Krzysztof Huben

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73.

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer. Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation. Two p53 homologues, p63 and p73, have been described in humans. Unlike p53, these proteins regulate developmental processes rather than genome stability. Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx. Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined. Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.

The addition of dialkyl phosphites and diphenylphosphine oxide on the triple bond of dimethyl acetylenedicarboxylate under solvent-free and microwave conditions

The addition of dialkyl phosphites or diphenylphosphine oxide to the triple bond of dialkyl acetylenedicarboxylate may result in mono- and/or bisadducts. The ratio of the products may be influenced by the molar ratio of the reactants and the conditions (temperature of microwave irradiation and reaction time). Stereospecific (3)J(PP) couplings were utilized in the assignments to the meso and racemic forms of the bisadducts. The bis(dialkylphosphonyl)-succinates were formed as a mixture of the corresponding meso form and racemate, while bis(diphenylphosphinoyl)-succinate was obtained as a racemate. The P-31-(CH)-C-12-(CH)-C-13-P-31 spin system of bis(dialkylphosphonyl)-succinates was subjected to a detailed NMR analysis supported by simulation.

The structure of cyclolinopeptide A in chloroform refined by RDC measurements

Three‐dimensional structures of molecules traditionally assigned from nuclear Overhauser effects and vicinal coupling constants are recently complemented by measurements of residual dipolar couplings. Residual dipolar couplings measured in a stretched poly(dimethylsiloxane) gel were used to determine the structure of cyclolinopeptide A in chloroform solution at −50 °C. After structure refinement, conformational details of main cluster were discussed in relation to crystal and nuclear Overhauser effect derived structures. Copyright

2,3-Bisphosphonosuccinic Acid Hexamethyl Ester [(CH3O)2(O)P-CH-COOCH3]2 – NMR-Spectroscopic Studies of ABX and [AM3R3X]2+Z6 Spin Systems

GRAPHICAL ABSTRACT Abstract Addition of dimethyl phosphite to the triple bond of dimethylacetylene-dicarboxylate by conventional routes or assisted by microwaves leads predominantly to the meso form of 2,3-bisphosphonosuccinic acid hexamethyl ester [(CH3O)2(O)P-CH-COOCH3]2. Molecular structure and dominant rotamer were deduced from multinuclear 31P{1H}-, 13C{1H}-, 1H-, 1H{31P}-NMR studies on ABX and [AM3R3X]2+Z6 spin systems. Vicinal coupling constants 3JPP, 3JPH, and 3JHH were used as stereochemically relevant parameters to deduce rotamer preferences via Karplus-type relationships.

Synthesis and biological activity of cyclolinopeptide A analogues modified with γ 4 -bis(homo-phenylalanine)

Cyclolinopeptide A (CLA), an immunosuppressive nonapeptide derived from linen seeds, was modified with S or R-γ4-bis(homo-phenylalanine) in positions 3 or 4, or both 3 and 4. These modifications changed the flexibility of new analogues and distribution of intramolecular hydrogen bonds. Analogues 11 c(Pro1-Pro2-Phe3-S-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9), 13 c(Pro1-Pro2-S-γ4-hhPhe3-R-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9) and 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) existed as a mixture of stable cis/trans isomers of Pro-Pro peptide bond. The comparison of the relative spatial orientations in crystal state of the two carbonyl groups, neighboring γ-amino acids, revealed conformational similarities to α-peptides. The addition of two -CH2- groups in γ-amino acids led to a more rigid conformation, although a more flexible one was expected. A significant difference in the relative orientation of the carbonyl groups was found for cyclic γ-peptides with a dominance of an antiparallel arrangement. As carbonyl groups may be engaged in the interactions with plausible receptors through hydrogen bonds, a similar biological activity of the modified peptides was expected. Our biological studies showed that certain cyclic, but not the corresponding linear peptides, lowered the viability of peripheral blood mononuclear cells (PBMC) at 100μg/mL concentration. The proliferation of PBMC induced by phytohemagglutinin A (PHA) was strongly inhibited by cyclic peptides only, in a dose-dependant manner. On the other hand, lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production in whole blood cell cultures was inhibited by both linear and cyclic peptides. Peptide 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) blocked the expression of caspase-3, inhibited the expression of caspases-8 and -9 in 24h culture of Jurkat cells, and caused DNA fragmentation in these cells, as an indicator of apoptosis. Thus, we revealed a new mechanism of immunosuppressive action of a nonapeptide.

α-Fluoro Phosphonocarboxylates: the NMR Analysis of the Heteronuclear ABMX Spin System

Abstract The α-fluorophosphonocarboxylate analogs of bisphosphonates have recently received attention due to their activity against Rab geranylgeranyl transferase, an enzyme implicated in a number of disease conditions. Most of the NMR data reported in the literature describing dialkyl α-fluorophosphonates are defined as first-order spectra. Here, we report a second-order pattern observed in 19F NMR spectrum, being a part of heteronuclear ABMX spin system. Such a spin system, CH2C(F)P, was subjected to NMR analysis supported by iteration. GRAPHICAL ABSTRACT

Photochemical transformations of 3,3,4,7-tetramethylpyrazolo[3,4-d]pyridazine in solution and frozen gas matrices

Photochemical transformations of pyrazolopyridazine 1 give isopropenylpyridazine 2 in addition to cyclopropapyridazine 3 in solution and to the ring-opened diazo isomer 4 observed in frozen gas matrices.