Tárraga Wilson A, Falomir-Lockhart Lisandro J, Garda Horacio A, Gonzalez Marina C
Instituto de Investigaciones Bioquímica de La Plata "Dr. Rodolfo R. Brenner" (INIBIOLP), Centro Científico Tecnológico-La Plata (CONICET), Calle 60 y 120 s/n, 1900 La Plata, Argentina.
Instituto de Investigaciones Bioquímica de La Plata "Dr. Rodolfo R. Brenner" (INIBIOLP), Centro Científico Tecnológico-La Plata (CONICET), Calle 60 y 120 s/n, 1900 La Plata, Argentina; Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115 s/n, 1900 La Plata, Argentina.
Biochim Biophys Acta Proteins Proteom. 2025 Aug 6;1873(6):141093. doi: 10.1016/j.bbapap.2025.141093.
Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein particles, conferring anti-atherogenic properties through reverse cholesterol transport. In its lipid-free state, apoA-I self-associates, a process implicated in normal physiology as well as in pathological conditions, such as amyloidosis. Although various mechanisms have been proposed to explain its self-association, there is still no consensus, and its functional implications remain unclear. Here, we employed a multi-parametric fluorescent probe to investigate apoA-I self-association. We used three single cysteine mutants located in different helixes: K107C (H4), K133C (H5), and F225C (H10); and labelled them with pyrene as detailed previously (Tárraga et al. Arch Biochem Biophys 699 (2021) 108748). Our original experiments revealed excimer emission between helixes H5 and H10 and polarity changes also in H4. By exploiting specific pyrene band emissions to monitor dimer association (via excimer formation) and microenvironment polarity (P-value), we tracked apoA-I oligomerization as a function of protein concentration. Several mathematical models of self-association were developed and compared using selection criteria to identify the simplest model reproducing apoA-I's complex behaviour in aqueous media: a Sequential Association submodel limited to a tetramer as the highest order oligomeric species and considering both dimers and tetramers as responsible for the excimer's emission. Multi-equilibria models enhanced the titration analysis, allowing estimation of association constants (Ka) and oligomeric species distribution. Our results support previous evidence that contacts among helixes, which stabilize discoidal HDL particles, are already present in lipid-free apoA-I, with dimeric species predominating, and possible tetrameric too. Further investigation of these species is essential to elucidate their physiological and pathological roles, such as in atherosclerosis.
载脂蛋白A-I（apoA-I）是高密度脂蛋白颗粒的主要蛋白质，通过逆向胆固醇转运赋予抗动脉粥样硬化特性。在其无脂状态下，apoA-I会自我缔合，这一过程与正常生理以及诸如淀粉样变性等病理状况有关。尽管已经提出了各种机制来解释其自我缔合，但仍未达成共识，其功能意义也仍不清楚。在这里，我们采用了一种多参数荧光探针来研究apoA-I的自我缔合。我们使用了位于不同螺旋中的三个单半胱氨酸突变体：K107C（H4）、K133C（H5）和F225C（H10）；并按照先前详细描述的方法（塔拉加等人，《生物化学与生物物理学报》699（2021）108748）用芘对它们进行标记。我们最初的实验揭示了H5和H10螺旋之间的准分子发射，以及H4螺旋中的极性变化。通过利用特定的芘带发射来监测二聚体缔合（通过准分子形成）和微环境极性（P值），我们跟踪了apoA-I寡聚化随蛋白质浓度的变化。开发了几种自我缔合的数学模型，并使用选择标准进行比较，以确定在水性介质中重现apoA-I复杂行为的最简单模型：一个顺序缔合子模型，限于四聚体作为最高阶寡聚物种，并将二聚体和四聚体都视为准分子发射的原因。多平衡模型增强了滴定分析，允许估计缔合常数（Ka）和寡聚物种分布。我们的结果支持了先前的证据，即稳定盘状高密度脂蛋白颗粒的螺旋之间的接触在无脂apoA-I中已经存在，二聚体物种占主导，也可能存在四聚体。对这些物种的进一步研究对于阐明它们在动脉粥样硬化等生理和病理作用至关重要。
