Chou Jordan S, Impens Francis, Gevaert Kris, Davies Peter L
Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6.
Biochim Biophys Acta. 2011 Jul;1814(7):864-72. doi: 10.1016/j.bbapap.2011.04.007. Epub 2011 Apr 28.
Calpains are Ca(2+)-dependent, intracellular cysteine proteases involved in many physiological functions. How calpains are activated in the cell is unknown because the average intracellular concentration of Ca(2+) is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Two of the proposed mechanisms by which calpains can overcome this Ca(2+) concentration differential are autoproteolysis (autolysis) and subunit dissociation, both of which could release constraints on the core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, we show that while the anchor helix is one of the first targets to be cut, this occurs in the same time-frame as several potentially inactivating cleavages in Domain III. Thus autolytic activation would overlap with inactivation. We also show that the small subunit does not dissociate from the large subunit, but is proteolyzed to a 40-45k heterodimer of Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit homodimer produced by subunit dissociation. We propose a model for m-calpain activation that does not involve either autolysis or subunit dissociation.
钙蛋白酶是依赖钙离子的细胞内半胱氨酸蛋白酶，参与多种生理功能。钙蛋白酶在细胞内如何被激活尚不清楚，因为细胞内钙离子的平均浓度比该酶在体外半最大激活所需浓度低几个数量级。关于钙蛋白酶能够克服这种钙离子浓度差异的两种推测机制是自蛋白水解（自溶）和亚基解离，这两种机制都可以通过破坏锚定螺旋与小亚基之间的联系来释放对核心区域的限制，从而使活性位点得以形成。通过测量钙蛋白酶不同位点的自溶速率，我们发现虽然锚定螺旋是最早被切割的靶点之一，但这与结构域III中几个潜在的失活切割发生在同一时间框架内。因此，自溶激活会与失活重叠。我们还表明，小亚基不会与大亚基解离，而是被蛋白水解为结构域IV和VI的40 - 45k异二聚体。以前很可能将这种自溶产生的异二聚体错误地鉴定为亚基解离产生的小亚基同二聚体。我们提出了一种不涉及自溶或亚基解离的μ-钙蛋白酶激活模型。
