ABSTRACT

Plasma cells depend on quality control of newly synthesized antibodies in the endoplasmic reticulum (ER) via macroautophagy/autophagy and proteasomal degradation. The cytosolic adaptor protein TFG (Trk-fused gene) regulates ER-Golgi transport, the secretory pathway and proteasome activity in non-immune cells. We show here that TFG is upregulated during lipopolysaccharide- and CpG-induced differentiation of B1 and B2 B cells into plasmablasts, with the highest expression of TFG in mature plasma cells. CRISPR-CAS9-mediated gene disruption of tfg in the B lymphoma cell line CH12 revealed increased apoptosis, which was reverted by BCL2 but even more by ectopic TFG expression. Loss of TFG disrupted ER structure, leading to an expanded ER and increased expression of ER stress genes. When compared to wild-type CH12 cells, tfg KO CH12 cells were more sensitive toward ER stress induced by tunicamycin, monensin and proteasome inhibition or by expression of an ER-bound immunoglobulin (Ig) μ heavy (µH) chain. CH12 tfg KO B cells displayed more total LC3, lower LC3-II turnover and increased numbers and size of autophagosomes. Tandem-fluorescent-LC3 revealed less accumulation of GFP-LC3 in starved and chloroquine-treated CH12 tfg KO B cells. The GFP:RFP ratio of tandem-fluorescent-LC3 was higher in tunicamycin-treated CH12 tfg KO B cells, suggesting less autophagy flux during induced ER stress. Based on these data, we suggest that TFG controls autophagy flux in CH12 B cells and propose that TFG is a survival factor that alleviates ER stress through the support of autophagy flux in activated B cells and mature plasma cells.Abbreviations: Ab, antibody; Ag, antigen; ASC, antibody-secreting cells; ATG, autophagy-related; BCR, B cell receptor; COPII, coat protein complex II; CpG, non-methylated CpG oligonucleotide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FO, follicular; GFP, green fluorescent protein; HC, heavy chain; Ig, immunoglobulin; IRES, internal ribosomal entry site; LC, light chain; MZ, marginal zone; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; TLR, toll-like receptor; UPR, unfolded protein response.

摘要

浆细胞依赖于通过巨自噬/自噬和蛋白酶体降解在内质网 (ER) 中新合成抗体的质量控制。胞浆衔接蛋白 TFG（Trk 融合基因）调节非免疫细胞中的 ER-高尔基体转运、分泌途径和蛋白酶体活性。我们在此显示，在脂多糖和 CpG 诱导的 B1 和 B2 B 细胞分化为浆母细胞期间，TFG 上调，其中 TFG 在成熟浆细胞中的表达最高。CRISPR-CAS9 介导的tfg基因破坏在 B 淋巴瘤细胞系 CH12 中显示细胞凋亡增加，这被 BCL2 逆转，但通过异位 TFG 表达甚至更多。TFG 的缺失破坏了 ER 结构，导致 ER 扩大和 ER 应激基因的表达增加。与野生型 CH12 细胞相比，tfg KO CH12 细胞对衣霉素、莫能菌素和蛋白酶体抑制或 ER 结合免疫球蛋白 (Ig) μ 重 (μH) 链的表达诱导的 ER 应激更敏感。CH12 tfg KO B 细胞显示出更多的总 LC3、更低的 LC3-II 转换和增加的自噬体数量和大小。串联荧光-LC3 显示在饥饿和氯喹处理的 CH12 tfg中 GFP-LC3 的积累较少KO B 细胞。在衣霉素处理的 CH12 tfg KO B 细胞中，串联荧光-LC3 的 GFP:RFP 比率较高，表明诱导的 ER 应激期间自噬通量较低。基于这些数据，我们建议 TFG 控制 CH12 B 细胞中的自噬通量，并提出 TFG 是一种生存因子，通过支持活化 B 细胞和成熟浆细胞中的自噬通量来缓解 ER 应激。缩写：Ab，抗体；Ag，抗原；ASC，抗体分泌细胞；ATG，自噬相关；BCR，B细胞受体；COPII，外壳蛋白复合物 II；CpG，非甲基化 CpG 寡核苷酸；ER，内质网；ERAD，ER 相关降解；FO，滤泡；GFP，绿色荧光蛋白；HC，重链；Ig，免疫球蛋白；IRES，内部核糖体进入位点；LC，轻链；MZ，边缘地带；NFKB，B细胞中κ轻多肽基因增强子的核因子；TLR，toll​​ 样受体；UPR，未折叠蛋白反应。