Design and characterization of α-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination

α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind α-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys4,10, d-Phe7–α-MSH4-13 analog. Structural analysis of the Re–peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate–metal–thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re–peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re–α-MSH analog. Characterization of the second-generation Re–peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding 99mTc- and 188ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine. In vivo, the 99mTcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of α-MSH analogs via 99mTc and 188Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties.

hCTR1: A human gene for copper uptake identified by complementation in yeast

The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.

The Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants1

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd2+ uptake as a model system. Radiotracer techniques were used to quantify unidirectional 109Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for 109Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd2+ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd2+ influx across the root-cell plasma membrane. The Cd2+ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd2+ influx, whereas the maximum initial velocity for Cd2+ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H+-ATPase did not play a role in the enhanced Cd2+ uptake. Expression studies with the Fe2+ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd2+ and Zn2+, in addition to Fe2+.

A novel iron-regulated metal transporter from plants identified by functional expression in yeast.

Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans. In Arabidopsis, IRT1 is expressed in roots, is induced by iron deficiency, and has altered regulation in plant lines bearing mutations that affect the iron uptake system. These results provide the first molecular insight into iron transport by plants.

The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation.

The yeast Saccharomyces cerevisiae has two separate systems for zinc uptake. One system has high affinity for substrate and is induced in zinc-deficient cells. The second system has lower affinity and is not highly regulated by zinc status. The ZRT1 gene encodes the transporter for the high-affinity system, called Zrt1p. The predicted amino acid sequence of Zrt1p is similar to that of Irt1p, a probable Fe(II) transporter from Arabidopsis thaliana. Like Irt1p, Zrt1p contains eight potential transmembrane domains and a possible metal-binding domain. Consistent with the proposed role of ZRT1 in zinc uptake, overexpressing this gene increased high-affinity uptake activity, whereas disrupting it eliminated that activity and resulted in poor growth of the mutant in zinc-limited media. Furthermore, ZRT1 mRNA levels and uptake activity were closely correlated, as was zinc-limited induction of a ZRT1-lacZ fusion. These results suggest that ZRT1 is regulated at the transcriptional level by the intracellular concentration of zinc. ZRT1 is an additional member of a growing family of metal transport proteins.