Horak, C. | Search bioRxiv

bioRxiv2025

Conformational and Functional Regulation of SET by Legumain Cleavage

The cysteine protease legumain typically localizes to the endolysosomal system, where it is an important player in the immune system. However, in the context of Alzheimers disease (AD), legumain has been shown to be translocated to the cytosol, where it cleaves SET, synonymously termed TAF-1 or I2PP2A, an inhibitor of protein phosphatase 2A. SET is primarily found in the nucleus, where it regulates gene transcription, cell cycle progression, and histone acetylation, but can also translocate to the cytoplasm where it regulates cell migration and is implicated in neuronal apoptosis in AD. In this study, we demonstrate that legumain cleaves SET at two major sites: Asn16 at the N-terminal end and Asn175 at the earmuff domain. Contrary to previous findings, our biochemical and crystallographic experiments reveal that the corresponding N- and C-terminal cleavage products remain bound in a stable complex, rather than dissociating. Additionally, we show that the C-terminal acidic stretch of SET is essential for its binding to histone 1, and that cleavage impairs this interaction. Finally, we demonstrate that SET positively modulates PP2A activity. This effect is however abolished upon cleavage by legumain.

molecular biology↗

bioRxiv2023

Engineering Regionally-Activated Drugs for Neuroscience

The brain is comprised of multiple regions performing distinct functions. Within each of these regions, there are multiple cell types that can affect brain physiology. Finally, within each cell there are multiple signaling pathways, that, when activated or inhibited, control the cells activity, and consequently the brain function. For these reasons, methods that can control the brain with regional, cell-type, and molecular precision have been widely used in neuroscience. However, so far, achieving sustained control over a brain region with that level of specificity relied either on gene delivery or placement of invasive devices. While gene therapy holds great promise, the risks of genomic integration, vector toxicity, vector-directed immune response, high cost, and gene delivery to the brain pose significant challenges. On the other hand, invasive devices enable site-specific delivery of drugs but can also surgically damage the modulated brain region, carrying risks of infection and hemorrhage. Here, we present a new approach that can provide multi-day, noninvasive, site-specific control over specific cell types in the brain without the need to use invasive devices or gene delivery. To achieve this, we introduce a new paradigm called Regionally Activated Interstitial Drugs, or RAID, which delivers a protein-based catalytic centers, or RAID enzymes, to the brain using focused ultrasound blood-brain barrier opening. This catalytic center is designed to attach to the interstitial space in the brain where it remains for days after initial delivery. While the catalytic center is present in the brain, it can locally process an inert BBB permeable prodrug into an active drug, resulting in localized therapy. Our proof-of concept studies demonstrated that the engineered RAID enzymes can retain activity in the brain parenchyma for several days, allowing for noninvasive site-specific induction of neuronal activity that was sufficiently potent to elicit behavioral effects. Overall, the RAID paradigm enabled noninvasive, tunable, temporally-re-solved, site-specific, non-genetic, neuromodulation over multiple days. The RAID paradigm is versatile and can be applied to any enzyme and prodrug pair to control various aspects of central nervous system physiology.

bioengineering↗