Griffin Jacob B, Rodriguez-Melendez Rocio, Dode Leonard, Wuytack Frank, Zempleni Janos
Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, 68583-0806, USA.
J Nutr Biochem. 2006 Apr;17(4):272-81. doi: 10.1016/j.jnutbio.2005.05.005. Epub 2005 Jun 13.
Protein folding in the endoplasmic reticulum (ER) depends on Ca(2+); uptake of Ca(2+) into the ER is mediated by sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3). The 5'-flanking region of the SERCA3 gene (ATP2A3) contains numerous binding sites for the transcription factors Sp1 and Sp3. Biotin affects the nuclear abundance of Sp1 and Sp3, which may act as transcriptional activators or repressors. Here we determined whether biotin affects the expression of the SERCA3 gene and, thus, protein folding in human lymphoid cells. Jurkat cells were cultured in media containing 0.025 nmol/L biotin (denoted "deficient") or 10 nmol/L biotin ("supplemented"). The transcriptional activity of the full-length human SERCA3 promoter was 50% lower in biotin-supplemented cells compared to biotin-deficient cells. Biotin-dependent repressors bind to elements located 731-1312 bp upstream from the transcription start site in the SERCA3 gene. The following suggest that low expression of SERCA3 in biotin-supplemented cells impaired folding of secretory proteins in the ER, triggering unfolded protein response: (i) sequestration of Ca(2+) in the ER decreased by 14-24% in response to biotin supplementation; (ii) secretion of interleukin-2 into the extracellular space decreased by 75% in response to biotin supplementation; (iii) the nuclear abundance of stress-induced transcription factors increased in response to biotin supplementation; and (iv) the abundance of stress-related proteins such ubiquitin activating enzyme 1, growth arrest and DNA damage 153 gene, X-box binding protein 1 and phosphorylated eukaryotic translation initiation factor 2alpha increased in response to biotin supplementation. Collectively, this study suggests that supplements containing pharmacological doses of biotin may cause cell stress by impairing protein folding in the ER.
内质网（ER）中的蛋白质折叠依赖于Ca(2+)；Ca(2+)进入内质网是由肌浆网/内质网Ca(2+)-ATP酶3（SERCA3）介导的。SERCA3基因（ATP2A3）的5'侧翼区域含有众多转录因子Sp1和Sp3的结合位点。生物素会影响Sp1和Sp3的核丰度，它们可能作为转录激活因子或抑制因子发挥作用。在此，我们确定生物素是否会影响SERCA3基因的表达，进而影响人类淋巴细胞中的蛋白质折叠。将Jurkat细胞培养在含有0.025 nmol/L生物素（称为“缺乏”）或10 nmol/L生物素（“补充”）的培养基中。与生物素缺乏的细胞相比，生物素补充的细胞中全长人SERCA3启动子转录活性降低了50%。生物素依赖性抑制因子与SERCA3基因转录起始位点上游731 - 1312 bp处的元件结合。以下情况表明，生物素补充的细胞中SERCA3的低表达损害了内质网中分泌蛋白的折叠，引发了未折叠蛋白反应：（i）补充生物素后，内质网中Ca(2+)的螯合减少了14 - 24%；（ii）补充生物素后，白细胞介素-2分泌到细胞外空间减少了75%；（iii）补充生物素后，应激诱导转录因子的核丰度增加；（iv）补充生物素后，泛素激活酶1、生长停滞和DNA损伤153基因、X盒结合蛋白1和磷酸化真核翻译起始因子2α等应激相关蛋白的丰度增加。总体而言，本研究表明，含有药理剂量生物素的补充剂可能通过损害内质网中的蛋白折叠而导致细胞应激。
