Introduction
Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of one molecule of pyrophosphate (PPi) to two molecules of phosphates (Pi). PPases are ubiquitous enzymes present in all cell types. PPases include membrane-bound V-PPases (vacuolar H+-translocating PPases) and soluble PPases. V-PPases use pyrophosphate (instead of ATP) as an energy source to transport H+ or Na+ across membranes in plants and some bacteria, archaea, and parasitic protists [1, 2]. Soluble PPases are divided into three families that differ in their sequences and structures [3-5]. PPases from families II and III are only found in prokaryotes, whereas Family I PPases exist in all kingdoms of life. Family I PPases are single-domain, Mg2+ dependent enzymes that exist as hexamers in prokaryotes and dimers in eukaryotes.
PPi is a metabolic product that is generated by many biosynthetic reactions for the synthesis of various important biomolecules, including proteins, nucleic acids, polysaccharides, and fatty acids [6]. The conversion of PPi to Pi by soluble PPases is an essential cellular process. On the one hand, the reaction is critical for maintaining cellular PPi homeostasis; on the other hand, the reaction provides a thermodynamic pull for the PPi-generating biosynthetic reactions because enzymatic hydrolysis of PPi is highly exergonic.
Soluble PPases are essential enzymes for growth and development. In E. coli, the ppa gene for PPase is essential for cell growth [7]. In fermenting yeast, PPase defects lead to cell cycle arrest and autophagic cell death [8]. In C. elegans, the PPase enzyme PYP-1 is required for larval development and intestinal function [9, 10]. In mammals, the PPase enzyme PPA1 has been reported to have the potential to regulate neurite growth in mouse neuroblastoma cells [11], to induce type I collagen synthesis and stimulate calcification by osteoblasts [12]. Increased expression and activity of cytosolic PPA1 in the liver of rat and mouse has been correlated with aging [13, 14].
Human possess two Family I PPases, PPA1 (289 aa) and PPA2 (305 aa) [15]. PPA2 is localized to the mitochondrion. The two human PPases share ~70% homology. PPA1 is a ubiquitous enzyme found in all tissues. It is found that PPA1 acts largely as a housekeeping gene [15], presumably due to its essential role in energy metabolism. More recently, the role of PPA1 in tumorigenesis has emerged [16-19]. Upregulated expression of PPA1 has been linked to many human malignant tumors including colorectal cancer [20-22], diffuse large B-cell lymphoma (DLBCL) [23], lung adenocarcinoma [17, 24], prostate cancer [25], hepatocellular carcinoma [19], breast cancer [26], gastric cancer[27, 28], intrahepatic cholangiocarcinoma (ICC) [29], and ovarian cancer [24, 30, 31]. In laryngeal squamous cell carcinoma (LSCC), metastases tumors showed an intense increase of PPA1 compared to primary tumors [32]. These data indicate that PPA1 protein is a potential prognostic biomarker for certain cancers.
The exact mechanism for the involvement of PPA1 overexpression in tumorigenesis is not fully understood. Dysregulated metabolism is an important hallmark of cancer cells [33]. Metabolic alterations caused by PPA1 overexpression may provide the extra energy and biomaterials for the cancer cells to maintain their high proliferation rate. In addition, it is found that PPA1 can directly dephosphorylate phosphorylated JNK1 (c-Jun N-terminal kinase) [22]. Functioning as a JNK phosphatase, PPA1 may regulate the activities of downstream effectors (including p53, β –catenin, Bcl-2, and Caspase-3) that control cell proliferation and apoptosis. In consistence with this speculated mechanism, we previously found that PPA1 overexpression in epithelial ovarian carcinoma (EOC) promoted the dephosphorylation and translocation of β-catenin [31]. In another study on EOC [30], it was found that PPA1 overexpression caused the decrease in p53 and increase in β-catenin expression level. Conversely, PPA1 knockdown led to the increase of p53 and decrease of β-catenin level.
While PPA1 overexpression enhances migration, invasion and proliferation of cancer cells [27], PPA1 knockdown (by RNAi mediated silencing) is shown to induce apoptosis and decrease proliferation in various cancer cells [22-24, 30, 31]. Results from these studies suggest that strategies for PPA1 down-regulation may have therapeutic potential for the treatment of certain cancers. PPA1 down-regulation might be achieved by mRNA knockdown through RNAi or RNA-targeting CRISPR systems. Alternatively, the PPA1 protein might represent a valuable target for the development of specific small-molecule inhibitors as anti-cancer drugs. Efforts had been made to target PPases from pathogenic organisms for small-molecule drug development. Anti-PPase small molecules were shown to have promising antibiotic activity against drug resistant strains of Staphylococcus aureus [34]. Small molecule inhibitors of the VSP1 (vacuolar soluble pyrophosphatase 1) protein from Trypanosoma brucei, the causative agent of African trypanosomiasis (HAT), provided a 40% protection from death in a mouse model of T. brucei infection [35]. Inhibition of PPase activity could also be achieved by targeting allosteric sites, as demonstrated by the discovery of allosteric and selective inhibitors of PPase from Mycobacterium tuberculosis [36].
In spite of the biological and physiopathological importance of human PPA1, there is no detailed study on the structure and catalytic mechanisms of this enzyme. Extensive structural and biochemical studies had been reported on Family I PPases from Escherichia coli (E-PPase) [37-39] and Saccharomyces cerevisiae (Y-PPase) [40-45]. X-ray crystallographic studies had also been carried out on soluble PPases from other species, including Thermus thermophilus (T-PPase) [46], Pyrococcus horikoshii (Pho-PPase) [47], human parasites Plasmodium falciparum (Pf-PPase) and Toxoplasma gondii (Tg-PPase) [48], Mycobacterium tuberculosis (Mt-PPase) [36, 49], Trypanosoma brucei brucei (also called vacuolar soluble protein TbbVSP1) [50], and S. japonicum (Sj-PPase, not published).
In this paper, we present the crystal structure of human PPA1. We also carried out modeling studies of PPA1 in complex with JNK derived peptides containing phosphotyrosine and phosphothreonine residues.