Ewa Kurowska

Binding of transition metal ions to albumin: sites, affinities and rates.

BACKGROUND Serum albumin is the most abundant protein in the blood and cerebrospinal fluid and plays a fundamental role in the distribution of essential transition metal ions in the human body. Human serum albumin (HSA) is an important physiological transporter of the essential metal ions Cu(2+), and Zn(2+) in the bloodstream. Its binding of metals like Ni(2+), Co(2+), or Cd(2+) can occur in vivo, but is only of toxicological relevance. Moreover, HSA is one of the main targets and hence most studied binding protein for metallodrugs based on complexes with Au, Pt and V. SCOPE OF REVIEW We discuss i) the four metal-binding sites so far described on HSA, their localization and metal preference, ii) the binding of the metal ions mentioned above, i.e. their stability constants and association/dissociation rates, their coordination chemistry and their selectivity versus the four binding sites iii) the methodology applied to study issues of items i and ii and iv) oligopeptide models of the N-terminal binding site. MAJOR CONCLUSIONS Albumin has four partially selective metal binding sites with well-defined metal preferences. It is an important regulator of the blood transport of physiological Cu(II) and Zn(II) and toxic Ni(II) and Cd(II). It is also an important target for metal-based drugs containing Pt(II), V(IV)O, and Au(I). GENERAL SIGNIFICANCE The thorough understanding of metal binding properties of serum albumin, including the competition of various metal ions for specific binding sites is important for biomedical issues, such as new disease markers and design of metal-based drugs. This article is part of a Special Issue entitled Serum Albumin.

Myelin Recovery in Multiple Sclerosis: The Challenge of Remyelination

Multiple sclerosis (MS) is the most common demyelinating and an autoimmune disease of the central nervous system characterized by immune-mediated myelin and axonal damage, and chronic axonal loss attributable to the absence of myelin sheaths. T cell subsets (Th1, Th2, Th17, CD8+, NKT, CD4+CD25+ T regulatory cells) and B cells are involved in this disorder, thus new MS therapies seek damage prevention by resetting multiple components of the immune system. The currently approved therapies are immunoregulatory and reduce the number and rate of lesion formation but are only partially effective. This review summarizes current understanding of the processes at issue: myelination, demyelination and remyelination—with emphasis upon myelin composition/architecture and oligodendrocyte maturation and differentiation. The translational options target oligodendrocyte protection and myelin repair in animal models and assess their relevance in human. Remyelination may be enhanced by signals that promote myelin formation and repair. The crucial question of why remyelination fails is approached is several ways by examining the role in remyelination of available MS medications and avenues being actively pursued to promote remyelination including: (i) cytokine-based immune-intervention (targeting calpain inhibition), (ii) antigen-based immunomodulation (targeting glycolipid-reactive iNKT cells and sphingoid mediated inflammation) and (iii) recombinant monoclonal antibodies-induced remyelination.

Revised Coordination Model and Stability Constants of Cu(II) Complexes of Tris Buffer

2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio.

Salivary histatin-5, a physiologically relevant ligand for Ni(II) ions

Histatins are a family of human salivary antimicrobial peptides. Histatin-5 (Hst-5, DSHAKRHHGYKRKFHEKHHSHRGY), a prominent member of this family contains an albumin-like, N-terminal Asp-Ser-His sequence, known to bind a Ni(II) ion in a square-planar geometry. Nickel is a strong allergen, and oral exposure to Ni(II) ions can elicit allergic reaction in sensitized persons. In contrast, prior oral exposure to nickel in non-sensitized persons can prevent sensitization. The fate of Ni(II) ions in saliva is obviously important for these processes, yet little is known about it. Using potentiometry, UV-visible titrations and circular dichroism, we determined stability constants for Ni(II) complexes of Hst-5 and two truncated analogs, 5Hst-5 (DSHAK) and 10Hst-5 (DSHAKRHHGY). The conditional binding constant at pH 7.4 for Hst-5 was 10(7.5±0.2), compared to the corresponding value for albumin, 10(6.8±0.3) (M. Sokołowska, A. Krężel, M. Dyba, Z. Szewczuk, W. Bal, Eur. J. Biochem. 269 (2002) 1323-1331). These values indicate that Hst-5 binds Ni(II) five times stronger than HSA. The simulated competition for Ni(II) between Hst-5 and albumin shows that significant amounts of Ni(II) ions may be carried by Hst-5 in vivo. Therefore, Hst-5 and other histatins should be considered as factors in nickel allergy and other forms of nickel toxicity.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated “donor” cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer.

The final frontier of pH and the undiscovered country beyond.

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K w = [H+]×[OH−].We used K w [in a reversed way] to calculate the number of undissociated H2O molecules required by this equilibrium constant to yield at least one of its daughter ions, H+ or OH− at a given pH. In this way we obtained a formula that relates pH to the minimal volume VpH required to provide a physical meaning to K w, (where N A is Avogadro’s number). For example, at pH 7 (neutral at 25°C) VpH = 16.6 aL. Any deviation from neutral pH results in a larger VpH value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H+ ions at equilibrium, thus the definition of pH based on K w is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H+ ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H+ ions as a general source for biochemical reactions. Consequences of this finding are discussed.

The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease

Elevated iron in the SNpc may play a key role in Parkinson’s disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

Chapter 3 - Recent Advances in Molecular Toxicology of Cadmium and Nickel

Abstract Cadmium (Cd) and nickel (Ni) are two toxic elements which are widespread in the human environment, but less recognized as hazardous by the general public. Herein, we describe molecular mechanisms of their toxicity toward humans, in the context of general chemical and toxicological properties of these metals. Following the introductory remarks, the routes of exposure are outlined. The next section covers the health hazards posed by Cd and Ni with the main stress placed on diseases like Cd-induced nephropathy, reproductive disorders due to Cd exposure, Cd-related chronic obstructive pulmonary disease, and Cd carcinogenesis. With respect to Ni, acute toxicity, Ni allergy, and Ni carcinogenicity were described. This overall description provides the basis for a detailed account of molecular mechanisms of Cd and Ni toxicity. They include the involvement of metallothioneins and their role in the transport of Cd(II) ions, and the role of oxidative damage and DNA repair inhibition in Cd carcinogenesis. The final section covered with respect to molecular mechanisms of Cd toxicity is its influence on cellular junctions. Molecular mechanisms of Ni toxicity are divided into subjects of Ni allergy and several mechanisms related to its carcinogenicity. The discussion is completed by the presentation of Ni and Cd interactions with zinc fingers as a possible common ground of their molecular toxicity.

Different effects of soluble and particulate guanylyl cyclases on expression of inflammatory cytokines in rat peripheral blood mononuclear cells.

Inflammation involves the cooperation of various cells and biologically active molecules. An important intracellular messenger molecule participating in the regulation of the process is cyclic GMP (cGMP), which is synthesized by guanylyl cyclases (GCs). The GC family comprises cytosolic (soluble) and membrane-bound (particulate) enzymes. The aim of this study was to determine whether and how the synthesis of cGMP by various forms of GC affects the expression of inflammatory cytokines depending on the activity of the transcription factors NF-κB (nuclear factor-κB) and AP-1 (activator protein-1). We established that in rat peripheral blood mononuclear cells (PBMCs), synthesis of cGMP was elevated by sodium nitroprusside (SNP), the activator of soluble GC, and by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), the activators of particulate GC-A and GC-B, respectively. Stimulation of various GCs differently affected the expressions of the cytokines IL-1β, IL-6, and TNF-α in control cells and in cells activated by bacterial endotoxin (LPS). In control PBMCs their expression was elevated by stimulation of soluble, but not particulate, GC. SNP caused an increase in NF-κB activity, but had no influence on the activity of AP-1. The cells treated with LPS decreased the expressions of IL-1β, IL-6, and TNF-α in response to stimulation of particulate GC-A, but not other guanylyl cyclases. This inhibitory effect was a result of suppression of the activities of NF-κB and AP-1. Both effects that of SNP and of ANP, were cGMP dependent, as shown using its membrane-permeable analog 8-Br-cGMP. The implementation of specific inhibitors showed that the stimulatory effect of SNP was mediated by soluble GC and cGMP-dependent protein kinase (PKG-I). However, PKG-I was not involved in the inhibition of NF-κB and AP-1 activities by ANP in LPS-activated cells. Taken together, these results for the first time indicate that various GCs and various cGMP-dependent signaling pathways can modulate the activity of AP-1 and/or NF-κB and thus affect the expressions of IL-1β, IL-6, and TNF-α, which play important roles in the development of inflammation.

Establishment of a cellular model to study TrkC-dependent neuritogenesis

The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (Ptight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.