Elucidation of the molecular mechanisms of myocardial autophagy regulation and MAPK pathway inactivation by SPRED proteins

心脏功能依赖于平衡的蛋白质周转。因此，调节蛋白质衰变对维持心脏功能至关重要。在这个项目中，我们将研究SPRED2的缺乏是否会导致自噬受损、心力衰竭和寿命缩短。SPRED2是ERK-MAPK信号在人类心脏中显著表达的细胞内抑制因子。spred2缺陷小鼠表现为心肌细胞肥大、心肌纤维化、电兴奋性受损和严重心律失常。从机制上讲，心肌细胞功能障碍是由ERK过度激活和自噬失调引起的，表现为囊泡、空泡结构的积聚和线粒体的退化。SPRED2-/-心脏的自噬通量降低反映在LC3-II/LC3-I比值降低和Atg7、Atg4B和Atg16L表达降低。此外，自噬体接头p62和NBR1以及溶酶体cathepind在SPRED2-/-心脏中积累。然而，SPRED2调控自噬的分子机制仍然是一个谜。SPRED2没有内在的酶活性，这使得自噬调节的另一种机制成为必要。在这项拨款申请中，我们希望详细研究必要的途径，以阐明SPRED2如何调节RTK信号所需的囊泡运输，以及心肌细胞自噬最终使RTK失活。阐明体内和体外的这些分子过程将与许多医学研究领域相关，因为多种疾病是由MAPK途径失活（“Rasopahties”）、细胞内运输和溶酶体缺乏融合（“溶酶体贮积病”）的紊乱引发的。

Cardiac functionality is dependent on a balanced protein turnover. Accordingly, regulated protein decay is critical to maintain cardiac function. In this project, we will investigate if deficiency of SPRED2, an intracellular repressor of ERK-MAPK signaling markedly expressed in human heart, results in impaired autophagy, heart failure, and shortened lifespan. SPRED2-deficient mice show cardiomyocyte hypertrophy, cardiac fibrosis, impaired electrical excitability, and severe arrhythmias. Mechanistically, cardiomyocyte dysfunction results from ERK hyperactivation and dysregulated autophagy, observed as accumulation of vesicles, vacuolar structures, and degenerated mitochondria. The diminished autophagic flux in SPRED2-/- hearts was reflected by a reduced LC3-II/LC3-I ratio and by decreased Atg7, Atg4B and Atg16L expression. Furthermore, the autophagosomal adaptors p62 and NBR1 and lysosomal CathepsinD accumulated in SPRED2-/- hearts. However, the molecular mechanisms how SPRED2 regulates autophagy remain enigmatic. SPRED2 has no intrinsic enzymatic activity, which makes an alternative mechanism of regulation of autophagy necessary. In this grant proposal, we would like to investigate in detail the pathways necessary to clarify how SPRED2 regulates vesicle transport required for RTK signaling but also final RTK inactivation by autophagy in cardiac myocytes. Elucidation of these molecular processes in vivo and ex vivo will be relevant in many areas of medical research, as a variety of diseases are triggered by disorders of MAPK pathway inactivation (“Rasopahties”), of intracellular transport, and lack of fusion of lysosomes (“Lysosomal Storage Diseases”).