Therapeutic aspect of 14-3-3 related trypanosome-specific modulation with phosphorylation and dephosphorylation.

我们之前已经证明布鲁氏锥虫14-3-3 I和II在细胞运动、细胞分裂和细胞周期中起关键作用（j.b oll . chem .）。280年,14085 - 14096)。本文报道了14-3-3异构体独特的二聚化模式；I既形成异二聚体也形成同二聚体，而II主要形成异二聚体。α-螺旋1-4负责二聚化，如哺乳动物的14-3-3。此外，我们发现这两种同工型的独特n端序列在锥虫科中存在，而在哺乳动物中不存在，是二聚化不可或缺的。我们进一步鉴定了14-3-3 II n端序列中的^4Phe、^6IIe、^7Pro和^<13>Leu是二聚化的关键氨基酸。14-3-3蛋白作为磷丝氨酸依赖的分子伴侣，在真核生物中识别保守的基序。然而，布鲁氏杆菌14-3-3控制细胞活动的确切机制尚未得到证实。本文报道布鲁氏杆菌14-3-3的蛋白质识别与其他真核生物不同；1)表面等离子体共振分析表明，14-3-3 I对保守的磷酸肽具有弱亲和力，而II则不具有。2)14-3-3II与许多蛋白相互作用，并不真正依赖磷酸化。我们进一步研究了保守基序是如何在培养的布鲁氏体中被广泛利用的，3)过度表达人类14-3-3异构体而不是突变形式，诱导生长速度急剧下降和形态变化，这在14-3-3 i和/或II敲低的细胞中观察到，4)携带保守识别基序的磷酸化蛋白的数量很少。新的途径被认为是磷酸化将发挥作用，即所谓的去磷酸化以镜像方式。

We have previously shown that Trypanosoma brucei 14-3-3 I and II play pivotal roles in motility, cytokinesis and cell cycle (J.Biol.Chem.280, 14085-14096). We here report the unique dimerization pattern of 14-3-3 isoforms ; I forms both heterodimer and homodimer, whereas II forms mainly heterodimer. The α-helices 1-4 are responsible for dimeraization as in the cases of mammalian 14-3-3. In addition, we found unique N-terminal sequences of both isoforms existed in Trypoanosomatidae but not in mammals are indispensable for dimeraization. We further characterized that amino acid ^4Phe, ^6IIe, ^7Pro and ^<13>Leu in the N-terminal sequence of 14-3-3 II are crucial for dimeraization. 14-3-3 proteins act as phoshoserine-dependent molecular chaperones which recognize well-conserved motifs among eukaryotes. However, the precise mechanism by which T.brucei 14-3-3 controls cellular events have yet to be proven. We report that the protein recognition of T.brucei 14-3-3 is different from other eukaryotes ; 1)the surface plasmon resonance analysis reveals that 14-3-3 I but not II possesses weak affinity to the conserved phosphopeptides. 2)14-3-3II interacts with many proteins, not truly phosphorylation-dependent. We further searched how the conserved motifs are utilized, extensively in cultured T.brucei, 3)over expression of the human 14-3-3 isoform but not the mutated form, induces a drastic reduction in a growth rates and morphological changes that are observed in 14-3-3I and/or II knockdown cells, 4)the number of phosphorylated proteins that carry the conserved recognition motifs are small in T.brucei. The new pathway was suggested that phosphorylation would play a roll as so called dephosphorylation in a mirroring fashion.