br Identification of the croaker mAR cDNA
Identification of the croaker mAR cDNA Testosterone (T) was observed to alter steroidogenesis through a nongenomic mechanism (not blocked by the transcription inhibitor, actinomycin D) in Atlantic croaker ovarian tissues (Braun and Thomas, 2003). This action of T was mimicked by T conjugated to BSA which indicates the action is mediated at the cell surface, and therefore suggests the presence of a mAR. Moreover, the androgen specificity of the androgen action differed from the steroid specificity of the nAR in this species, which suggests the presence of a novel mAR (Braun and Thomas, 2003, Sperry and Thomas, 1999). Subsequently, extensive biochemical characterization of androgen binding to croaker ovarian membranes clearly demonstrated that its binding characteristics differed from that of the nAR (Braun and Thomas, 2004). A modification of the successful procedure used to discover the nucleotide sequence of spotted seatrout membrane progestin receptor alpha (mPRα) cDNA (Thomas et al., 2002, Zhu et al., 2003) was used to identify the croaker mAR. It involved solubilization of the receptor and partial purification of the protein on a DEAE column. A single protein band (40kDa) in the [3H]-T binding fraction was detected by PAGE which was used to generate antisera in mice. The polyclonal mouse antibody was then used in a receptor capture assay to screen a croaker ovarian cDNA library. A positive clone was identified with an open reading frame of 933bp encoding a protein with a predicted molecular mass of 33kDa (Berg et al., 2014). Sequence analysis of the croaker cDNA showed it had high nucleotide and amino Anti-Inflammatory Peptide 1 sequence identities with ZIP9s which are members the ZIP (SLC39A) zinc transporter family. The croaker ZIP9 is closely related to teleost ZIP9s with 80–95% nucleotide and amino acid identities. However, topology analysis predicted croaker ZIP9 has 7 transmembrane domains, whereas other vertebrate ZIP9s are predicted to have 8 transmembrane domains. Further structural analysis showed that a single isoleucine amino acid at position 38 from the N-terminal end of the croaker protein resulted in a prediction of 7 TMs. It is concluded from these analyses that the croaker cDNA is a ZIP9.
Zinc functions and zinc transport Zinc is an essential metal that has many critical functions in vertebrates (Maret, 2013) and exerts pleiotropic, zinc-specific, and often cell-specific effects on morphogenesis, growth, differentiation and development (Kim et al., 2010). Zinc is an integral component of 10% (∼3000) of human proteins and is essential for their proper structure and function (Maret, 2013). Zinc acts as a catalytic cofactor for over 300 enzymes and also acts as an intracellular signaling molecule (Kochanczyk et al., 2015). Furthermore, zinc has been shown to be involved in the regulation of important processes such as apoptosis, cell proliferation and growth (Chimienti et al., 2001). Zinc is transported in the extracellular space as a highly charged molecule and cannot cross over the plasma membrane by passive diffusion. Therefore, a large number of different zinc transporter proteins are located in the cell and mitochondrial membranes in order to transport zinc across these membranes into the cytoplasm. Zinc imbalance in physiological systems can lead to numerous adverse effects. In humans, insufficient levels of zinc have been shown to induce a wide variety of diseases including growth retardation, asthma, diabetes, cancer, and Alzheimer’s disease (Devirgiliis et al., 2007, Jansen et al., 2009, Kambe et al., 2014). In contrast, high levels of zinc have also been shown to have deleterious effects on the health of vertebrate animals including hemolytic anemia, hepatic dysfunction, and acute renal failure (Maret and Sandstead, 2006, Cummings, 2009). Because zinc imbalance has deleterious impacts on many critical biological processes, the regulation of zinc homeostasis is crucial for maintaining physiological functions in vertebrates. Zinc homeostasis is regulated by a superfamily of proteins responsible for maintaining an equilibrium of zinc efflux, uptake and storage.