Vitte Anne-Laure, Jalinot Pierre
LBMC, UMR5239 CNRS - ENS de Lyon, IFR 128 Biosciences Lyon Gerland 46 Allée d'Italie, 69364 Lyon cedex 07, France.
BMC Biotechnol. 2008 Feb 29;8:24. doi: 10.1186/1472-6750-8-24.
We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form.
Several bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway.
Our observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.
我们之前开发了一种小的杂合蛋白，其由与融合至Tat蛋白转导结构域（PTD）的七肽相连的SUMO-1组成。该七肽基序是从随机序列文库中筛选出来的，用于特异性结合HIV-1调节蛋白Tat或Rev。这些构建体，命名为SHP，能够进入原代淋巴细胞，其中一些还能抑制HIV-1复制。鉴于这些阳性结果以及文献中的其他数据，我们进一步测试了在其N端与各种PTD相连的泛素或SUMO-1将与其双甘氨酸基序下游融合的蛋白质或肽递送至细胞内的能力。在该系统中，预期细胞内的泛素或SUMO-1水解酶会从货物多肽上切割下PTD-Ub或PTD-SUMO-1模块，从而使其以未修饰的形式被递送。
构建了几种细菌表达载体，以产生从N端到C端包含以下结构的模块化蛋白：FLAG表位、蛋白酶切割位点、PTD、人泛素或SUMO-1，以及GFP或HA表位。测试了九种不同的PTD，包括Tat碱性结构域、野生型或带有各种突变的类型，以及精氨酸或赖氨酸片段。观察到其中一些PTD，主要是Tat PTD以及七个或九个残基长的聚精氨酸基序，会导致杂合蛋白与细胞结合，但这些构建体均未被递送至细胞质。这一结论来自生化和免疫荧光研究，也源于从未观察到泛素或SUMO-1被蛋白酶切割后产生的游离货物蛋白这一事实。然而，与我们之前的观察结果一致，如在SHP构建体中那样，将双甘氨酸基序突变为丙氨酸-精氨酸，通过对活细胞的免疫荧光观察和细胞分级分离分析表明可实现细胞质进入。这一过程源自非内吞途径。
我们的观察结果表明，将SUMO-1与肽-PTD模块融合可产生一种稳定的杂合蛋白，该蛋白易于在细菌中产生且能有效进入细胞，但这种特性需要对SUMO-1末端的双甘氨酸基序进行突变，从而损害单独肽的递送。
