Impact of titin post-translational modifications on muscle contractile function

在其生命周期中，心肌细胞或心肌细胞(CMC)必须在结构上、机械上和功能上适应来自其环境的多种生理信号和生物力学约束。特殊的机械敏感和活性蛋白是巨噬细胞对环境的功能性反应的中心，如肌动蛋白、肌球蛋白和其他。通过横跨半个肌球收缩单位(即肌节)的长度，巨大的蛋白质titin(~3MDA)作为一个被动的分子弹簧来限制肌节的伸展范围，防止富含肌球蛋白的粗丝滑出富含肌动蛋白的细丝。位于肌节I带区域的肌动蛋白在肌肉弹性中起着重要的作用。Titin弹性区有几种异构体，由串联排列的Ig结构域和充当熵弹簧的大的非结构结构域组成，即富含脯氨酸、谷氨酸、缬氨酸和赖氨酸的PEVK结构域，以及N2B和N2A结构域。PEVK和N2B结构域的磷酸化和去磷酸化被证明影响单个Titin分子的弹性性质，这反过来被认为可以微调肌节的收缩活性。我们假设，受系统和组织环境的影响，其他翻译后修饰也可以调节Titin和其他肌肉蛋白的弹性性质。这种环境修饰包括糖酵解副产物，如乙二醛和甲基乙醛的存在，它们通过与赖氨酸和精氨酸残基反应对蛋白质进行非酶修饰，从而产生各种修饰，包括蛋白质交联物和羧甲基赖氨酸和羧乙基赖氨酸加合物。除了二羰基衍生的蛋白质交联物的直接破坏作用外，羧甲基赖氨酸和羧乙基赖氨酸的修饰也在蛋白质的物理化学性质上具有显著的破坏潜力，因为它们将碱性残基转化为带负电荷的侧链，类似于磷酸化。肌球蛋白、肌动蛋白、α-肌动蛋白、肌球蛋白、肌球蛋白和肌动蛋白等丰富的肌小球收缩蛋白是直接影响肌细胞机械功能的二羰基修饰的主要候选者。由于其弹性区域中有大量的赖氨酸残基，因此肌动蛋白特别容易发生糖基化。有趣的是，本质上无序的PEVK结构域具有高密度的暴露赖氨酸残基，预计特别容易发生这样的修饰。因此，我们研究计划的总体目标是评估二羰基衍生的PTM对CMCs收缩蛋白的影响。本节目将重点介绍Titin的机械性能及其在CMS收缩功能中的作用。

Over their lifetime, cardiac muscle cells or cardiomyocytes (CMCs) must structurally, mechanically and functionally adapt to a multitude of physiological cues and biomechanical constraints from their environment. Specialized mechano-sensitive and -active proteins are central to the functional response of CMCs to their environment, as exemplified by titin, actin, myosin and others. By spanning the length of half a contractile unit of CMCs (i.e. a sarcomere), the giant protein titin (~3 MDa) acts as a passive molecular spring to limit sarcomere extension range, preventing the myosin-rich thick filaments from slipping outside the actin-rich thin filaments. The portion of titin located in the I-band region of the sarcomere plays an important role in muscle elasticity. Several isoforms of the titin elastic region exist, consisting of tandem arranged Ig domains and large unstructured domains acting as entropic springs, namely the PEVK domain, rich in proline, glutamate, valine and lysine, and the N2B, and N2A domains. Phosphorylation and dephosphorylation of the PEVK and N2B domains was demonstrated to impact the elastic properties of individual titin molecules, which in turn are believed to fine-tune the contractile activity of sarcomeres. We hypothesize that other post-translational modifications, influenced by systemic and tissue environment, could also modulate the elastic properties of titin and other muscle proteins. Such environmental modifications include the presence of dicarbonyl by-products of glycolysis, such as glyoxal and methylglyoxal which produce non-enzymatic modifications on proteins by reacting with lysine and arginine residues to produce a variety of modification including proteins crosslinks and carbonyl adducts such as carboxymethyl-lysine and carboxyethyl-lysine. In addition to the direct disruptive effects of dicarbonyl-derived protein crosslinkings, carboxymethyl-lysine and carboxyethyl-lysine modifications also have a significant disruptive potential in proteins physicochemical properties since they convert basic residues into negatively charged side chains, similarly to phosphorylation. Abundant sarcomeric contractile proteins such as myosin, actin, alpha-actinin, nebulin, myomesin and titin are prime candidates to dicarbonyl-derived modifications which could have a direct downstream impact on CMCs mechanical function. With his numerous lysine residues in its elastic regions, titin could be particularly susceptible to glycation. Interestingly, the intrinsically disordered PEVK domain, with its high density of exposed lysine residues, is expected to be particularly prone to such modifications. Therefore, the general goal of our research program is to assess the impact of dicarbonyles-derived PTM on CMCs contractile proteins. This program will focus on the mechanical properties of titin and its role in the contractile functions of CMS.