Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics

Abstract

In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality-control systems composed by various proteins. Among them, UDP-glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure. This study used pseudo-misfolded glycoproteins, comprising a modified dihydrofolate reductase with artificial pyrene-cysteine moiety on the protein surface (pDHFR) and Man9 GlcNAc2 -methotrexate (M9-MTX). All five M9-MTX/pDHFR complexes, with a pyrene group at different positions, were found to be good substrates of UGGT, irrespective of the site of pyrene modification. These results suggest UGGT's mode of substrate recognition is fuzzy, thus allowing various glycoproteins to be accommodated in the folding cycle.

Small-Molecule Hydrophobic Tagging: A Promising Strategy of Druglike Technology for Targeted Protein Degradation

“…Similar to fulvestrant-induced surface hydrophobicity, hydrophobic fragments within HyT play a crucial role in promoting protein ubiquitination and degradation. Various hydrophobic tags have been reported thus far, including adamantane, tert -butyl carbamate-protected arginine (Boc 3 Arg), fluorene, pyrene, carborane, menthoxyacetyl, and norbornene . These tags can be employed to induce degradation of pathogenesis-related proteins, including some previously deemed undruggable targets.…”

Applications of Bacterial Degrons and Degraders — Toward Targeted Protein Degradation in Bacteria

“…A variation on the use of small molecules to induce TPD is a method called hydrophobic tagging. Hydrophobic stretches are often exposed in unfolded proteins, and can be recognized by protein quality control pathways and result in protein degradation (Hachisu et al, 2016). Hydrophobic tags (HyTs) are chimeric compounds designed to have high hydrophobicity and low molecular weight (Neklesa et al, 2011).…”

Chemical‐Synthesis‐Based Approach to Glycoprotein Functions in the Endoplasmic Reticulum

“…The glycan‐protein complex formed by conjugation of M9‐Gly‐MTX with DHFR (M9‐MTX‐DHFR) was far less reactive than M9‐MTX, reflecting that the hydrophobic substituent was masked by bounding to the folded protein [28a] . On the other hand, when the pyrene‐modified DHFR variants were employed, resultant complexes [M9‐MTX‐DHFR(Pyr)] restored the reactivity toward UGGT [57] (Figure 8), well supporting the postulation that UGGT recognizes surface‐exposed hydrophobicity of client glycoproteins. Several lines of evidence that strongly support this notion have been obtained by experiments that employed synthetic glycoproteins, as will be discussed in 3.8 .…”