Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Humaira Adnan,Clifford Lingwood,Clare Che,Hyun-Joo Park,Chetankumar Tailor,Mustafa Kamani,Beth Binnington,Zhenbo Zhang,George Spitalny,Ludger Johannes

doi:10.1371/journal.pone.0166948

Humaira Adnan, Clifford Lingwood + Show 8 more

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https://doi.org/10.1371/journal.pone.0166948

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Abstract

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2–4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD–exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

Highlights

Endoplasmic reticulum associated degradation (ERAD) is a cellular quality control mechanism by which the three dimensional folding of nascent polypeptides is sampled for aberrant features [1]
Band b accumulated by ~5 fold after VT treatment but no band c was detected in these HeLa cells
ERAD is a key component of homeostasis, and while several ERAD inhibitors have been developed [69] and tested for therapeutic effect [70,71,72], they have to contend with the adverse effects of induction of endoplasmic reticulum (ER) stress [73] and the unfolded protein response (UPR)[74]

Summary

Introduction

Endoplasmic reticulum associated degradation (ERAD) is a cellular quality control mechanism by which the three dimensional folding of nascent polypeptides is sampled for aberrant features [1]. Members of the Derlin protein family are central and the role of reverse transit of the Sec translocon in ERAD has become contentious[14,15], the Sec translocon [16,17,18] may yet be involved This translocon is selectively hijacked by the A subunit of various plant and bacterial protein subunit toxins, which require cytosolic access for A subunit induction of cellular damage [19,20,21]. Since the A subunit is a translocon substrate, any ERAD inhibition would be temporary and lost once translocated This provides impetus to study the potential efficacy of toxoid rescue of ERAD substrates. Approach to develop highly bioactive vectors for the targeted, rapid, temporary blockade of ERAD and potential benign amelioration of any ERAD-based disease These toxoids were without overt cell culture cytopathology (apoptosis). Given their distinct mechanism of action, these toxoids may be able to complement other misfolded protein rescue strategies [37,38]

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