Active-site cysteine 215 sulfonation targets protein tyrosine phosphatase PTP1B for Cullin1 E3 ligase-mediated degradation

Living organisms utilize oxygen to maintain the cellular homeostasis throughout their lifespan. A unique feature of oxygen metabolism within a living cell is the production of reactive oxygen species (ROS), which can be generated by a diverse array of extracellular stimuli including physiological ligands [1,2] and extracellular matrix-induced engagement of integrin [3]. This transient generation of ROS is a hallmark to activate crucial signaling pathways [4], including cellular growth, differentiation, senescence, adhesion, and programmed cell death [5]. In addition, constitutive generation of ROS takes place in all types of cell as the by-products of oxidative phosphorylation, which is a key function of mitochondria for ATP synthesis [6]. Physiologically generated ROS can be rapidly removed or scavenged by a variety of antioxidants through a series of enzymatic reactions [7]. However, overflowed ROS may be accumulated intracellularly, thus promoting oxidative modifications on proteins [8].

Among 20 amino acids, cysteine (Cys) and methionine (Met), both containing a sulfur atom, are two residues susceptible to ROS-mediated oxidation [9,10]. Moreover, the Cys residue with a low pKa characteristic at neutral pH that often functions as the catalytic center in thiol enzymes is highly reactive to H2O2 [11]. ROS-promoted post-translational modifications (PTMs) on Cys residue can be ranged from the reversible to irreversible state, depending on the degree of oxidants in the vicinity [12]. According to existing knowledge, Cys residues with sulfenic acid (-SOH) and sulfenamide (-SN) are reversibly oxidized, whereas sulfinic acid (-SO2H) and sulfonic acid (-SO3H) belong to the category of irreversible oxidation [12]. In a living cell, there are approximately 1–2% Cys residues subjected to sulfinated or sulfonated modification [13]. It is believed that the vast majority of such oxidized Cys residues are located at the active site of thiol enzymes, including protein tyrosine phosphatases (PTPs), due to the unique feature of low pKa [14]. Thiol enzymes carry out catalysis exclusively depending on the active-site Cys residue in the reduced form, which functions as a nucleophile to target substrates [15]. Once irreversible oxidation occurs at the active-site Cys, thiol enzymes become permanently inactive as the nucleophilic characteristic no longer exists. Based on this notion, thiol enzymes in the terminally oxidized state should be removed by a cellular process in order to maintain the proteomic homeostasis. It is particularly important for the non-mitotic cells such as cardiomyocytes to develop a robust pathway that degrades irreversibly oxidized proteins, thus minimizing ROS-induced proteotoxicity and cell death [16]. A recent study supported the hypothesis that a sulfonic acid moiety attached to Cys residues might act as a signaling tag, which facilities subsequent protein degradation [17]. The same study further suggested that proteasome and autophagy pathways may be responsible for the elimination of Cys-sulfonated proteins [17].

PTPs comprise a super-family of thiol enzymes that play a critical role in phosphotyrosine (pY) signaling homeostasis under physiological conditions [18,19]. In order to prevent the disturbance of pY signaling networks that are often seen in human diseases [20,21], the enzymatic activity of PTPs in every cell throughout the body must be regulated precisely at all times. For this, the low pKa active-site Cys of PTPs should be switched back and forth between the reduced form and the reversibly oxidized form [22,23]. It has been shown that PTPs are reversibly oxidized in cell's response to various ligands [22,23], leading to transient inhibition of their activity. The reversibly oxidized Cys may be reduced to a free thiol through the action of cellular oxidoreductases such as glutathione or thioredoxin reductase [24,25]. On the other hand, it is seemingly inevitable that the active-site Cys of PTPs is also susceptible to irreversible oxidation, rendering permanent inactivation of these enzymes. When exposed to a high level of ROS in the vicinity, reversibly oxidized Cys is further pushed to the formation of sulfinic or sulfonic acid, resulting in a permanent loss of PTP activity [26]. To date, it remains elusive whether irreversibly oxidized PTPs may occur under physiological conditions. Moreover, it is completely unknown how such terminally inactivated PTPs can be removed efficiently from a healthy cell.

In this study, we hypothesized that sulfonation of the active-site Cys on PTPs is a physiologically relevant PTM. Using cardiomyoblast H9c2 cells as a model, we examined whether terminally oxidized PTPs are degraded by ubiquitin proteasome system (UPS)-mediated process. We further explored the causal relationship between Cys sulfonation-induced conformational change and PTP ubiquitination. A screening on yeast proteome chip suggested a candidate of E3 ligase responsible for ubiquitination of Cys-sulfonated PTPs. Such finding was subsequently validated by cell-based analysis. We further examined whether accumulation of terminally oxidized PTPs may lead to cytotoxicity. These data highlight a novel mechanism that facilitates PTP homeostasis in cells with constitutive ROS production.

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