An integrated mass spectrometry approach to study heparin structure-bioactivity

 说明(申请人提供)：肝素相关糖胺聚糖(GAG)在再生医学领域有着巨大的前景，也是一种基本上未被开发的治疗药物来源，用于治疗包括癌症和神经退行性疾病在内的一系列其他医学上重要的疾病。然而，它们在临床上的实际应用仍然局限于抗凝血剂，而在其他领域的进展仍然相对较小。这些生物聚合物对医学乃至更广泛的结构生物学提出的巨大挑战在很大程度上是由于它们巨大的结构异质性，这反映在它们在调节血管生成、细胞黏附、胚胎发生、炎症、转移和伤口愈合方面的多重作用。尽管进行了广泛的研究工作，但对各种结构特征如何调节这些生物聚合物的功能仍然缺乏明确的了解，可以说是因为调查是使用锁和钥匙模型的框架设计的，这一范式在结构生物学应用于蛋白质方面取得了巨大成功。然而，这种方法没有认识到GAG表现出的显著更高的结构多样性具有功能重要性。这项拟议的研究试图通过改变明确定义的空间互补结合位点的概念，并强调多价静电的更大作用，来改变GAG/蛋白质相互作用领域的主流范式。除了揭示GAG/蛋白质相互作用的决定因素并了解它们如何微调结合过程外，我们还将获得肝素与几种治疗相关蛋白质相互作用机制的高分辨率和动态描述。这将既不会将结构空间限制为几个精确定义的Gag分子，也不会将构象空间减少到几个可从晶体结构中获得的静态快照。为了实现这一目标，我们将对蛋白质/GAG相互作用采用一种“整体”的方法，这种方法包括肝素样GAG的结构多样性，而不是普遍接受的方法，即将研究范围限制在通过合成获得的少数几个分子上。我们将使用自下而上和自上而下相结合的方法来研究蛋白质/Gag的相互作用，前者集中于通过亲和分离获得的相对定义明确的短肝素低聚物的子集，而后者旨在研究完整的 肝素。这将揭示GAG与几种治疗相关蛋白质相互作用的结构特性。气相离子化学(电子捕获和碰撞激活)的新应用将被开发用于表征大的多相Gag/蛋白质复合体。最后，与H/D交换研究并行的原子水平蒙特卡罗模拟将导致对相互作用过程的高分辨率和动态描述。这种关于GAG/蛋白质相互作用的新观点将使肝素样GAG的治疗潜力在再生医学、炎症、神经退行性疾病和肿瘤学等领域得到更有效的开发。

 DESCRIPTION (provided by applicant): Heparin-related glycosaminoglycans (GAGs) hold enormous promise in the field of regenerative medicine, and also are a largely unexploited source of therapeutic agents to address a range of other medically important conditions including cancer and neurodegenerative diseases. Nevertheless, their practical utilization in the clinic is still confined to anticoagulants, while progress in other areas remains relatively modest The great challenge presented by these biopolymers to medicine and, more broadly, to structural biology is due in large part to their enormous structural heterogeneity, which is mirrored by their multiple roles in modulating angiogenesis, cell adhesion, embryogenesis, inflammation, metastasis and wound healing. Despite extensive research efforts, clear understanding of how various structural features modulate function of these biopolymers remains wanting, arguably because the inquiry was designed using the framework of the lock-and-key model, a paradigm that had been tremendously successful in structural biology applied to proteins. However, this approach fails to recognize that the dramatically higher level of structural diversity exhibited by GAGs has functional importance. The proposed research seeks to shift the prevailing paradigm in the field of GAG/protein interactions by moving away from the notion of well-defined sterically complementary binding sites and emphasizing a greater role of polyvalent electrostatics. In addition to revealing the determinants of GAG/protein interactions and understanding how they fine-tune the binding process, we will obtain a high-resolution and dynamic description of the mechanism of heparin interaction with several therapeutically relevant proteins. This will be done without either limiting the structural space to a few precisel defined GAG molecules or reducing the conformational space to a few static snapshots available from crystal structures. Towards this goal, we will adopt a "holistic" approach to protein/GAG interactions that embraces the structural diversity of heparin-like GAGs, as opposed to the commonly accepted approach which limits the scope of inquiry to a few molecules accessible through synthesis. We will use a combination of bottom-up and top-down approaches to study protein/GAG interactions, the former focusing on relatively well-defined subsets of short heparin oligomers obtained by affinity separations, and the latter aimed at intact heparin. This will reveal the structural properties governing the interactions of GAGs with several therapeutically relevant proteins. Novel applications of gas-phase ion chemistry (electron capture and collisional activation) will be developed for characterization of large heterogeneous GAG/protein complexes. Finally, atomic-level Monte Carlo simulations in parallel with H/D exchange studies will lead to a high-resolution and dynamic depiction of the interaction process. This new view of GAG/protein interactions will allow the therapeutic potential of heparin-like GAGs to be exploited more efficiently in areas as diverse as regenerative medicine, inflammation, neurodegenerative disorders and oncology.