Authors:

Jaralynn Morellano

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About Paper:

The Ubiquitin C-terminal Hydrolase UCH37/UCHL5 is a deubiquitinating enzyme (DUB) associated with the 26S proteasome. It plays a key role in protein degradation by cleaving branchpoints for Lys48 linkages within polyubiquitinated substrates prior to degradation. Biochemical studies have shown there is a noncanonical ubiquitin binding site (S1' site) for this linkage specificity that lies on the opposite face of the canonical binding site (S1 site). The canonical and noncanonical nanobodies are developed by yeast display to bind to the S1 and S1' site, allowing for the inhibition of multivalent ubiquitin binding. Studies surrounding Lys48 branchpoints are particularly of interest in cancer due to its integral role in protein degradation. UCH37 can cleave Lys48 branchpoints to save proteins from degradation. The proteasome subunit Rpn13 brings UCH37 out of its autoinhibited state and fully activates cleavage activity. Our experiments aim to determine the structural basis of multivalent nanobody association with UCH37. We formed a UCH37 C88A Rpn13 DEUBAD ncNb complex through a combination of column affinity purification and size exclusion chromatography. Currently, crystal screens are being incubated and observed for crystal growth. As an alternative we have also turned to protein cryogenic electron microscopy. This structure will allow us to observe how different nanobodies of similar sequences differentiate between the distinct ubiquitin binding sites of UCH37. Keywords: Deubiquitinating Enzyme (DUB); UCH37/UCHL5; Nanobodies; Inhibition; Multivalent Ubiquitin Binding

Source:

Purdue University / 2024

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Co-authors:

Jaralynn Morellano