Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy.
J Thromb Haemost. 2018 Oct;16(10):2035-2043. doi: 10.1111/jth.14236. Epub 2018 Aug 9.
Essentials Missense mutations often impair protein folding, and thus intracellular trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved intracellular trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype.
Background Missense mutations often impair protein folding and intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), intracellular trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, 100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired intracellular trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.
错义突变常导致蛋白质折叠异常，从而影响细胞内运输和分泌。曾用具有分子伴侣样活性的小分子化合物处理重型 B 型血友病的细胞模型。苯丁酸钠可改善常见的 p.R294Q 突变的细胞内运输和分泌。p.R294Q 的凝血活酶活性水平（约 3%）升高可改善出血表型。
背景：错义突变常导致蛋白质折叠和细胞内加工异常，具有分子伴侣样活性的小分子化合物可改善这种异常。然而，在凝血功能障碍中，这种方法的应用较少，因为即使是功能水平的适度升高也可能具有治疗意义。目的：通过分子伴侣样化合物，恢复与 B 型血友病（HB）相关的错义突变影响的因子 IX（FIX）变体的表达。
研究重组（r）FIX 变体的表达，并在使用分子伴侣样化合物处理细胞前后，通过 ELISA 检测分泌水平，通过免疫荧光检测细胞内运输，通过凝血酶原活性测定法检测活性。
我们选择了最常见的 HB 突变（p.R294Q，约 100 例患者）作为模型，与其他与严重/中度 B 型 HB 相关的常见突变相比。免疫荧光研究显示 rFIX 变体在内质网中滞留，在高尔基体中几乎没有定位，这表明其细胞内运输受损。与患者的凝血表型一致，所有错义突变均导致分泌减少（<1%野生型 rFIX）。苯丁酸钠（NaPBA）可定量改善高尔基体运输，并依赖剂量促进分泌（从 0.3±0.1%增加至 1.5±0.3%），仅 rFIX-294Q 变体有此作用。值得注意的是，该变体显示出特定的凝血酶原活性，在所有处理条件下均高于野生型 rFIX（约 2.0 倍）。重要的是，凝血酶原活性同时增加至（3.0±0.9%）水平，如果患者达到此水平，可改善出血表型。
总之，我们的数据详细说明了 I 型 HB 的分子机制，并将 NaPBA 作为负担得起的“个体化”治疗药物候选物，用于治疗高频 p.R294Q 突变患者，这些患者替代疗法的可及性较低。
