Biology subjects

Dall, E.

Publications and source records attributed to Dall, E..

4 recordsLinked to original sources

bioRxiv2025

Positional Scanning and Computational Modeling Reveal Determinants of Legumain Transpeptidase Activity

ABSTRACTLegumains are cysteine proteases that, in addition to their canonical hydrolase function, can act as peptide ligases or transpeptidases. In humans, this activity becomes particularly relevant under pathophysiological conditions, where legumain relocalizes to near-neutral pH compartments favoring ligation/transpeptidation over hydrolysis. Here, we combined in vitro positional scanning with in silico substrate profiling to elucidate the substrate determinants governing human legumain-mediated peptide cyclization. We identified glycine residues at P1'' and P1' and basic residues at P2'/P2'' as key determinants of human legumain-mediated peptide cyclization. Guided by these insights, we designed an optimized substrate exhibiting substantially enhanced cyclization efficiency. Computational analysis not only recapitulated the experimental observations but also predicted a covalent inhibition mechanism involving a P1' cysteine, revealed a kcat-tuning switch embedded within the substrate, and highlighted its potential for developing high-performance fluorogenic substrates. Collectively, these findings advance the mechanistic understanding of legumains transpeptidase activity and provide a framework for developing selective probes and inhibitors across the legumain family and related cysteine proteases. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/693912v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@121ffcborg.highwire.dtl.DTLVardef@120a587org.highwire.dtl.DTLVardef@536fc7org.highwire.dtl.DTLVardef@1cfaa67_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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

Arabidopsis thaliana phytocystatin 6 forms functional oligomer and amyloid fibril states

Cystatins not only encode a high functional variability because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils, however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Non-covalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The non-covalent dimeric form of AtCYT6 retained activity against its target proteases, papain, and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to AtCYT6s N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications in plant protease regulation.

biochemistry↗

bioRxiv2023

Phytocystatin 6 is a context-dependent, tight-binding inhibitor of Arabidopsis thaliana legumain isoform β

Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform {beta} (AtLEG{beta}) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEG{beta} and papain-like cysteine proteases through two cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEG{beta}. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEG{beta} suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEG{beta} regulation and its broader physiological significance.

biochemistry↗