ReviewThe functional interactome of GSTP: A regulatory biomolecular network at the interface with the Nrf2 adaption response to oxidative stress
Introduction
Glutathione S-transferases (GSTs) are a family of ubiquitous proteins represented from unicellular organism to mammalians. The different members of this family show characteristic subcellular distribution and function (reviewed in Ref. [1]). Cytosolic GSTs, namely Alpha, Mu, Pi, Sigma, Theta, Zeta and Omega, are dimeric proteins abundantly expressed in tissues. These are pleiotropic proteins extensively investigated as detoxification enzymes (reviewed in Ref. [2]). However, the specificity for electrophilic substrates in the enzymatic conjugation reaction with reduced glutathione (GSH) is rather low for some of these isoenzymes, thus pointing to a redundancy in the detoxification response of this group of proteins that may explain the importance of the GST-dependent line of defence in cellular systems. Moreover, early evidence demonstrated that GST functions extend beyond just GSH conjugation to include binding activity toward small molecules (ligandin function) and macromolecules, peroxidase activity, isomerization, disulphide interchange and thiolysis. More recently some members of this family of enzymes have been described as critical components of signal transduction pathways, an aspect that will be extensively reviewed in this paper as far as GSTP activity concerns.
Constitutive expression of cytosolic GSTs is abundant in tissues with intense metabolic activity, such as liver, lung and kidney. In mouse liver this subfamily of proteins has been calculated to constitutively represent 5–8% of total cellular proteins, with GSTP as the most abundant form (from 2 to 3 fold the levels of A or M form) a significant fraction of total proteins ([3]. GSTs are very abundant proteins also in human liver even if the proportion between isoforms appears to change between human and mouse hepatocytes, with a low constitutive expression of GSTP in human cells ([3] and references therein). Gene induction properties may bring GSTs to much higher proportions. Several reasons for such an abundant and dynamic expression of cytosolic GST genes can be proposed. Former, the need of maintaining both at the constitutive and stress-stimulated state a sufficient potential for detoxification and “redox buffering” of cellular systems, a condition that reaches its maximal level of expression in some tumors (extensively reviewed in Refs. [4], [5]). This is to face toxicological challenges coming from endogenous and exogenous stressors, with the latter encompassing a broad group of xenobiotics and carcinogens with electrophilic properties that are known to be substrates of the GSH-conjugating activity of GSTs [2]. Transcriptional inducers of endogenous origin may include reactive products of the free radical-mediated damage of lipids, amino acids and proteins, such as some hydroperoxides and α,β-unsaturated aldehydes [6]. These are small molecules that function as non-catalytic ligands or as catalytic substrates of the GSH-conjugating activity of GST proteins.
Other and more recently identified reasons for such an important occurrence of cytosolic GSTs at the cellular level may include a putative role as components of the redox sensing and signaling platform of the cell. GSTP has been the most investigated signaling protein in this family of proteins. Its monomeric form shows the characteristic propensity to physically interact with other proteins, a process that results in regulatory effects, some of which are GSH-dependent [7]. Heterodimeric complexes of GSTP protein have been suggested to form with other GST subunits, such as GSTM monomers, some cellular kinases and peroxiredoxin 6 (Prdx 6) [7]. Besides its cytosolic localization, GSTP protein was also identified in mitochondria and the nucleus [8], [9] thus pointing to a broad influence of GSTP interactions on signal transduction and detoxification processes of different cellular compartments.
It is therefore arguable that this form of GST is an active player of the cellular interactome, i.e., the regulatory biomolecular network orchestrated through the dynamic interaction of classes of molecules in a cell [10], [11].
Redox-dependent effects can be easily anticipated on macromolecular targets of these interactions in virtue of the reported activity of GSTP in the reduction, thiolation and possibly S-nitrosylation of Cys moieties in proteins (reviewed in Ref. [12]).
The functional interactome of GSTP is expected to experience physiological or pathological changes that impact on cellular phenotypes. In fact, transcriptional and biochemical features of GSTP are compatible with significant changes in gene expression mechanisms, protein binding and redox properties and oligomerization state, which together provide wide versatility to its interaction properties. GSTP gene induction during cancer cell transformation and drug resistance have been proposed to represent an exquisite example of the biological relevance of such a dynamic character of GSTP interactions. Functional consequences are the modified activity of cellular kinases and stress adaption genes associated with the activation of anti-apoptotic and proliferative pathways, and with the metabolic changes of cancer cells [5], [7]. As a consequence, polymorphic characteristics and transcriptional regulation of GSTP gene are currently investigated in diagnostic and therapeutic protocols of several types of cancers, and could even be considered to explore the toxicological risk of populations exposed to environmental carcinogens [13].
GSTP gene expression is highly inducible and is finely tuned at the transcriptional level by the activation of nuclear factors that sense electrophilic stressors of different origin and nature on the plasmalemma and the cytosol, then migrating to the nucleus to bind specific sequences of GST promoter region, such as the antioxidant responsive element (ARE) [4]. Nuclear factor erythroid 2-related factor (Nrf2) is one of the most important transcription factors recognized to stimulate the induction of GSTP as well as of other cytosolic GSTs and phase II genes (reviewed in Refs. [2], [12]). Importantly, that process has been found to become constitutive in some drug-resistant cancers [4].
This dynamics of expression combined with the molecular “interactivity” and catalytic role of GSTP may produce consequences that translate from subcellular compartments to tissues and even to inter-organ and systemic processes. For instance, a marked GSTP overexpression in some tissues combined with an increased availability of electrophilic substrates may impact on the inter-organ metabolism and redox of GSH. This is a relatively common event during conditions of severe toxicity and oxidative stress such as in the case of chronic kidney disease (CKD) and hyperbilirubinemic patients [14], [15], [16]. Blood cells and possibly in other tissues of these patients show a chronic transcriptional activation of GSTP gene that is expected to increase the cellular turnover of GSH and the consumption rate of reducing equivalents through the GSH/GSSG redox couple. The risk of shortage for this reducing substrate may thus increase, at least in some tissues [17]. The resulting molecular defect is detrimental to GSH-dependent cell protection and detoxification mechanisms, and may contribute to explain the impaired signaling of tissues and the increased allostatic load and frailty of CKD patients.
After decades of studies on the non-catalytic binding properties of cytosolic GSTs toward small molecules [18], [19], macromolecule binding of GSTs is thus emerging as a new and stimulating subject of investigation. The capability of GSTP, and possibly of other cytosolic GSTs, to interact physically, chemically (mainly at the redox level) and functionally with cellular proteins with roles in signal transduction and key cellular processes, is discussed in this paper. Providing an overview of the literature published so far on the functional interactome of GSTP, it becomes evident that this protein represents a major node in the network of biomolecular interactions that govern the physiological (adaption) and pathological (for instance tumorigenic) response to cellular stresses. To strengthen that functional interpretation, recent work by our group suggests that the stress-related network of GSTP interactions includes its upstream transcriptional regulator Nrf2 [20]. Methodological aspects of cellular interactome investigation are also briefly discussed.
Section snippets
Interaction database exploration
Molecular features and functional implications of regulatory biomolecular networks have been thoroughly revised elsewhere [21], [22]. These are characterized by a series of molecular nodes that interact at the physical and/or chemical level (the interactome) to produce a concerted outcome (the functional interactome). Varying the nature and number of molecular interactions, the network may transit to different states of molecular expression. Nodes represented by proteins or even small
Pathway analysis of GSTP interactions
Pathway interpretation of most relevant protein–protein interactions of human GSTP1 performed with String DB (Fig. 2) points to a role for this protein as a node that bridges MAPK signaling and the GSH-dependent regulation of Prdx6 with the detoxification of reactive oxygen species (ROS), cytochrome P450-dependent metabolism of xenobiotics, cell cycle regulation, chemical carcinogenesis and tumorigenesis pathways.
Interaction proteomics methods
Physical interactions between proteins, or protein–protein interactions, are essential to virtually all the molecular aspects of life; for that reason, proteomics methods aimed at exploring these interactions are becoming increasingly popular and important in biochemistry and molecular biology labs. In recent years, these methods nowadays collectively identified with the name of “interaction proteomics”, have been used to discover protein interactions and characterize the molecular aspects of
Conclusions
Interaction proteomics methods used to explore the cellular interactome have been extensively applied in the exploration of the GST interaction network. The regulatory interactions observed between the monomeric forms of cytosolic GSTs and other cellular proteins convincingly suggest a role for these proteins as molecular chaperones of signaling and redox-sensitive proteins. In the case of GSTP, experimental data clearly demonstrate that signaling and regulatory functions coexist with the
Acknowledgments
Part of this work was supported by the grant programs of the Italian Ministry of University, National Technology Agrifood Cluster, Health and Nutrition program, PROS.IT project (CTN01_00230_413096).
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2019, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :The so far reported interactions of GSTP1 involve proteins with different cellular localizations, molecular characteristics and biological role (Fig. 3B) [73]. Recent examination of the literature suggested that main hotspots of this interactome have a strategic distribution on cellular pathways associated with inflammatory and stress response processes (Fig. 4) [46]. This would favour a functional coordination between these processes that is important in normal tissues to control inflammation and to produce an efficient adaptive response to inflammatory stimuli.