Tài liệu Báo cáo khoa học: Bacitracin is not a specific inhibitor of protein disulfide isomerase pptx

Bacitracin is not a specific inhibitor of protein disulfide

isomerase

Anna-Riikka Karala and Lloyd W. Ruddock

Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland

Introduction

Protein disulfide isomerase (PDI) is an endoplasmic

reticulum (ER)-resident protein catalyst that helps

newly translated polypeptide chains to fold and form

native disulfide bonds [1]. PDI can catalyze the oxida￾tion of two cysteines to form a disulfide bond, as well

as the reduction and isomerization of disulfide bonds

in peptides and proteins. PDI has four structural thior￾edoxin-like domains, a, b, b¢, and a¢, a linker region x

between the b¢ and a¢ domains, and a C-terminal acidic

extension. The a and a¢ domains contain the CGHC

active site motif, and are sufficient alone to perform

thiol–disulfide exchange reactions in simple substrates

[2]. The b¢ domain has the principal peptide and non￾native protein-binding site, and is required for isomeri￾zation reactions [2–4], whereas the b domain is of

unknown function.

PDI is one of a family of 20 PDI-like proteins

identified in the ER [1]. These proteins contain one or

more domains that are similar to the domains of PDI,

and many have been shown to catalyze thiol–disulfide

exchange reactions. However, their specific roles,

substrate specificities and mechanisms of cooperation

with other catalysts and chaperones in the cell are not

yet clear.

Besides PDI being abundant in the ER, several stud￾ies have shown non-ER locations for PDI family mem￾bers [5]. PDI inhibitors and specific antibodies have

often been used to discover the function of PDI-like

proteins, especially outside the ER. Bacitracin is a

commonly used inhibitor in these studies, and it is usu￾ally considered to be a specific inhibitor of PDI activ￾ity [6–8]. However, in vitro evidence for the action of

bacitracin as an inhibitor of PDI is scarce, and evi￾dence of its specificity for PDI is nonexistent. Bacitracin

is also used medicinally to prevent infections in small

cuts and burns and to treat gastrointestinal infections.

In addition, it is used as an animal feed additive for

disease prevention and growth promotion in farm

animals. For all of these functions, the effects are

unrelated to PDI inhibition. Commercially available

Keywords

bacitracin; chaperone; protein disulfide

isomerase; protein folding; thiol–disulfide

exchange

Correspondence

L. W. Ruddock, University of Oulu,

Department of Biochemistry, PO Box 3000,

University of Oulu, Oulu 90014, Finland

Fax: +358 8 5531141

Tel: +358 8 5531683

E-mail: [email protected]

(Received 12 August 2009, revised 3 March

2010, accepted 19 March 2010)

doi:10.1111/j.1742-4658.2010.07660.x

To successfully dissect molecular pathways in vivo, there is often a need to

use specific inhibitors. Bacitracin is very widely used as an inhibitor of pro￾tein disulfide isomerase (PDI) in vivo. However, the specificity of action of

an inhibitor for a protein-folding catalyst cannot be determined in vivo.

Furthermore, in vitro evidence for the specificity of bacitracin for PDI is

scarce, and the mechanism of inhibition is unknown. Here, we present

in vitro data showing that 1 mm bacitracin has no significant effect on the

ability of PDI to introduce or isomerize disulfide bonds in a folding protein

or on its ability to act as a chaperone. Where bacitracin has an effect on

PDI activity, the effect is relatively minor and appears to be via competition

of substrate binding. Whereas 1 mm bacitracin has minimal effects on PDI,

it has significant effects on both noncatalyzed protein folding and on other

molecular chaperones. These results suggest that the use of bacitracin as a

specific inhibitor of PDI in cellular systems requires urgent re-evaluation.

Abbreviations

BPTI, bovine pancreatic trypsin inhibitor; CM, carboxymethyl; ER, endoplasmic reticulum; PDI, protein disulfide isomerase.

2454 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS