Xiao Huanhuan, Li Mofei, Zhong Yongwang, Patel Avani, Xu Rui, Zhang Chenyu, Athey Thomas W, Fang Shengyun, Xu Tianjun, Du Shaojun
Department of Biochemistry and Molecular Biology, Institute of Marine and Environmental Technology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
College of Life Sciences, Tianjin Normal University, Tianjin, China.
FASEB J. 2025 Jan 15;39(1):e70283. doi: 10.1096/fj.202401875R.
Molecular chaperones play critical roles in post-translational maintenance in protein homeostasis. Previous studies have shown that loss of Smyd1b function results in defective myofibril organization and dramatic upregulation of heat shock protein gene (hsp) expression in muscle cells of zebrafish embryos. To investigate the molecular mechanisms and functional importance of this stress response, we characterized changes of gene expression in smyd1b knockdown and knockout embryos using RNA-seq. The results showed that the top upregulated genes encode mostly cytosolic heat shock proteins. Co-IP assay revealed that the upregulated cytosolic Hsp70s associate with myosin chaperone UNC45b which is critical for myosin protein folding and sarcomere assembly. Strikingly, several hsp70 genes also display muscle-specific upregulation in response to heat shock-induced stress in zebrafish embryos. To investigate the regulation of hsp gene upregulation and its functional significance in muscle cells, we generated heat shock factor 1 (hsf) knockout zebrafish mutants and analyzed hsp gene expression and muscle phenotype in the smyd1bsingle and hsf1;smyd1b double-mutant embryos. The results showed that knockout of hsf1 blocked the hsp gene upregulation and worsened the muscle defects in smyd1b mutant embryos. Moreover, we demonstrated that Hsf1 is essential for fish survival under heat shock (HS) conditions. Together, these studies uncover a correlation between Smyd1b deficiency and the Hsf1-activated heat shock response (HSR) in regulating muscle protein homeostasis and myofibril assembly and demonstrate that the Hsf1-mediated hsp gene upregulation is vital for the survival of zebrafish larvae under thermal stress conditions.
分子伴侣在蛋白质稳态的翻译后维持中发挥着关键作用。先前的研究表明，Smyd1b功能丧失会导致斑马鱼胚胎肌肉细胞中肌原纤维组织缺陷以及热休克蛋白基因（hsp）表达显著上调。为了研究这种应激反应的分子机制和功能重要性，我们使用RNA测序对smyd1b基因敲低和敲除胚胎中的基因表达变化进行了表征。结果表明，上调最明显的基因大多编码胞质热休克蛋白。免疫共沉淀分析显示，上调的胞质Hsp70与肌球蛋白伴侣UNC45b相关联，UNC45b对肌球蛋白蛋白折叠和肌节组装至关重要。引人注目的是，几个hsp70基因在斑马鱼胚胎中对热休克诱导的应激也表现出肌肉特异性上调。为了研究hsp基因上调的调控及其在肌肉细胞中的功能意义，我们构建了热休克因子1（hsf）基因敲除的斑马鱼突变体，并分析了smyd1b单突变和hsf1;smyd1b双突变胚胎中的hsp基因表达和肌肉表型。结果表明，敲除hsf1会阻断hsp基因上调，并使smyd1b突变胚胎中的肌肉缺陷恶化。此外，我们证明Hsf1对于鱼类在热休克（HS）条件下的存活至关重要。总之，这些研究揭示了Smyd1b缺陷与Hsf1激活的热休克反应（HSR）在调节肌肉蛋白质稳态和肌原纤维组装之间的相关性，并证明Hsf1介导的hsp基因上调对于斑马鱼幼虫在热应激条件下的存活至关重要。
