Vargas Gabriela, Cortés Omar, Arias-Muñoz Eloisa, Hernández Sergio, Cerda-Troncoso Cristobal, Hernández Laura, González Alexis E, Tatham Michael H, Bustamante Hianara A, Retamal Claudio, Cancino Jorge, Varas-Godoy Manuel, Hay Ronald T, Rojas-Fernández Alejandro, Cavieres Viviana A, Burgos Patricia V
Centro de Biología Celular y Biomedicina (CEBICEM), Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago, Chile.
Centro de Envejecimiento y Regeneración (CARE-UC), Facultad de Ciencias Biológicas, Pontificia Universidad Católica, Santiago, Chile.
Front Cell Dev Biol. 2022 Mar 2;10:743287. doi: 10.3389/fcell.2022.743287. eCollection 2022.
Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.
巨自噬和泛素蛋白酶体系统在维持细胞稳态中作为一个相互连接的网络发挥作用。事实上，营养剥夺时巨自噬的有效激活是由蛋白酶体对预先存在的蛋白质进行降解来维持的。然而，目前尚不清楚蛋白酶体为激活巨自噬而降解的具体底物。通过定量蛋白质组学分析，我们鉴定了几种在饥饿时独立于ATG5表达而下调的蛋白质。其中，最显著的是HERPUD1，一种内质网（ER）膜蛋白，其表达水平较低，且已知在正常条件下会被蛋白酶体降解。相反，在内质网应激下，HERPUD1的水平由于其蛋白酶体降解受阻而迅速增加。因此，我们探讨了HERPUD1的稳定性是否可作为自噬的负调节因子。在这项研究中，我们表达了一个缺失泛素样结构域（UBL）的HERPUD1版本，已知该结构域对其蛋白酶体降解至关重要。与野生型HERPUD1-WT相比，我们发现缺失UBL的版本对基础自噬和诱导自噬均产生负作用。出乎意料的是，我们发现稳定的HERPUD1促进内质网重塑，而不依赖于未折叠蛋白反应的激活，观察到类似于先前描述的管状内质网重排的堆叠管状结构增加。重要的是，UBL内的磷酸模拟S59D突变模拟了缺失UBL版本所观察到的表型，包括HERPUD1稳定性增加、内质网重塑以及对自噬的负作用。此外，我们发现缺失UBL的版本和HERPUD1-S59D会导致细胞大小增加，而HERPUD1-S59D还会导致细胞核大小增加。有趣的是，通过缺失UBL和磷酸模拟S59D版本进行的内质网重塑导致溶酶体数量和功能增加。此外，缺失UBL的版本和磷酸模拟S59D版本建立了紧密的内质网-溶酶体网络，存在内质网-溶酶体膜接触位点的扩展斑块，这种情况表明在应激条件下细胞存活率增加。总之，我们提出稳定的HERPUD1下调巨自噬，转而有利于内质网和溶酶体之间的紧密相互作用，从而影响药物-细胞应激存活。
