Decreased Erythrocyte CCS Content is a Biomarker of Copper Overload in Rats

Copper (Cu) is an essential trace metal that is toxic in excess. It is therefore important to be able to accurately assess Cu deficiency or overload. Cu chaperone for Cu/Zn superoxide dismutase (CCS) protein expression is elevated in tissues of Cu-deficient animals. Increased CCS content in erythrocytes is particularly sensitive to decreased Cu status. Given the lack of a non-invasive, sensitive and specific biomarker for the assessment of Cu excess, we investigated whether CCS expression in erythrocytes reflects Cu overload. Rats were fed diets containing normal or high levels of Cu for 13 weeks. Diets contained 6.3 ± 0.6 (Cu-N), 985 ± 14 (Cu-1000) or 1944 ± 19 (Cu-2000) mg Cu/kg diet. Rats showed a variable response to the high Cu diets. Some rats showed severe Cu toxicity, while other rats showed no visible signs of toxicity and grew normally. Also, some rats had high levels of Cu in liver, whereas others had liver Cu concentrations within the normal range. Erythrocyte CCS protein expression was 30% lower in Cu-2000 rats compared to Cu-N rats (P < 0.05). Notably, only rats that accumulated high levels of Cu in liver had lower erythrocyte CCS (47% reduction, P < 0.05) compared to rats fed normal levels of Cu. Together, these data indicate that decreased erythrocyte CCS content is associated with Cu overload in rats and should be evaluated further as a potential biomarker for assessing Cu excess in humans

Adaptor Protein Sorting Nexin 17 Regulates Amyloid Precursor Protein Trafficking and Processing in the Early Endosomes*

Accumulation of extracellular amyloid β peptide (Aβ), generated from amyloid precursor protein (APP) processing by β- and γ-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Aβ from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in Aβ production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to Aβ. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to Aβ

Endogenous tassel-specific small RNAs-mediated RNA interference enables a novel glyphosate-inducible male sterility system for commercial production of hybrid seed in Zea mays L.

Hybrid crops produce higher yields than their inbred parents due to heterosis. For high purity of hybrid seeds, it is critical to eliminate self-pollination. Manual or mechanical removal of male parts (such as detasseling in maize) is labor-intensive, fuel and time-consuming, and can cause physical damage to female plants, resulting in significant seed yield reductions. Many male-sterility systems either require a maintainer for male-sterile line propagation or are often affected by environmental factors. Roundup® Hybridization System (RHS) utilizes glyphosate to induce male sterility, which effectively eliminates the need for maintainer lines and removal of male parts for commercial hybrid seed production. The first-generation RHS (RHS1) is based on low expression of a glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in pollen. This report presents the second-generation RHS (RHS2) technology built on RNA interference (RNAi) combined with CP4 EPSPS. It utilizes maize endogenous male tissue-specific small interfering RNAs (mts-siRNAs) to trigger cleavage of the CP4 EPSPS mRNA specifically in tassels, resulting in glyphosate-sensitive male cells due to lack of the CP4 EPSPS protein. Male sterility is then induced by glyphosate application at the stages critical for pollen development, and the male-sterile plants are used as the female parent to produce hybrid seed. The endogenous mts-siRNAs are conserved across maize germplasms, and the inducible male sterility was replicated in representative germplasms through introgression of a CP4 EPSPS transgene containing the mts-siRNA target sequence. This technology combines the relative simplicity and convenience of a systemic herbicide spray methodology with targeted protein expression to create an inducible male sterility system for industrial production of row crop hybrid seeds in an environmentally-independent manner

Copper Transporter 2 Content Is Lower in Liver and Heart of Copper-Deficient Rats

Copper (Cu) transporter 2 (Ctr2) is a transmembrane protein that transports Cu across cell membranes and increases cytosolic Cu levels. Experiments using cell lines have suggested that Ctr2 expression is regulated by Cu status. The importance of changes in Ctr2 expression is underscored by recent studies demonstrating that lower Ctr2 content in cells increases the cellular uptake of platinum-containing cancer drugs and toxicity to the drugs. In this study, we examined whether Ctr2 expression is altered by a nutritional Cu deficiency in vivo. Ctr2 mRNA and protein in liver and heart from rats fed a normal (Cu-N), moderately deficient (Cu-M) or deficient (Cu-D) Cu diet was measured. Rats fed the Cu-deficient diets showed a dose-dependent decrease in liver Ctr2 protein compared to Cu-N rats. Ctr2 protein was 42% and 85% lower in Cu-M and Cu-D rats, respectively. Liver Ctr2 mRNA was 50% lower in Cu-D rats and unaffected in Cu-M rats. In heart, Ctr2 protein was only lower in Cu-D rats (46% lower). These data show that Cu deficiency decreases Ctr2 content in vivo

Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1*

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals