Role of protein unfolding in endosomal escape

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With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Professor Schepartz from the University of California, Berkeley is studying the process that enables certain proteins to traffic efficiently across endosomal membranes into the cytosol. This process is often referred to as “endosomal escape”. Endosomal escape is poorly understood and represents the fundamental bottleneck limiting the intracellular utility of protein therapeutics. Schepartz will test the hypothesis that endosomal escape demands protein and/or delivery agent unfolding and seeks to identify the biophysical criteria – the design rules – that make a protein “deliverable”. Were such design rules known, scientists could develop and prepare peptides and proteins with confidence that they would reach their intended intracellular location. The single molecule technique known as fluorescence correlation spectroscopy (FCS) is the very best way to monitor intracellular trafficking, as it is the only method that quantifies endosomal escape directly and without amplification. Yet the set-up is tricky and the math is complicated; both limit its adoption in academia and industry. The Schepartz team proposes two highly complementary activities (an FCS BootCamp and BASIS lesson for 5th graders) to extend the impact of their science beyond established communities. Both include elements related to Innovation, Informal Science Communication, Higher Education/Professional Development, and Broadening Participation. Most proteins and peptides circumnavigate a complex path to traffic into the cytosol or nucleus. Although some cyclic peptides diffuse passively through biological membranes, these molecules are the exception, not the rule. For most peptides and proteins, the journey to the cytosol involves endocytosis and demands not just endocytic uptake but also endosomal escape. Most molecules described as “cell-penetrating peptides” (CPPs) escape endosomes with efficiencies of 10%. As a result, although there has been an enormous amount of outstanding work on the basic science of peptidomimetics and artificial proteins, the utility of these molecules largely ends at the cell surface. ZF5.3 is unlike most CPPs. The polypeptide known as ZF5.3 is a 28-amino acid, non-toxic, de novo-designed mini-protein that traffics with unprecedented efficiency ( 50%) to the cytosol and nucleus, even when covalently fused to a protein cargo. The remarkable activity of ZF5.3 requires a penta-arginine motif–five Arg residues displayed in a defined array on an ⍺-helix–and a unique portal created by the HOPS (homotypic fusion and protein sorting) complex, a multimeric protein complex associated with endosomal transport. This proposal will test the hypothesis that endosomal escape demands protein and/or delivery agent unfolding and seeks to identify the biophysical criteria that make a protein “deliverable”.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.