Engineering Synthetic Biomolecular Condensates for Targeted Autophagy Modulation

/ ABSTRACT The modulation of autophagy is a promising target for drug discovery due to its role in various diseases. Current approaches often result in drugs with unanticipated effects due to the complexity of autophagy signaling networks, highlighting the need for novel strategies. Biomolecular condensates (BMCs), membrane-less organelles formed by phase separation, play a key role in selectively disposing of unwanted proteins via autophagy. Selective autophagy receptors (SARs) function as BMCs, recruiting degradation-bound cargo to phagophores. For example, p62/SQSTM1 forms BMCs to regulate autophagosome biogenesis by stabilizing phagophores, a biophysical regulatory mechanism that correlates the physicochemical properties of BMCs with autophagy efficacy. Despite advances, the regulatory mechanisms of BMCs in autophagy remain largely unexplored due to their compositional complexity and transient nature. This poses a barrier to systematically studying the biophysical interactions during autophagic engulfment. We propose to engineer a novel BMC-based tool with tunable physicochemical properties using PopTag, a synthetic BMC platform developed in our lab. This will create an orthogonal SAR-like BMC capable of supporting autophagosome biogenesis and selective cargo recruitment. Our approach involves leveraging PopTag's modular domain architecture, which includes the 'condenser' (drives condensation), the 'tuner' (modulates properties), and the 'actor' (enables recruitment). This platform allows us to disentangle signaling-based regulatory mechanisms from biophysical ones, enabling detailed investigation of BMC properties and autophagy. We will first Investigate the physicochemical properties of p62/SQSTM1. Using confocal microscopy, fluorescence recovery after photobleaching (FRAP), and microelectrophoresis, we will establish the homeostatic range of surface tension, viscosity, and surface electrostatics for p62/SQSTM1-mediated autophagosome biogenesis. We will also assay disease-relevant mutations to identify abnormal physical properties (Aim 1). Next, we will engineer an orthogonal BMC-based platform for selective autophagy. Using rational protein design, we will create an autophagy-targeted PopTag (AutoPop) and optimize its physicochemical properties to enhance autophagosome biogenesis and cargo recruitment (Aim 2). This proposal aims to establish the foundational physicochemical properties required for autophagosome biogenesis and develop an orthogonal BMC-based tool for investigating the cellular biophysics of autophagic engulfment, paving the way for innovative therapeutic strategies targeting the biophysical aspects of autophagy. Project Number: 1R21AG093393-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Keren Lasker | Institution: SCRIPPS RESEARCH INSTITUTE, THE, LA JOLLA, CA | Award Amount: $506,000 | Activity Code: R21 | Study Section: Enabling Bioanalytical and Imaging Technologies Study Section[EBIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11120516