Mitko Dimitrov

Microheterogeneity of parsley plastocyanin

A procedure for isolation of two iso‐plastocyanins from parsley has been described here. Three consecutive Chromatographic steps on DE‐52‐Whatman cellulose were applied for isolation of two total plastocyanin (PC) fractions, oxidized [PC(II)] and reduced [PC(I)]. By chromatofocusing of PC(II) on Polybuffer exchanger 74 two different plastocyanins, designated as plastocyanin a (PCa) and plastocyanin b (PCb), were obtained. The isoelectric points (pI) of PCa and PCb at 10°C are 4.16 and 4.14, respectively. The complete amino acid sequences of PCa and PCb were determined. The two iso‐proteins consist of 97 amino acid residues and differ only at sequence position 53, where Glu in PCa is replaced by Asp in PCb.

Complete amino acid sequence of poplar plastocyanin b

A second type of plastocyanin, plastocyanin b (PCb) was isolated from leaves of poplar Populus nigra var. Italica. The complete amino acid sequence of 99 residues in the single polypeptide chain of plastocyanin b has been determined: DVLLGADDG SLAFVPSEFS GEKIVFK NNAGFPHN FDED PSGV D SKISMSEE DLLNAKGETF EVALS KGEY FYCSPHQGA GMVGKV VN The sequence obviously demonstrates, that PCb, in comparison with the known plastocyanin, plastocyanin a (PCa), has 12 amino acid replacements (underlined letters): Ile 1→Val, Ile 21‐Ser 22‐Pro 23→Val‐Pro‐Ala, Ile 39‐Val 40→Val‐Leu, Ser 45‐Ile 46→ Ala‐Val, Ala 52→Val, Asn 76→Asp, Ser 81→Thr and Thr 97→Ile. The replacements at positions 40 and 76 of PCb are probably essential for differences in its redox and electrochemical properties, respectively.

Structural comparison of the poplar plastocyanin isoforms PCa and PCb sheds new light on the role of the copper site geometry in interactions with redox partners in oxygenic photosynthesis.

Plastocyanin (PC) from poplar leaves is present in two isoforms, PCa and PCb, which differ in sequence by amino acid replacements at locations remote from the copper center and simultaneously act in the photosynthetic electron-transport chain. We describe ultra-high resolution structures of PCa and high-resolution structures of PCb, both under oxidizing and reducing conditions at pH 4, 6 and 8. The docking on cytochrome f and photosystem I, respectively, has been modeled for both isoforms. PCa and PCb exhibit closely similar overall and active-site structures, except for a difference in the relative orientation of the acidic patches. The isoforms exhibit substantial differences in the dependence of the reduced (Cu(I)) geometry on pH. In PCa, the decrease in pH causes a gradual dissociation of His87 from Cu(I) at low pH, probably adopting a neutral tautomeric state. In PCb, the histidine remains covalently bound to Cu(I) and may adopt a doubly protonated state at low pH. The fact that both isoforms have similar although not identical functions in photosynthetic electron flows suggests that the His87 imidazole does not play a crucial role for the pathway of electron transport from cytochrome f to oxidized PC.

Cross-Border Cooperation in Southeastern Europe

This article examines the current status, limits, prospects, and policies of cross-border cooperation in the border zone of Albania, Bulgaria, FYROM (Former Yugoslav Republic of Macedonia), and Greece on the basis of a survey sample of 291 manufacturing firms located near the borders in all four countries. The analysis suggests that border region firms have a higher level of interaction than the respective average national firms in all countries and that trade relations and economic cooperation eventually depend on the level of specialization and the size of the markets. It also suggests that barriers to cooperation are important and can negatively affect the performance of border region firms. Overall, firms are less concerned about the quality of infrastructure and more concerned about the general or the financial conditions prevailing in each country, indicating that the best policy for cross-border cooperation, rather than improvements in infrastructure, may be the development of the economies in the region and the improvements in their economic environment.

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.

Inhibition of Notch pathway arrests PTEN-deficient advanced prostate cancer by triggering p27-driven cellular senescence.

Activation of NOTCH signalling is associated with advanced prostate cancer and treatment resistance in prostate cancer patients. However, the mechanism that drives NOTCH activation in prostate cancer remains still elusive. Moreover, preclinical evidence of the therapeutic efficacy of NOTCH inhibitors in prostate cancer is lacking. Here, we provide evidence that PTEN loss in prostate tumours upregulates the expression of ADAM17, thereby activating NOTCH signalling. Using prostate conditional inactivation of both Pten and Notch1 along with preclinical trials carried out in Pten-null prostate conditional mouse models, we demonstrate that Pten-deficient prostate tumours are addicted to the NOTCH signalling. Importantly, we find that pharmacological inhibition of γ-secretase promotes growth arrest in both Pten-null and Pten/Trp53-null prostate tumours by triggering cellular senescence. Altogether, our findings describe a novel pro-tumorigenic network that links PTEN loss to ADAM17 and NOTCH signalling, thus providing the rational for the use of γ-secretase inhibitors in advanced prostate cancer patients.

Generation of monoclonal antibody fragments binding the native γ-secretase complex for use in structural studies.

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimers disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.

Shedding of neurexin 3β ectodomain by ADAM10 releases a soluble fragment that affects the development of newborn neurons.

Neurexins are transmembrane synaptic cell adhesion molecules involved in the development and maturation of neuronal synapses. In the present study, we report that Nrxn3β is processed by the metalloproteases ADAM10, ADAM17, and by the intramembrane-cleaving protease γ-secretase, producing secreted neurexin3β (sNrxn3β) and a single intracellular domain (Nrxn3β-ICD). We further completed the full characterization of the sites at which Nrxn3β is processed by these proteases. Supporting the physiological relevance of the Nrxn3β processing, we demonstrate in vivo a significant effect of the secreted shedding product sNrxn3β on the morphological development of adult newborn neurons in the mouse hippocampus. We show that sNrxn3β produced by the cells of the dentate gyrus increases the spine density of newborn neurons whereas sNrxn3β produced by the newborn neuron itself affects the number of its mossy fiber terminal extensions. These results support a pivotal role of sNrxn3β in plasticity and network remodeling during neuronal development.

Some physicochemical peculiarities of poplar plastocyanins a and b.

The redox potentials of poplar plastocyanins a and b (PCa, PCb) were determined by spectro photometric titrations of their reduced forms with [Fe(CN)6]3-. It was found that the two isoforms have the following millimolar extinction coefficients ε597, equilibrium constants Keq of one-electron exchange with [Fe(CN)6]4-/[Fe(CN)6]3-, and standard electron potentials E0′: PCa: ε597 = (4.72 ± 0.08) mM-1 cm-1, Keq = 0.133 ± 0.009, E0′ = (354 ± 11) mV; PCb: ε597 = (5.23 ± 0.16) mM-1 cm-1, Keq = 0.175 ± 0.010, E0′ = (363 ± 12) mV. The pH dependence of the redox potential of PCb was studied too. It was found, that the value of E0′ for PCb is constant in the pH range 6.5 - 9.5, but decreases in the range 4.8 - 6.5. On the whole, the dependence resembles that of PC from some well-known plant species, including poplar PCa. The changes of E0′ in the pH-dependent region for poplar PCb, however, are smaller and are 13 mV per pH unit, whereas in the other well-known plant species the changes are about 50 - 60 mV per pH unit. It has been assumed that the weaker pH dependence of E0′ of PCb accounts for some structural differences between PCa and PCb

Changes in the content of poplar isoplastocyanins a and b during vegetation cycle

Changes in the content of isoplastocyanins a (PCa) and b (PCb) in Populus nigra leaves were studied during complete vegetation cycle (from April 30 to October 28). Measurements were made every 7 days. The procedure included extraction and purification of total protein followed by analytical isoelectric focusing and densitometry of obtained isophoregrams. The positions of PCa and PCb were determined by comparison with the positions of the highly purified two isoforms. The PCb/PCa ratio was calculated from the areas of the corresponding densitometric peaks. The amount of PCb was one half of PCa amount in the earliest stage of the leaf formation as well as during the longer part of the summer, e.g., the second half of July, August, and September. During the most active period of the poplar growth, e.g., June and the first half of July, the ratio was about or above unity. This result could be important for understanding the specific functional significance of PCa and PCb.