Campioni Mara, Severino Anna, Manente Lucrezia, De Luca Antonio, La Porta Raffaele, Vitiello Antonio, Fiore Paola, Toldo Stefano, Spugnini Enrico P, Paggi Marco G, Baldi Alfonso
Department of Biochemistry, Section of Pathology, Second University of Naples, Naples, Italy.
Front Biosci (Elite Ed). 2011 Jun 1;3(4):1493-9. doi: 10.2741/e350.
The human heat shock protein HtrA1, a member of the HtrA family of serine proteases, is a evolutionarily highly conserved factor which displays a widespread pattern of expression. The yeast two-hybrid technique was employed to identify new cellular proteins physically interacting with HtrA1, and thus potential targets of this serine protease. An enzymatically inactive HtrA1 point mutant, HtrA1-S328A, was generated and used as bait in a yeast two-hybrid system. Fifty-two plasmids were isolated from primary positive yeast clones. Subsequent sequencing and BLAST analysis revealed cDNAs encoding for 13 different proteins. These putative binding partners of HtrA1 appeared to be a) components of extracellular matrix; b) factors related to signal pathways, and c) unknown proteins. Among the 13 positive clones identified and reported here, it is worth of note that the interaction of HtrA1 with tubulin and collagen (extracellular matrix proteins) and with tuberin (cytoplasmic protein) is confirmed by other studies, and this further supports previous findings in which HtrA1 can be found active as an intracytoplasmic protein or as secreted protein as well.
人类热休克蛋白HtrA1是丝氨酸蛋白酶HtrA家族的成员，是一种在进化上高度保守的因子，其表达模式广泛。采用酵母双杂交技术来鉴定与HtrA1发生物理相互作用的新的细胞蛋白，从而确定这种丝氨酸蛋白酶的潜在靶点。构建了一种无酶活性的HtrA1点突变体HtrA1-S328A，并将其用作酵母双杂交系统中的诱饵。从初级阳性酵母克隆中分离出52个质粒。随后的测序和BLAST分析揭示了编码13种不同蛋白质的cDNA。这些HtrA1的假定结合伴侣似乎是：a）细胞外基质成分；b）与信号通路相关的因子；c）未知蛋白质。在本文鉴定并报道的13个阳性克隆中，值得注意的是，其他研究证实了HtrA1与微管蛋白和胶原蛋白（细胞外基质蛋白）以及与结节性硬化蛋白（细胞质蛋白）之间的相互作用，这进一步支持了先前的研究结果，即在细胞质内或分泌蛋白中均可发现有活性的HtrA1。
