Tài liệu Báo cáo khoa học: Post-translational modification of the deubiquitinating enzyme otubain 1

Post-translational modification of the deubiquitinating

enzyme otubain 1 modulates active RhoA levels and

susceptibility to Yersinia invasion

Mariola J. Edelmann, Holger B. Kramer, Mikael Altun and Benedikt M. Kessler

Department of Clinical Medicine, University of Oxford, UK

Introduction

The genus Yersinia consists of three pathogenic species

that are agents of a variety of diseases, one of which

was historically the cause of major pandemics. These

include the bubonic plague caused by Yersinia pestis,

mesenteric adenitis and septicaemia caused by

Yersinia pseudotuberculosis and gastroenteritis caused

Keywords

deubiquitinating enzymes; otubain 1;

phosphorylation; RhoA; YpkA

Correspondence

B. M. Kessler, Henry Wellcome Building for

Molecular Physiology, Nuffield Department

of Clinical Medicine, University of Oxford,

Roosevelt Drive, Oxford OX3 7BN, UK

Fax: +44 1865 287 787

Tel: +44 1865 287 799

E-mail: [email protected]

(Received 24 November 2009, revised 17

March 2010, accepted 29 March 2010)

doi:10.1111/j.1742-4658.2010.07665.x

Microbial pathogens exploit the ubiquitin system to facilitate infection and

manipulate the immune responses of the host. In this study, susceptibility

to Yersinia enterocolitica and Yersinia pseudotuberculosis invasion was

found to be increased upon overexpression of the deubiquitinating enzyme

otubain 1 (OTUB1), a member of the ovarian tumour domain-containing

protein family. Conversely, OTUB1 knockdown interfered with Yersinia

invasion in HEK293T cells as well as in primary monocytes. This effect

was attributed to a modulation of bacterial uptake. We demonstrate that

the Yersinia-encoded virulence factor YpkA (YopO) kinase interacts with a

post-translationally modified form of OTUB1 that contains multiple phos￾phorylation sites. OTUB1, YpkA and the small GTPase ras homologue

gene family member A (RhoA) were found to be part of the same protein

complex, suggesting that RhoA levels are modulated by OTUB1. Our

results show that OTUB1 is able to stabilize active RhoA prior to invasion,

which is concomitant with an increase in bacterial uptake. This effect is

modulated by post-translational modifications of OTUB1, suggesting a

new entry point for manipulating Yersinia interactions with the host.

Structured digital abstract

l MINT-7717124: ypkA (uniprotkb:Q05608) physically interacts (MI:0915) with OTUB1 (uni￾protkb:Q96FW1) by anti bait coimmunoprecipitation (MI:0006)

l MINT-7717229: rhoA (uniprotkb:P61586) physically interacts (MI:0915) with OTUB1 (uni￾protkb:Q96FW1) by affinity chromatography technology (MI:0004)

l MINT-7717075, MINT-7717207, MINT-7717193, MINT-7717170: ypkA (uniprotkb:Q56921)

physically interacts (MI:0915) with OTUB1 (uniprotkb:Q96FW1) by anti tag coimmunopre￾cipitation (MI:0007)

l MINT-7717390: ypkA (uniprotkb:Q56921) physically interacts (MI:0914) with OTUB1 (uni￾protkb:Q96FW1) and RhoA (uniprotkb:P61586) by anti tag coimmunoprecipitation (MI:0007)

Abbreviations

HA-Ub-Br2, hemagglutinin-tagged ubiquitin-bromide; MOI, multiplicity of infection; OTUB1, otubain 1; Rac1, ras-related C3 botulinum toxin

substrate 1; RhoA, ras homolog gene family member A; USP, ubiquitin-specific protease; Yop, Yersinia outer protein; YpkA ⁄ YopO, Yersinia

serine ⁄ threonine kinase.

FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2515