Salotra P, Ralhan R, Sreenivas G
Molecular Biology Laboratory, Institute of Pathology (ICMR), New Delhi, India.
Int J Biochem Cell Biol. 2000 Mar;32(3):309-16. doi: 10.1016/s1357-2725(99)00134-x.
In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.
在利什曼原虫等寄生虫中，由于温度升高是其生命周期中不可或缺的一部分，因此研究热应激诱导的分子事件极具意义。已知蛋白质磷酸化可控制真核细胞增殖和分化的主要步骤。对原生动物细胞内信号系统的研究相对较新。我们研究了热休克对杜氏利什曼原虫前鞭毛体蛋白质磷酸化状态的影响。使用寄生虫的[32P] - 正磷酸盐标记和单克隆抗磷酸酪氨酸抗体进行免疫印迹，检测总蛋白质磷酸化模式和酪氨酸残基的特异性磷酸化。在24℃时被磷酸化的杜氏利什曼原虫主要蛋白质的表观分子量为110、105、66 - 68、55、36 - 40和20 kDa。热休克（从24℃升至37℃）导致有毒前鞭毛体中大多数磷蛋白的磷酸化显著降低。另一方面，无毒前鞭毛体在热应激下蛋白质磷酸化未显示任何降低。在有毒和无毒寄生虫中，推测大小为105 - 110 kDa的蛋白质中均可检测到酪氨酸残基的主要磷酸化。对于有毒寄生虫，热休克导致这两种蛋白质中的磷酸酪氨酸水平降低，而在无毒寄生虫中未检测到这种降低。仅在有毒寄生虫中观察到的响应热应激的蛋白质磷酸化状态的显著改变，包括含酪氨酸蛋白质的改变，表明蛋白质磷酸化/去磷酸化的调节可能在寄生虫进入哺乳动物宿主时遇到热休克后的信号转导途径中发挥作用。
