pH regulation of cell surface receptors

摘要 理解GPCR活性的质子门控（H+-门控）是信号生物学的一个重要新前沿。 GPCR在各种情况下，如核内体、肿瘤、 突触发炎组织和免疫反应然而，这些信号调节的程度 GPCR功能代表了我们对细胞传感，通信和生物学的理解中的一个重大差距。 药物发现。我们研究计划的目标是为GPCRome的大部分人提供这种见解。 一个主要的目标是开发pH智能纳米抗体，使直接跟踪和 在正常和酸化条件下调节GPCR功能，长期计划是开发 最有前途的精密工具应用于治疗。我们在这些努力中取得了很大成功。 我们已经成功地设计了必要的纳米抗体发现平台，并使用 已知的GPCR-纳米抗体对。我们还通过计算设计和制造了新的合成 我们已经开始针对不同GPCR池筛选纳米抗体库。因为我们的 图书馆规模大，命中率高，我们发现了一个瓶颈，在摆盘，编目，挑选， 和表征携带推定的纳米抗体命中的酵母菌落。我们要的仪器， Mini Qpix小足迹微生物菌落采集器将克服这一瓶颈，使我们能够扩大 我们处理成百上千的纳米抗体的过程。这种能力将大大加快 发现GPCR的纳米抗体调节剂，并有可能阐明纳米抗体的多样性 以训练用于从头纳米抗体设计的人工智能算法所需的深度和规模。 因此，NIGMS对我们的仪器请求的支持将促进该领域的实质性飞跃。

ABSTRACT Understanding proton-gating (H+-gating) of GPCR activity is a major new frontier in signaling biology. GPCRs regularly encounter coincident H+ signals in a variety of contexts, such as endosomes, tumors, synapses, inflamed tissue, and immune responses. However, the extent to which these signals regulate GPCR function represents a significant gap in our understanding of cellular sensing, communication, and drug discovery. The goal of our research program is to provide this insight for much of the GPCRome. A major goal is the development of pH-intelligent nanobodies that enable the direct tracking and regulation of GPCR function in normal and acidified conditions, with a longer-term plan is to develop the most promising of these precision tools into therapeutics. We have had much success in these efforts. We have successfully engineered the necessary nanobody discovery platform and validated it using known GPCR-nanobody pairs. We have also computationally designed and manufactured new synthetic nanobody libraries which we have begun to screen against pools of diverse GPCRs. Because of our large library sizes and high hit rates, we have discovered a bottleneck in the plating, cataloging, picking, and characterization of yeast colonies carrying putative nanobody hits. The instrument we are requesting, a Mini Qpix small footprint microbial colony picker, will overcome this bottleneck and enable us to expand our processing from hundreds to thousands of nanobody hits. Such capabilities would greatly accelerate the discovery of nanobody regulators of GPCRs and have the potential to illuminate nanobody diversity at a depth and scale necessary for training artificial intelligence algorithms for de novo nanobody design. As such, NIGMS support for our instrument request would catalyze a substantial leap forward in the field.