Arsenic binding to gap junction protein connexin 43 alters cell to cell communication and tissue homeostasis

Hussain, Afaq (2018) Arsenic binding to gap junction protein connexin 43 alters cell to cell communication and tissue homeostasis. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Chronic exposure to arsenic pollution in groundwater is one of the largest environmental health hazards in the world. The toxicity of trivalent arsenicals primarily occurs due to its interaction with sulfhydryl groups in proteins. Arsenic binding to protein may alter its conformation, function, as well as its recruitment and interaction with other functional proteins leading to tissue damage. Therefore, there has been much emphasis on studies of arsenic bound proteins, for the purpose of understanding the origins of toxicity and to explore therapeutics. Here we study the dynamic effect of arsenic on connexin 43 (Cx43). Cx43 has nine Cys residues, six of which are in the extracellular domain and three in the intracellular domain, whose alteration deeply perturbs the cell to cell communication vital for maintaining tissue homeostasis. Gap junction proteins are known to have a tumor suppressive role and their sustained down-regulation can confer a loss of tumor suppressive action, leading to the progression of carcinogenesis. In this study, we focused on the effects of arsenic toxicity on gap junction proteins. In silico molecular modeling, in vitro cell culture, and in vivo mouse studies comparing arsenic-treated and untreated conditions show distinct results. Gap junction communication is severely disrupted by arsenic due to reduced availability of unaltered Cx43 in the membrane-bound form. In silico and Inductively Coupled Plasma Mass Spectrometry studies revealed the interaction of arsenic with Cx43 possibly through surface exposed cysteines, thereby capping the thiol groups that form disulfide bonds in the tertiary structure. This leads to disruption of Cx43 oligomerization, and altered Cx43 is incompetent for transportation to the membrane surface, often forming aggregates primarily localizing in the endoplasmic reticulum. In vivo studies show a reduction in expression of Cx43 in lung and kidney tissues of Swiss Albino mice post arsenic exposure through drinking water. The decrease in Cx43 mediated gap junctions was found with a concomitant abnormal compaction of lung tissue, decreased staining in the liver tissues and cortical chronic nephritis in kidney tissues. Loss of functional Cx43 on the cell surface has a deleterious effect on cellular homeostasis leading to selective vulnerability to cell death and tissue damage. The Connexin expression and/or function of connexins have been observed to be decreased in most tumor cell lines and in solid tissue tumors, including melanomas. Our study revealed that arsenic causes disruption of gap junction intercellular communication both in vivo and in vitro. This is the first study of its kind, where the mechanism of disruption of gap junction intercellular communication by arsenic has been deciphered. A better understanding of the mechanism(s) of action of arsenic-induced alteration of GJIC and alteration of gap junction proteins (Cxs) will help in deciphering the risks associated with exposure to arsenic.

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