Rationale: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified ubiquitin-conjugating enzyme E2 variant 1 (Ube2v1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. Objective: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant alphaB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect. Methods and Results: Neonatal rat ventricular cardiomyocytes (NRVMs) were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing NRVMs. Ubiquitin proteasome system (UPS) performance was analyzed using a UPS reporter protein. Ube2v1 knockdown improved UPS performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using adeno-associated virus 9 (AAV9) to deliver multiplexed single guide RNAs (sgRNAs) against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function and prolonged lifespan in vivo. Conclusions: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

中文翻译：

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Ube2v1通过部分地通过K63泛素化调节泛素蛋白酶体系统的性能来积极调节蛋白质聚集。

理由：蛋白质质量控​​制不严格会导致心脏中蛋白毒性的细胞内蛋白质聚集，从而导致心脏病和心力衰竭。定义参与者并了解心脏蛋白质聚集的潜在机制对于寻找治疗靶点至关重要。我们在全基因组筛选中确定了泛素结合酶E2变体1（Ube2v1），以鉴定聚集过程的新型效应子。但是，其在心肌细胞中的作用尚不确定。目的：评估Ube2v1是否调节由突变αB结晶蛋白（CryABR120G）的心肌细胞表达引起的蛋白质聚集，并确定Ube2v1如何发挥其作用。方法和结果：用表达野生型CryAB（CryABWT）或CryABR120G的腺病毒感染新生的大鼠心室心肌细胞（NRVM）。随后，进行了功能丧失和功能获得实验。Ube2v1敲低降低了CryABR120G表达引起的聚集体积累。过度表达的Ube2v1促进了表达CryABWT和CryABR120G的NRVM中聚集体的形成。使用UPS报告蛋白分析泛素蛋白酶体系统（UPS）的性能。Ube2v1组合件可改善UPS性能，并促进CryABR120G心肌细胞中不溶性泛素化蛋白的降解，但不会改变自噬通量。由Ube2v1表达调节的Lys（K）63-连接的泛素化增强了蛋白质聚集，并有助于Ube2v1调节蛋白质聚集体的功能。通过使用腺相关病毒9（AAV9）在心脏特异性Cas9小鼠中针对Ube2v1传递多重单向导RNA（sgRNA）敲除心肌细胞中的Ube2v1，可减轻CryABR120G诱导的蛋白聚集，改善心脏功能并延长体内寿命。结论：Ube2v1在蛋白质聚集体形成中起重要作用，部分是通过在蛋白毒性刺激过程中增强K63泛素化来实现的。Ube2v1的抑制通过增强的泛素蛋白酶体系统性能（而非自噬）降低了CryABR120G诱导的聚集体形成，并可能提供治疗心脏蛋白病的新治疗靶标。