Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii

Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins that have been described in yeast, animals, and more recently in plants. In this study, we report the molecular characterization of autophagy in the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the carboxyl-terminal conserved glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such as nutrient limitation, oxidative stress, or the accumulation of misfolded proteins in the endoplasmic reticulum caused an increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy activation. Furthermore, rapamycin-mediated inhibition of the Target of Rapamycin signaling pathway, a major regulator of autophagy in eukaryotes, results in identical effects on CrATG8 and a relocalization of this protein in Chlamydomonas cells similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to stress conditions by inducing autophagy via Target of Rapamycin signaling modulation.Protein turnover is essential for the adaptation of cells to variable environmental conditions. Similar to other eukaryotes, plants have developed two distinct mechanisms to regulate protein degradation, a selective ubiquitin/26S proteasome pathway (Vierstra, 2009) and macroautophagy (hereafter referred to as autophagy), a nonselective membrane-trafficking process (Bassham, 2009). During autophagy, a large number of cytosolic components, including entire organelles, organelle fragments, and protein complexes, are enclosed in bulk within a double-membrane structure known as the autophagosome and delivered to the vacuole/lysosome for degradation to recycle needed nutrients or degrade toxic components (Xie and Klionsky, 2007; Nakatogawa et al., 2009). The autophagosomes appear to arise from isolation membranes usually observed in close proximity to the vacuole called the preautophagosomal structure (PAS). These membranes expand and fuse to encircle portions of the cytoplasm, generating an autophagosome that is targeted to the vacuole. The outer membrane of the autophagosome then fuses with the vacuole membrane, and the remaining vesicle, known as the autophagic body, is finally released to the vacuole for its degradation (Xie and Klionsky, 2007).The evolutionary conservation of autophagy among eukaryotes indicates that structural and regulatory components of this cellular process must be also conserved. Accordingly, a significant number of autophagy-related (ATG) genes that participate in autophagy regulation and autophagosome formation have been identified, initially through genetic approaches in yeast and subsequently in higher eukaryotes, including mammals, insects, protozoa, and plants (Bassham et al., 2006; Bassham, 2007; Meijer et al., 2007). In yeast, two protein conjugation systems composed of the ubiquitin-like proteins ATG8 and ATG12 and the three enzymes ATG3, ATG7, and ATG10 play an essential role in autophagosome formation and seem to be conserved through evolution (Geng and Klionsky, 2008). ATG8 becomes modified with the lipid molecule phosphatidylethanolamine (PE) by the consecutive action of the ATG7 and ATG3 enzymes in a process mechanistically similar to ubiquitination (Ichimura et al., 2000). Prior to this modification, ATG8 must be cleaved by the Cys protease ATG4 to expose a C-terminal Gly residue that is conjugated to PE (Kirisako et al., 2000; Kim et al., 2001). ATG12 becomes covalently attached to the ATG5 protein in a conjugation reaction that is catalyzed by ATG7 and ATG10 (Mizushima et al., 1998). ATG8-PE and ATG12-ATG5 conjugates localize to autophagy-related membranes and are required for the initiation and expansion of autophagosomal membrane and hemifusion of this membrane with the vacuolar membrane (Hanada et al., 2007; Nakatogawa et al., 2007, 2009; Fujita et al., 2008; Geng and Klionsky, 2008; Xie et al., 2008).Our understanding of the autophagy process has substantially increased with the development of specific markers for autophagy. In plants, two markers for autophagosomes have been described, the monodansylcadaverine dye and GFP-ATG8 fusion protein (Yoshimoto et al., 2004; Contento et al., 2005; Thompson et al., 2005). As in other species, binding of ATG8 to autophagosomes has been used to monitor autophagy in plants. In contrast to yeast, where a single ATG8 gene is present, plants appear to contain a small gene family with several ATG8 isoforms, suggesting that autophagy is more complex in these photosynthetic organisms. For example, Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) encode nine and five ATG8 genes, respectively (Doelling et al., 2002; Hanaoka et al., 2002; Ketelaar et al., 2004; Chung et al., 2009). However, despite the high complexity of the ATG8-conjugating system in plants, important findings have been recently reported on the molecular characterization of autophagy using ATG8 as an autophagy marker in these organisms. The use of specific markers for autophagy in plants has revealed that this process is active at a basal level under normal growth and is induced upon nitrogen- or carbon-limiting conditions as well as in response to oxidative stress (Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005, 2007; Chung et al., 2009). Reverse genetic approaches have also been applied to a number of Arabidopsis ATG genes using T-DNA insertional mutants or RNA interference in order to investigate the physiological roles of autophagy in plants. The initial characterization of autophagy-deficient plants demonstrated that the ATG system is not essential under nutrient-rich conditions. However, a detailed analysis of these mutants indicated that autophagy is required for the proper response of the plant to nutrient limitation or pathogen infection. Plants lacking the ATG4, ATG5, ATG7, ATG9, or ATG10 gene display premature leaf senescence and are hypersensitive to nitrogen or carbon limitation (Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005; Phillips et al., 2008). Arabidopsis plants with reduced levels of ATG18, which is required for autophagosome formation, are more sensitive to methyl viologen treatment and accumulate high levels of oxidized proteins, demonstrating that autophagic processes participate in the response of the plant to oxidative stress (Xiong et al., 2005, 2007). Plants deficient in the autophagy genes ATG6/Beclin1, ATG3, ATG7, and ATG9 exhibit unrestricted hypersensitive response lesions in response to pathogen infection (Liu et al., 2005; Hofius et al., 2009). These findings implicate autophagy as a prosurvival mechanism to restrict programmed cell death associated with the pathogen-induced hypersensitive response in plants. Arabidopsis ATG6 has also been shown to mediate pollen germination in a manner independent of autophagy (Fujiki et al., 2007).As mentioned above, autophagy is triggered among other factors by a reduction in the availability of nutrients. This starvation signal is transmitted to the autophagic machinery by important regulatory factors, including the Ser/Thr kinases Target of Rapamycin (TOR), ATG1, and SNF1 and the phosphatidylinositol 3-kinase ATG6/Beclin1 (Diaz-Troya et al., 2008b; Bassham, 2009; Cebollero and Reggiori, 2009). TOR has been identified as a negative regulator of autophagy in yeast, mammals, and fruit flies (Diaz-Troya et al., 2008b). The pharmacological inhibition of TOR by rapamycin leads to autophagy activation through a mechanism that requires the activation of the ATG1 kinase (Kamada et al., 2000). It has been recently demonstrated in mammals and fruit flies that a rapamycin-sensitive TOR signaling complex termed TORC1 directly phosphorylates and inhibits the ATG1 kinase and its regulatory protein ATG13 (Chang and Neufeld, 2009; Hosokawa et al., 2009; Jung et al., 2009). These regulatory proteins are conserved in plants, although except for ATG6 (Liu et al., 2005), there is no direct evidence for regulation of autophagy by these signaling pathways.The unicellular green alga Chlamydomonas reinhardtii has been used as a model for the study of important cellular and metabolic processes in photosynthetic organisms (Harris, 2001). More recently, Chlamydomonas has also been proposed as a useful system for the characterization of the TOR signaling pathway in photosynthetic eukaryotes based on the finding that, unlike plants, Chlamydomonas cell growth is sensitive to rapamycin (Crespo et al., 2005; Diaz-Troya et al., 2008a). Treatment of Chlamydomonas cells with rapamycin results in a pronounced increase of vacuole size that resembles autophagy-like processes (Crespo et al., 2005). However, a role of TOR in autophagy regulation could not be demonstrated due to the absence of an autophagy marker in Chlamydomonas. Actually, no studies have been reported on any autophagy-related protein in green algae, despite the high conservation of ATG genes in Chlamydomonas (Diaz-Troya et al., 2008b).This study reports the molecular and cellular characterization of autophagy in the green alga Chlamydomonas. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) may be used as a specific autophagy marker. Nutrient limitation and cell aging trigger an autophagic response that can be traced as an increase at the level of CrATG8, the detection of modified forms of CrATG8, and a change in the cellular localization of this protein. Furthermore, we demonstrate that autophagy is inhibited by a rapamycin-sensitive TOR cascade in Chlamydomonas.     

Copper response element and Crr1-dependent Ni(2+)-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii

The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni²⁺-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes.     

Tumor suppressor PALB2 maintains redox and mitochondrial homeostasis in the brain and cooperates with ATG7/autophagy to suppress neurodegeneration

The PALB2 tumor suppressor plays key roles in DNA repair and has been implicated in redox homeostasis. Autophagy maintains mitochondrial quality, mitigates oxidative stress and suppresses neurodegeneration. Here we show that Palb2 deletion in the mouse brain leads to mild motor deficits and that co-deletion of Palb2 with the essential autophagy gene Atg7 accelerates and exacerbates neurodegeneration induced by ATG7 loss. Palb2 deletion leads to elevated DNA damage, oxidative stress and mitochondrial markers, especially in Purkinje cells, and co-deletion of Palb2 and Atg7 results in accelerated Purkinje cell loss. Further analyses suggest that the accelerated Purkinje cell loss and severe neurodegeneration in the double deletion mice are due to excessive oxidative stress and mitochondrial dysfunction, rather than DNA damage, and partially dependent on p53 activity. Our studies uncover a role of PALB2 in mitochondrial homeostasis and a cooperation between PALB2 and ATG7/autophagy in maintaining redox and mitochondrial homeostasis essential for neuronal survival.     

Biomarkers of oxidative stress in the fungal strain Humicola lutea under copper exposure

The fungal strain Humicola lutea 103 was used as a model organism to examine the relationship between copper toxicity and oxidative stress in low eukaryotes such as filamentous fungi. Spores or submerged cultures were treated with different copper concentrations and the oxidative stress-inducing agent paraquat (PQ). Oxidative stress biomarkers such as reactive oxygen species (ROS), cyanide-resistant respiration, protein carbonyls, reserve carbohydrates, and antioxidant defence were identified in cells treated or not treated with either copper ions or PQ. Copper inhibited the growth and conidiospore formation of H. lutea 103 in a concentration-dependent manner. This treatment also resulted in increased superoxide anion radical formation. Copper stress was furthermore accompanied by transient accumulation of trehalose and glycogen, as well as increased protein carbonyl content. Compared to control cultures, copper-treated mycelia demonstrated a marked increase in the activity of protective enzymes (superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase). These increased antioxidant enzyme activities were blocked by inhibitors of protein synthesis, suggesting that de novo enzyme formation was involved. Biomarker response to the heavy metal was similar to treatment with known ROS generators such as PQ. The observed hyper-oxidative status and increased oxidative damage suggest a relationship between acute metal treatment and oxidative stress in fungal cells.     

Francisella tularensis Harvests Nutrients Derived via ATG5-Independent Autophagy to Support Intracellular Growth

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.     

ATG4B promotes colorectal cancer growth independent of autophagic flux

Autophagy is reported to suppress tumor proliferation, whereas deficiency of autophagy is associated with tumorigenesis. ATG4B is a deubiquitin-like protease that plays dual roles in the core machinery of autophagy; however, little is known about the role of ATG4B on autophagy and proliferation in tumor cells. In this study, we found that ATG4B knockdown induced autophagic flux and reduced CCND1 expression to inhibit G₁/S phase transition of cell cycle in colorectal cancer cell lines, indicating functional dominance of ATG4B on autophagy inhibition and tumor proliferation in cancer cells. Interestingly, based on the genetic and pharmacological ablation of autophagy, the growth arrest induced by silencing ATG4B was independent of autophagic flux. Moreover, dephosphorylation of MTOR was involved in reduced CCND1 expression and G₁/S phase transition in both cells and xenograft tumors with depletion of ATG4B. Furthermore, ATG4B expression was significantly increased in tumor cells of colorectal cancer patients compared with adjacent normal cells. The elevated expression of ATG4B was highly correlated with CCND1 expression, consistently supporting the notion that ATG4B might contribute to MTOR-CCND1 signaling for G₁/S phase transition in colorectal cancer cells. Thus, we report that ATG4B independently plays a role as a positive regulator on tumor proliferation and a negative regulator on autophagy in colorectal cancer cells. These results suggest that ATG4B is a potential biomarker and drug target for cancer therapy.     

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.     

The Essential Autophagy Gene ATG7 Modulates Organ Fibrosis via Regulation of Endothelial-to-Mesenchymal Transition

Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFβ signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.     

Molecular identification,differential expression and protective roles of iron/manganese superoxide dismutases in the green algae Closterium ehrenbergii against metal stress

The green microalgae Closterium ehrenbergii is an ideal organism for ecotoxicology assessments; however, its toxicogenomics has been insufficiently examined. Here, we identified three iron/manganese superoxide dismutase (SOD) genes (designated as CeFeSOD1, CeFeSOD2, and CeMnSOD) from C. ehrenbergii and examined their expressional patterns for four metals (iron, manganese, copper, and nickel). These genes encoded 362, 224, and 245 amino acids, respectively; signal-peptide analysis showed that they were differentially located in chloroplasts, cytosol, or mitochondria. Real-time PCRs revealed differential expression patterns according to metal and doses. Interestingly, CeSODs displayed no noticeable changes to treatment with their corresponding cofactor metals, iron or manganese, even at high doses. However, they were obviously up-regulated under toxic metal (copper and nickel) exposure, exhibiting approximately 10.8- and 4.4-fold increases, respectively. Copper (0.2 mg/L) dramatically stimulated intracellular reactive oxygen species (ROS) formation, increased SOD activity, and reduced photosynthetic efficiency in C. ehrenbergii. These results suggest that CeFeSODs and CeMnSOD might be involved in protecting cells against damage and oxidative stress caused by non-cofactor metals, such as copper and nickel. These genes were sensitively responsive at levels well below the EC₅₀, showing that they can be used as molecular biomarkers to assess the toxicity of specific metal contaminants.     

The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16⁻ and ATG9⁻/16⁻ cells and compare them to the previously reported ATG9⁻ mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9⁻ and ATG16⁻ cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9⁻/16⁻ double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9⁻/16⁻ cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9⁻/16⁻ cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.     

Proteomic analysis of the response of an acidophilic strain of Chlamydomonas sp. (Chlorophyta) to natural metal‐rich water

A proteomic approach including 2‐DE and MALDI‐TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas sp. isolated from an extreme acidic environment, Río Tinto (southwest Spain). We have analyzed the soluble proteome obtained from whole cells growing on metal‐rich natural acidic water from the river in comparison with the same strain growing in artificial BG‐11 media. The most drastic effect was the decrease in the abundance of the ribulose‐1,5‐biphosphate carboxylase as well as other enzymes related to photosynthesis. However, phytochrome B, phosphoribulokinase, and phosphoglycerate kinase were upregulated when cells were grown in metal‐rich acidic water. Besides, increased accumulation of two Hsps, Hsp70 and Hsp90 as well as other stress‐related enzymes were also found in the cells growing in natural acidic water. These results suggest that naturally occurring metal‐rich water induces a stress response in acidophilic Chlamydomonas forcing algal cells to reorganize their metabolic pathways as an adaptive response to these environmental conditions.     

Hierarchal Autophagic Divergence of Hematopoietic System

Autophagy is integral to hematopoiesis and protects against leukemogenesis. However, the fundamentals of the required molecular machinery have yet to be fully explored. Using conditional mouse models to create strategic defects in the hematopoietic hierarchy, we have shown that recovery capacities in stem cells and somatic cells differ if autophagy is impaired or flawed. An in vivo Atg7 deletion in hematopoietic stem cells completely ablates the autophagic response, leading to irreversible and ultimately lethal hematopoiesis. However, while no adverse phenotype is manifested in vivo by Atg7-deficient myeloid cells, they maintain active autophagy that is sensitive to brefeldin A, an inhibitor targeting Golgi-derived membranes destined for autophagosome formation in alternative autophagy. Removing Rab9, a key regulatory protein, in alternative autophagy, disables autophagy altogether in Atg7-deficient macrophages. Functional analysis indicates that ATG7-dependent canonical autophagy is physiologically active in both hematopoietic stem cells and in terminally differentiated hematopoietic cells; however, only terminally differentiated cells such as macrophages are rescued by alternative autophagy if canonical autophagy is ineffective. Thus, it appears that hematopoietic stem cells rely solely on ATG7-dependent canonical autophagy, whereas terminally differentiated or somatic cells are capable of alternative autophagy in the event that ATG7-mediated autophagy is dysfunctional. These findings offer new insight into the transformational trajectory of hematopoietic stem cells, which in our view renders the autophagic machinery in stem cells more vulnerable to disruption.     

Coordination of Ni2+ and Cu2+ to metal ion binding domains of E. coli SlyD protein

The C-terminal region of Escherichia coli SlyD is unstructured and extremely rich in potential metal-binding amino acids, especially in histidine residues. SlyD is able to bind two to seven nickel ions per molecule, in a variety of coordination geometries and coordination numbers. This protein contributes to the insertion of nickel into the hydrogenase precursor protein and it has a peptidyl-prolyl cis/trans-isomerase activity which can be regulated through nickel ions. This inspired us to undertake systematic studies on the coordination ability of two histidine-rich peptides from the C-terminus of the SlyD protein with nickel. Also, it is known that histidine-rich regions are part of a Cu^2 + binding domain involved in copper uptake under conditions of metal starvation in vivo in other bacteria. For this reason we decided to examine the complex formation of Ac-AHGHVHGAHDHHHD-NH₂ and Ac-GHGHDHGHEHG-NH₂ fragments with copper ions, which are also reference metal ions in this study. Experiments were performed in a DMSO/water 30:70 solvent. The Ac-AHGHVHGAHDHHHD-NH₂ and Ac-GHGHDHGHEHG-NH₂ fragments were synthesized and their interactions with Ni^2 + and Cu^2 + ions were studied by potentiometric, mass spectrometric, UV-vis, CD, EPR, and NMR spectroscopic techniques in solution. The results show that the Ac-GHGHDHGHEHG-NH₂ fragment forms equimolar complexes with both nickel and copper ions. At physiological pH, the metal ion is bound only through nitrogens from imidazole sidechain of histidine residues. On the contrary, Ac-AHGHVHGAHDHHHD-NH₂ binds 2 metal ions per molecule, at pH range 5 to 7, even if the 1:2 metal:peptide ratios were used. NMR studies indicate the involvement of all His residues in this pH-range in metal binding of the latter peptide. At higher pH, the stoichiometry changes to 1:1 and the His residues are displaced by amide nitrogens.