Stepensky Polina, Chacón-Flores Montserrat, Kim Katherine H, Abuzaitoun Omar, Bautista-Santos Arnulfo, Simanovsky Natalia, Siliqi Dritan, Altamura Davide, Méndez-Godoy Alfonso, Gijsbers Abril, Naser Eddin Adeeb, Dor Talia, Charrow Joel, Sánchez-Puig Nuria, Elpeleg Orly
Department of Pediatric Hematology, Oncology and Bone Marrow Transplantation, Hadassah, Hebrew University Medical Center, Jerusalem, Israel.
Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, México.
J Med Genet. 2017 Aug;54(8):558-566. doi: 10.1136/jmedgenet-2016-104366. Epub 2017 Mar 22.
For the final step of the maturation of the ribosome, the nascent 40S and 60S subunits are exported from the nucleus to the cell cytoplasm. To prevent premature association of these ribosomal subunits, eukaryotic initiation factor 6 (eIF6) binds the 60S subunit within the nucleus. Its release in the cytoplasm requires the interaction of EFL1 and SDBS proteins. In Shwachman-Diamond syndrome (SDS), a defective SDBS protein prevents eIF6 eviction, inhibiting its recycle to the nucleus and subsequent formation of the active 80S ribosome.
This study aims to identify the molecular basis of an SDS-like disease, manifested by pancytopenia, exocrine pancreatic insufficiency and skeletal abnormalities in six patients from three unrelated families.
Whole exome analysis was used for mutation identification. Fluorescence microscopy studies assessed the localisation of Tif6-GFP, the yeast eIF6 homologue, in yeast WT and mutant cells. Human and yeast EFL1 proteins, WT and mutants, were expressed in BCY123 strain, and circular dichroism and small-angle X-ray scattering were used to assess the folding and flexibility of these proteins. Green malachite colorimetric assay was performed to determine the GTPase activity of WT and Efl1 mutants.
Four patients were homozygous for p.R1095Q variant and two patients were homozygous for p.M882K variant in . Residue R1095 and M882 are conserved across species. Neither the GTPase activity of the mutant proteins nor its activation by the SDBD protein or the 60S ribosomal subunit were affected. Complementation of yeast cells with the mutants rescued the slow growth phenotype. Nonetheless, Tif6-GFP was relocalised to the cytoplasm in mutant yeast cells in contrast to its nuclear localisation in WT cells.
Mutations in clinically manifest as SDS-like phenotype. Similar to the molecular pathology of SDS, mutant EFL1 proteins do not promote the release of cytoplasmic Tif6 from the 60S subunit, likely preventing the formation of mature ribosomes.
对于核糖体成熟的最后一步，新生的40S和60S亚基从细胞核输出到细胞质。为防止这些核糖体亚基过早结合，真核起始因子6（eIF6）在细胞核内与60S亚基结合。其在细胞质中的释放需要EFL1和SDBS蛋白的相互作用。在施瓦赫曼-戴蒙德综合征（SDS）中，有缺陷的SDBS蛋白会阻止eIF6的排出，抑制其循环回到细胞核以及随后活性80S核糖体的形成。
本研究旨在确定一种类似SDS疾病的分子基础，该疾病在来自三个无亲缘关系家庭的六名患者中表现为全血细胞减少、外分泌胰腺功能不全和骨骼异常。
采用全外显子组分析进行突变鉴定。荧光显微镜研究评估了酵母eIF6同源物Tif6-GFP在酵母野生型和突变细胞中的定位。野生型和突变型的人类及酵母EFL1蛋白在BCY123菌株中表达，利用圆二色性和小角X射线散射来评估这些蛋白的折叠和灵活性。进行孔雀石绿比色法以测定野生型和Efl1突变体的GTP酶活性。
4名患者在 中为p.R1095Q变体纯合子，2名患者为p.M882K变体纯合子。R1095和M882残基在物种间保守。突变蛋白的GTP酶活性及其被SDBD蛋白或60S核糖体亚基激活的情况均未受影响。用 突变体对酵母细胞进行互补挽救了生长缓慢的表型。尽管如此，与野生型细胞中Tif6-GFP定位于细胞核不同，在突变酵母细胞中它重新定位于细胞质。
中的突变临床上表现为类似SDS的表型。与SDS的分子病理学相似，突变的EFL1蛋白不促进细胞质中的Tif6从60S亚基释放，可能会阻止成熟核糖体的形成。
