Systems analysis of protein modification and cellular responses induced by electrophile stress

Abstract

Biological electrophiles result from oxidative metabolism of exogenous compounds or endogenous cellular constituents, and they contribute to pathophysiologies such as toxicity and carcinogenicity. The chemical toxicology of electrophiles is dominated by covalent addition to intracellular nucleophiles. Reaction with DNA leads to the production of adducts that block replication or induce mutations. The chemistry and biology of electrophile-DNA reactions have been extensively studied, providing in many cases a detailed understanding of the relation between adduct structure and mutational consequences. By contrast, the linkage between protein modification and cellular response is poorly understood. In this Account, we describe our efforts to define the chemistry of protein modification and its biological consequences using lipid-derived alpha,beta-unsaturated aldehydes as model electrophiles. In our global approach, two large data sets are analyzed: one represents the identity of proteins modified over a wide range of electrophile concentrations, and the second comprises changes in gene expression observed under similar conditions. Informatics tools show theoretical connections based primarily on transcription factors hypothetically shared between the two data sets, downstream of adducted proteins and upstream of affected genes. This method highlights potential electrophile-sensitive signaling pathways and transcriptional processes for further evaluation. Peroxidation of cellular phospholipids generates a complex mixture of both membrane-bound and diffusible electrophiles. The latter include reactive species such as malondialdehyde, 4-oxononenal, and 4-hydroxynonenal (HNE). Enriching HNE-adducted proteins for proteomic analysis was a technical challenge, solved with click chemistry that generated biotin-tagged protein adducts. For this purpose, HNE analogues bearing terminal azide or alkyne functionalities were synthesized. Cellular lysates were first exposed to a single type of HNE analogue (azido- or alkynyl-HNE), and then click reactions were performed against the cognate alkynyl- and azido-biotin derivative. The resulting biotin-labeled proteins were captured and enriched over a streptavidin matrix for subsequent mass spectrometric analysis. We thereby identified a multitude of HNE targets. Simultaneous microarray analysis of changes in gene expression triggered by HNE also produced an abundance of data. Functional analysis of both data sets generated the hypothesis that an important pathway of cellular response derives from electrophile modification of protein chaperones, resulting in the release of transcription factors that are their clients. Informatic analysis of the protein modification and microarray data sets identified several transcription factors as potential mediators of the cellular response to HNE-adducted proteins. Among these, heat shock factor 1 (HSF1) was confirmed as a sensitive and robust effector of HNE-induced changes in gene expression. Activation of HSF1 appears, in part, to be mediated by the electrophilic adduction of Hsp70 and Hsp90, which normally maintain HSF1 in an inactive cytosolic complex. The identification of HSF1 as a mediator of biological effects downstream of HSF1 has provided new opportunities for research, illustrating the potential of our systems-based approach. Accordingly, we characterized HSF1-mediated gene expression in protecting against electrophile-induced toxicity. Among the genes induced by HSF1, Bcl-2- associated athanogene 3 (BAG3) is notable for its actions in promoting cell survival through stabilization of antiapoptotic Bcl-2 proteins, appearing to have a critical role in mediating cellular protection against electrophile-induced death.

Figure 1

Role of electrophile generation in cellular damage and carcinogenesis.

Figure 2

Inhibition of NF-κB signaling by reactive electrophile modification of IκB kinase.

Figure 3

Generation of membrane-bound and diffusible electrophiles from glycerophospholipid peroxidation.

Figure 4

Approach to relating electrophile modification of proteins to transcriptional responses. Inventories of modified proteins and changes in gene expression are determined in parallel experiments. Potential linkages between the two data sets are established by informatic analysis of signal transduction networks. This allows formulation of hypotheses relating modification of individual proteins to altered gene expression.

Figure 5

HNE reactions with amino acid side chains.

Figure 6

Method for adduct capture and analysis utilizing click chemistry compatible HNE analogues.

Figure 7

Network relating protein modification and gene expression changes induced by HNE. The dots at the bottom of the diagram represent genes that are upregulated (red) or downregulated (green) by HNE treatment. These genes are connected to the cis regulatory elements that control their expression, which are connected to the transcription factors that bind to them. Lines drawn from protein targets of HNE modification to the transcription factors controlling gene expression represent linkage by direct interaction or via signal transduction pathways that establish a plausible link between protein modification and altered transcriptional activity. The linkages representing control of the heat shock response are highlighted in red. This analysis was provided by Bing Zhang using data from refs (15) and (21).

Figure 8

Comparative viabilities of HSF1- and Nrf2-silenced RKO cells treated with HNE.

Figure 9

Proposed role for HSF1-enhanced BAG3 and Hsp72 expression in mitigating cell death based on increased levels of antiapoptotic client proteins.