Arsenic Modulates Posttranslational S-Nitrosylation and Translational Proteome in Keratinocytes

Arsenic is a class I human carcinogen (such as inducing skin cancer) by its prominent chemical interaction with protein thio (-SH) group. Therefore, arsenic may compromise protein S-nitrosylation by competing the -SH binding activity. In the present study, we aimed to understand the influence of arsenic on protein S-nitrosylation and the following proteomic changes. By using primary human skin keratinocyte, we found that arsenic treatment decreased the level of protein S-nitrosylation. This was coincident to the decent expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). By using LC-MS/MS, around twenty S-nitrosoproteins were detected in the biotin-switched eluent. With the interest that arsenic not only regulates posttranslational S-nitrosylation but also separately affects protein's translation expression, we performed two-dimensional gel electrophoresis and found that 8 proteins were significantly decreased during arsenic treatment. Whether these decreased proteins are the consequence of protein S-nitrosylation will be further investigated. Taken together, these results provide a finding that arsenic can deplete the binding activity of NO and therefore reduce protein S-nitrosylation.


Introduction
Protein S-nitrosylation, the covalent attachment of nitric oxide (NO) to protein thio (-SH) group, is an important posttranslational modification that affects a wide variety of proteins for cellular signaling in normal physiology and a broad spectrum of human diseases [1,2]. S-nitrosylation signaling controls a number of cellular processes, such as proteinprotein interactions [3], nuclear transcriptions [4], and membrane-associated proteins activation [5,6]. Pathophysiology is correlated with hypo-or hyper-S-nitrosylation of specific protein targets rather than a general cellular insult due to not only the loss of or enhanced nitric oxide synthase activity but also the denitrosylation by a major denitrosylase, S-nitrosoglutathione reductase (GSNOR) [1]. Abnormal protein S-nitrosylation causes many diseases such as cardiovascular, musculoskeletal, and neurological dysfunction [7]. Furthermore, autophagy, a vacuolar degradation for longlived and aggregate-prone proteins, plays an important role in neurodegeneration. Inhibition of autophagy by S-nitrosylation results in stress-mediated protein aggregation in neurodegenerative diseases [8,9].
S-nitrosylation is also associated with cancer [10]. Potential mechanisms of S-nitrosylation in carcinogenesis are focused on apoptosis and DNA repair [11]. Survival of tumor cell could be induced by inactivation of proapoptotic signaling 2 The Scientific World Journal or activation of antiapoptotic pathways [12]. Inhibition of caspase protease by protein nitrosylation promotes extended survival of malignant cells [13]. For example, nitrosylation of caspase 9 inhibits the mitochondrial pathway of apoptosis in cholangiocarcinoma cell line [14]. Additionally, p53 induces apoptotic cell death and causes cell cycle arrest in response to various stresses [15]. S-nitrosylation of p53 suppresses p53-mediated apoptosis in colon carcinogenesis [16]. Bcl-2, a major anti-apoptotic regulatory protein, was regulated by S-nitrosylation in various carcinoma tissues [11,17,18]. S-nitrosylation is shown to modulate the activity, stability, and cellular localization of key DNA repair proteins, including O(6)-alkylguanine-DNAalkyltransferase (AGT), 8-oxoguanine glycosylase (Ogg1), apurinic-apyrimidinic endonuclease 1 (APE1), and DNAdependent protein kinase catalytic subunit (DNA-PKcs) [19]. Inactivation of AGT by S-nitrosylation is found in hepatocarcinogenesis [20]. Moreover, S-nitrosylated APE1 export from the nucleus to the cytoplasm is described in colon adenomas, breast cancer, and hepatocellular carcinomas [21]. Also, S-nitrosylated DNA-PKcs shows increased transcriptional expression and activity in HEK-293 [22].
It is well known that arsenic, a human carcinogen, showed its chemical carcinogenesis activity by interaction with protein -SH groups. By its -SH binding activity, therefore, arsenic may compromise protein S-nitrosylations in cells. In addition, it is reported that arsenic has a significant effect on NO production in the endothelium [23]. Arsenic remains one of the most concerned environmental toxicants, which has been classified as a group I human carcinogen by the International Agency for Research on Cancer (IARC). Epidemiological studies demonstrated that long-term exposure to inorganic arsenic through either ingestion or inhalation is associated with an increased risk of malignant cancers in the urinary bladder, lung, and especially skin, since arsenic tends to concentrate in keratinocytes [24]. However, the role of S-nitrosylation induced by arsenic in skin carcinogenesis remains unclear. In the present study, we hypothesize that arsenic can alter protein S-nitrosylation and NO production in skin keratinocyte. Our contribution is identifying interactions between arsenic and S-nitrosylation axis in keratinocytes that can provide the novel molecular and pharmacological strategies for potential clinical applications.

Human Keratinocyte Culture.
Freshly obtained prepuce specimens were used to cultivate the primary human cultured keratinocytes. Briefly, normal human prepuce specimens were washed with PBS, then cut into small pieces, and incubated in medium containing 0.25% trypsin overnight at 4 ∘ C. The epidermal sheet was lifted from the dermis using a fine forceps. The epidermal cells were pelleted by centrifugation (500 ×g, 10 min) and dispersed into individual cells by repeated aspiration with a pipette. Isolated keratinocytes must be cultured in commercialized keratinocyte serum free medium (Invitrogen, Carlsbad, USA) at 37 ∘ C in a humidified incubator with 5% CO 2 atmosphere with or without sodium arsenite (Sigma, St. Louis, USA) treatment (10 M). This primary keratinocyte culture protocol using human skin samples has been approved by Institutional Review Board of Kaohsiung Medical University Hospital (KMUH-IRB-960119).

Arsenic Reduces Protein S-Nitrosylation.
Biotin switch is now the most popular methodology for identifying S-nitrosoproteins in various tissues [22]. In the present study, the procedure of biotin switch was simply indicated (Figure 1(a)). By treatment with 10 M of arsenic for 1 hour, at least three groups of protein S-nitrosylation were significantly decreased in keratinocytes (Figure 1(b)). These biotinylated proteins, that is, NO-bound S-nitrosylated proteins, were purified from streptavidin conjugated agarose and then analyzed by LC-MS/MS. Totally, 20 and 13 S-nitrosoproteins were separately identified in the control and arsenic treatments. This was coincident to the decreased NO production and the reduced S-nitrosylated proteins.

Arsenic Decreases the Expressions of eNOS and iNOS.
It has been reported that NO in keratinocytes was produced 4 The Scientific World Journal   . By using ImageMaster software, the proteins with decreased expression (<0.7 fold) were indicated. (b) The relative expression levels of these decreased proteins were statistically calculated from three repeats.
through the activation of eNOS and iNOS [23]. Therefore, in the present study, we examined the expressions of both enzymes in the existence of arsenic. As shown in Figures 2(a) and 2(b), the significant decreases of their expressions were observed.

Arsenic Modulates Translational Proteome in Ker-
atinocytes. In addition to elucidating those posttranslational S-nitrosylated proteins, we further investigated the proteins changed in their expression levels. By using 2-DE, 8 proteins showed a dramatic decrease in the expression level ( Figures  3(a) and 3(b)). These identified proteins are annotated function in (Table 1). More specifically, HNRNPK is a pre-mRNAbinding protein, which has been identified to be involved in arsenic-induced apoptosis in acute myeloid leukemia cells [26]. PSMD13 is proteasome protein regulating the degradation of ubiquitinated proteins, which has been identified in fetal fibroblasts from glutathione deficient mouse using arsenic-induced apoptosis model by cDNA microarray [27]. RPLP0 is a ribosomal protein, in general conditions, constantly expressed in cells (house-keeping gene). The functional interactions between arsenic and RPLP0 required further investigations. CTSD is a protease active in intracellular protein breakdown. Increased CTSD has been identified in arsenic-treated human lymphoblastoid cell lines correlated with autophagy [28]. However, here, we found arsenic treatment decreased CTSD in human keratinocytes. NDUFS3 is a subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). Decreased NDUFS3 has been identified in hepatoma cell line [29]. Indeed, our previous study has identified the dose response between arsenic and NDUFS3 in human keratinocytes. Low concentrations 6 The Scientific World Journal (0.1-1.0 M) of arsenic increased NDUFS3 associated with keratinocyte proliferation. High concentration (5.0 M) of arsenic decreased NDUFS3 correlated with cell death [30]. This decreased NDUFS3 was represented in our current data. ECHS1 is a catalytic enzyme in mitochondrial fatty acid betaoxidation pathway; however, direct evidence between ECHS1 and arsenic is not yet clarified. To summarize, from our proteomic data, altered apoptosis and mitochondrial metabolic functions were the dominant effects of arsenic on human keratinocytes.

Conclusion
In the current study, we concluded that arsenic can compete with nitric oxide in binding cysteine residues so that the protein S-nitrosylation is inhibited. Whether the decreased S-nitrosylation is correlated to arsenic-induced pathology attracts a great attention in the further study. In addition, the homeostasis of cellular apoptosis and mitochondrial dysfunctions is worthy of further study.