Calbindin-D<Subscript>28K</Subscript> acts as a calcium-dependent chaperone suppressing α-synuclein fibrillation <Emphasis Type="Italic">in vitro</Emphasis>

α-Synuclein, a natively unfolded protein aggregation which is implicated in the pathogenesis of Parkinson’s disease and several other neurodegenerative diseases, is known to interact with a great number of unrelated proteins. Some of these proteins, such as β-synuclein and DJ-1, were shown to inhibit α-synuclein aggregation in vitro and in vivo therefore acting as chaperones. Since calbindin-D_28K is co-localized with Ca²⁺ neuronal membrane pumps, and since α-synuclein is also found in the membrane proximity, these two proteins can potentially interact in vivo. Here we show that calbindin-D28K interacts with α-synuclein and inhibits its fibrillation in a calcium-dependent manner, therefore potentially acting as a calcium-dependent chaperone.     

Motor-cargo interactions: the key to transport specificity

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.     

Identification of Nedd4 E3 ubiquitin ligase as a binding partner and regulator of MAK-V protein kinase

MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.     

Similarity Between Naturally Occurring Modified Desialylated, Electronegative and Aortic Low Density Lipoprotein

The sialic acid content of electronegative low density lipoprotein (LDL) and LDL isolated from human aortic intima was measured. Sialic acid level in electronegative LDL of healthy subjects was 1.7-fold lower than in native LDL. Sialic acid content in electronegative LDL of coronary atherosclerosis patients was 3-fold lower than in native LDL. Lipoproteins isolated from grossly normal human aortic intima and from fatty streaks contained 20-56% less sialic acid as compared to blood plasma LDL. A negative correlation was established between the ability of electronegative and aortic LDL to stimulate lipid accumulation in cells cultured from uninvolved human aortic intima and lipoprotein sialic acid content. The results obtained indicate that electronegative and aortic LDLs have a low sialic acid content, i.e., are desialylated lipoproteins. Considered together with the fact that all known atherogenic LDLs have similar characteristics, our findings suggest that modified LDLs are the same lipoprotein particles subjected to multiple modification.     

Mechanisms of Candida Resistance to Antimycotics and Promising Ways to Overcome It: The Role of Probiotics

Probiotics and Antimicrobial Proteins - Pathogenic Candida and infections caused by those species are now considered as a serious threat to public health. The treatment of candidiasis is...     

Recoverin is a zinc-binding protein

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.     

The effect of macromolecular crowding on protein aggregation and amyloid fibril formation

Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein alpha-synuclein. The aggregation of alpha-synuclein has been implicated as a critical factor in development of Parkinson's disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate alpha-synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to approximately 100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson's disease by epidemiological studies, are shown to accelerate alpha-synuclein fibrillation under conditions of molecular crowding.     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.