Enhanced Cellular Uptake and Photodynamic Effect with Amphiphilic Fluorinated Porphyrins: The Role of Sulfoester Groups and the Nature of Reactive Oxygen Species.

A class of amphiphilic photosensitizers for photodynamic therapy (PDT) was developed. Sulfonate esters of modified porphyrins bearing-F substituents in the ortho positions of the phenyl rings have adequate properties for PDT, including absorption in the red, increased cellular uptake, favorable intracellular localization, low cytotoxicity, and high phototoxicity against A549 (human lung adenocarcinoma) and CT26 (murine colon carcinoma) cells. Moreover, the role of type I and type II photochemical processes was assessed by fluorescent probes specific for various reactive oxygen species (ROS). The photodynamic effect is improved not only by enhanced cellular uptake but also by the high generation of both singlet oxygen and oxygen-centered radicals. All of the presented results support the idea that the rational design of photosensitizers for PDT can be further improved by better understanding the determinants affecting its therapeutic efficiency and explain how smart structural modifications can make them suitable photosensitizers for application in PDT.

Resistance of Cu(Aß4-16) to Copper Capture by Metallothionein-3 Supports a Function for the Aß4-42 Peptide as a Synaptic Cu(II) Scavenger.

Aß4-42 is a major species of Aß peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu(II) with an affinity approximately 3000 times higher than the commonly studied Aß1-42 and Aß1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu(II) from Aß1-40 when acting in its native Zn7 MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7 MT-3 against Aß toxicity. In this work, we used the truncated model peptides Aß1-16 and Aß4-16 to demonstrate that the high-affinity Cu(II) complex of Aß4-16 is resistant to Zn7 MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aß4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aß4-42 as a Cu(II) scavenger in the synaptic cleft.

Local and systemic regulation of sulfur homeostasis in roots of Arabidopsis thaliana.

Nutrients are limiting for plant growth and vigour. Hence, nutrient uptake and homeostasis must be adjusted to the needs of the plant according to developmental stages and environmental conditions. A split-root system was applied to analyse the systemic and local response of Arabidopsis thaliana to sulfur starvation. Arabidopsis thaliana plants in which only one root half was starved while the other root half was supplied with sulfate were analysed at the metabolic and transcriptional level. No systemic induction of sulfate uptake or expression of sulfate starvation marker genes was observed in split-roots sufficiently supplied with sulfate. Our data suggest that no activation of sulfur uptake takes part in sulfur-supplied root patches when the general sulfur status declines. When comparing roots of fully sulfate-starved plants with sulfate-starved split-root roots, expression of several potentially OAS responsive genes was attenuated in split-roots depending on the shoot sulfate status and the local root O-acetylserine concentration. In contrast, high-affinity sulfate transporters displayed similar expression in sulphate-starved split-roots and the corresponding controls. Feeding of (35) SO(4) (2-) to the shoot or to either part of a split-root system revealed that sulfate is the most prominent mobile sulfur-containing compound within the plant. Hence, we postulate a model whereby the soil sulfate availability regulates the sulfate uptake system of roots while the shoot sulfur status modulates the local O-acetylserine response in the root by passive 'plant sulfur status-dependent' transport of sulfate.

From methodological limitations to the function of metallothioneins - a guide to approaches for determining weak, moderate, and tight affinity zinc sites.

Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low-picomolar affinity in the α and ß domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nanomolar to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4-6MTs species in cellular zinc buffering in a wide picomolar to nanomolar range of free Zn(II) concentrations. Until today, there was no clear agreement on the presence of differentiated or only tight zinc sites. Here, we present a series of spectroscopic, mass spectrometry-based, and enzymatic competition experiments that reveal how weak, moderate, or high-affinity ligands interact with human MT2, with special attention to the determination of Zn(II) affinities. The results show that the simplification of the stability model is the major reason for determining significantly different stability data that obscured the actual MTs function. Therefore, we emphasize that different metal affinities are the single most important reason for their presumed function, which changed over the years from tight binding and, thus, storage to one that is highly dynamic.

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2.

Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M4S11 (α) and M3S9 (ß) clusters. Recent discoveries indicate that Zn(ii) ions are bound with MT2 with the range from nano- to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(ii)-depleted MT2 species. These forms serve as Zn(ii) donors or acceptors and are formed under varying cellular free Zn(ii) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(ii) binding mechanism in human MT2 with high resolution with respect to particular Zn(ii) binding sites, and provide structural insights into Zn(ii)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the α-domain is formed sequentially in the first stages, followed by the formation of the ß-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the ß-domain upon applying the seventh Zn(ii) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(ii) ion associates with the ß-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(ii) binding to the isolated ß-domain differs significantly from the whole protein, which explains its previously observed different Zn(ii)-binding properties. MD results obtained for Zn(ii) binding to the whole protein and isolated ß-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.

Relationship between the architecture of zinc coordination and zinc binding affinity in proteins--insights into zinc regulation.

Zinc proteins are an integral component of the proteome of all domains of life. Zn(II), one of the most widespread transition elements, serves multiple functions in proteins, such as a catalytic co-factor, a structural center and a signaling component. The mechanism by which proteins associate with and dissociate from Zn(II) and the factors that modulate their affinity and stability remain incompletely understood. In this article, we aim to address how zinc binding sites present in proteins differ in their architecture and how their structural arrangement is associated with protein function, thermodynamic and kinetic stability, reactivity, as well as zinc-dependent regulation. Here, we emphasize that the concentration-dependent functionality of the interprotein zinc binding site may serve as another factor regulating the relationship between cellular Zn(II) availability and protein function.