NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I

Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues. Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively. Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed. However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).     

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44‐expressing cancer cells

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44⁺ human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide‐field and low phototoxic hyperspectral imaging system has been successful for performing spectral three‐dimensional (3D) localization and spectroscopic identification of CD44‐targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic‐based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub‐cellular activity with the use of NPs as spectral sensors. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Characterization of the neuron-specific L1-CAM cytoplasmic tail: naturally disordered in solution it exercises different binding modes for different adaptor proteins

L1, a highly conserved transmembrane glycoprotein member of the immunoglobulin superfamily of cell adhesion molecules, mediates many developmental processes in the nervous system. Here we present the biophysical characterization and the binding properties of the least structurally defined part of this receptor: its cytoplasmic tail (CT). We have shown by analytical ultracentrifugation and dynamic light scattering experiments that it is mostly monomeric and unstructured in aqueous solution. We have defined by nuclear magnetic resonance the molecular details of L1-CT binding to two major targets: a membrane-cytoskeletal linker (MCL), ezrin, and an endocytosis mediator, AP2. Surprisingly, in addition to the two previously identified ezrin binding motifs, the juxtamembrane and the (1176)YRSLE regions, we have discovered a third one, a part of which has been previously associated with binding to another MCL, ankyrin. For the L1 interaction with AP2 we have determined the precise interaction region surrounding the (1176)YRSLE binding site and that this overlaps with the second ezrin binding site. In addition, we have shown that the juxtamembrane region of L1-CT has some binding affinity to AP2-mu2, although the specificity of this interaction needs further investigation. These data indicate that L1-CT belongs to the class of intrinsically disordered proteins. Endogenous flexibility of L1-CT might play an important role in dynamic regulation of intracellular signaling: the ability of cytoplasmic tails to accommodate different targets has the potential to fine-tune signal transduction via cell surface receptors.     

Quaternary structure of alpha-crystallin is necessary for the binding of unfolded proteins: a surface plasmon resonance study

The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein. Our data indicate that quaternary structure of alpha-crystallin is necessary for its chaperone-like activity.     

Involvement of mtDNA damage in free fatty acid-induced apoptosis

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.     

Folding and Self-Assembly of the TatA Translocation Pore Based on a Charge Zipper Mechanism

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Highlights? Charge zippers as a new concept for folding and assembly of membrane proteins ? 3D structure of TatA pore explains translocation of folded proteins across membrane ? Ladders of salt bridges connect an amphiphilic palisade that can span the bilayer ? MD simulations and specific charge mutations support the charge zipper model     

Identification of novel PARP-1 inhibitors by structure-based virtual screening

Poly(ADP-ribose)polymerase-1 (PARP-1) is an abundant and ubiquitous chromatin-bound nuclear protein. PARP-1, a DNA repair enzyme, has been in the limelight as a chemotherapeutic target. In this study, we demonstrated the successful use of structure-based virtual screening to identify inhibitors of PARP-1 from Otava databases comprised of nearly 260,000 compounds. Five novel inhibitors belonging to thienopyrimidinone, isoquinolinoquinazolinone, pyrroloquinazolinone, and cyclopentenothienopyrimidinone scaffolds revealed inhibitory potencies with IC₅₀ values ranged from 9.57 μM to 0.72 μM. Structural features relevant to the activity of these novel compounds within the active site of PARP-1 are discussed in detail and will guide future SAR investigation on these scaffolds.     

Structure-based discovery of novel flavonol inhibitors of human protein kinase CK2

Serine/threonine protein kinase CK2 controls vast variety of fundamental processes in cell life; however, despite long period of study, its functional role is not completely determined. CK2 has a significant pathogenic potential and its activity is strictly associated with the development of various kinds of disorders. There are a growing number of facts that inhibitors of CK2 could be used as pharmaceutical agents for the cancer treatment, viral infections, and inflammatory diseases. In this article, we report structural and biological data on the novel synthetic flavonol derivatives, 3-hydroxy-4'-carboxyflavones, possessing a high inhibitory activity toward CK2. With the aid of combinatorial organic synthesis, molecular modeling techniques and biochemical in vitro tests, we studied the structure-activity relationships of flavonol derivatives and developed binding model describing their key intermolecular interactions with the CK2 ATP-binding site. Obtained data show that the synthetic 3-hydroxy-4'-carboxyflavones possess the highest activity among flavonol inhibitors of CK2 known till date.