative to the cytosol was detected by
ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells
expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had
reduced Caprin-1 levels and slightly reduced condensate sizes. This
unexpected finding may suggest that binding of the USP10-derived SLiM to
NTF2 reduces the propensity of G3BP1 to enter condensates.

G3BP是一类被称为应激颗粒（stress granules, SGs）的应激诱导蛋白质-RNA相互作用网络中的核心节点。与应激颗粒相关的蛋白质Caprin-1与USP10可互斥结合G3BP1的NTF2结构域（NTF2 domain），分别促进与抑制应激颗粒的形成。本文中，我们解析了G3BP1-NTF2与Caprin-1衍生的短线性基序（short linear motif, SLiM）结合形成复合物的晶体结构。经生化与生物物理结合实验验证，Caprin-1可结合USP10未覆盖的第三个NTF2结合位点，并与该位点内的组氨酸31（His-31）与组氨酸62（His-62）发生相互作用。纳米差示扫描荧光法（nano-differential scanning fluorimetry）检测发现，G3BP1-NTF2在酸性pH条件下的热稳定性降低。相较于结合Caprin-1，结合USP10对该去稳定化效应的抵消效果更为显著。从人类U2OS细胞中免疫沉淀获得的G3BP1/USP10复合物，相较G3BP1/Caprin-1复合物更耐受酸性缓冲液洗涤。通过对融合于G3BP1的pHluorin2进行比率荧光分析，我们检测到细胞凝聚体的pH相较于胞质溶胶降低了约0.5个单位。表达具有更高G3BP1结合亲和力的Caprin-1/FGDF嵌合蛋白的细胞，其Caprin-1表达水平降低，且细胞凝聚体的尺寸略有减小。这一意外发现表明，USP10衍生的SLiM与NTF2的结合，会降低G3BP1进入细胞凝聚体的倾向。