Forward and reverse motion of single RecBCD molecules on DNA

RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.     

COX-2 in inflammation and resolution

Aspirin and the other NSAIDs have popularized the notion of inhibiting prostaglandins as a common anti-inflammatory strategy based on the erroneous premise that all eicosanoids are, within the context of inflammation, generally detrimental. However, our fascination with aspirin and the emergence of COX-2 has shown a more affable side to lipid mediators based on our increasing interest in the endogenous control of acute inflammation and in factors that mediate its resolution. Epilipoxins, for instance, are produced from aspirin's acetylation of COX-2 and together with Resolvins and COX-2-derived prostaglandins of the D(2) and J(2) series represent an increasingly important family of immunoregulatory lipid mediators with strong implications for disease control and drug discovery.     

Structure–activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC₅₀ = 11.9 μM) in cell culture without any apparent host-toxicity.     

Aβ(1-40) Forms Five Distinct Amyloid Structures whose β-Sheet Contents and Fibril Stabilities Are Correlated

The ability of a single polypeptide sequence to grow into multiple stable amyloid fibrils sets these aggregates apart from most native globular proteins. The existence of multiple amyloid forms is the basis for strain effects in yeast prion biology, and might contribute to variations in Alzheimer's disease pathology. However, the structural basis for amyloid polymorphism is poorly understood. We report here five structurally distinct fibrillar aggregates of the Alzheimer's plaque peptide Aβ(1-40), as well as a non-fibrillar aggregate induced by Zn²⁺. Each of these conformational forms exhibits a unique profile of physical properties, and all the fibrillar forms breed true in elongation reactions under a common set of growth conditions. Consistent with their defining cross-β structure, we find that in this series the amyloid fibrils containing more extensive β-sheet exhibit greater stability. At the same time, side chain packing outside of the β-sheet regions contributes to stability, and to differences of stability between polymorphic forms. Stability comparison is facilitated by the unique feature that the free energy of the monomer (equivalent to the unfolded state in a protein folding reaction) does not vary, and hence can be ignored, in the comparison of ΔG° of elongation values for each polymorphic fibril obtained under a single set of conditions.     

Structure formation in short designed peptides probed by proteolytic cleavage

The formation of local structure, in short peptides has been probed by examining cleavage patterns and rates of proteolysis of designed sequences with a high tendency to form beta-hairpin structures. Three model sequences which bear fluorescence donor and acceptor groups have been investigated: [see text]. Fluorescence resonance energy transfer (FRET) provides a convenient probe for peptide cleavage. MALDI mass spectrometry has been used to probe sites of cleavage and CD spectroscopy to access the overall backbone conformation using analog sequences, which lack strongly absorbing donor and acceptor groups. The proteases trypsin, subtilisin, collagenase, elastase, proteinase K and thermolysin were used for proteolysis and the rates of cleavage determined. Peptide 3 is the most susceptible to cleavage by all the enzymes except thermolysin, which cleaves all three peptides at comparable rates. Peptides 1 and 2 are completely resistant to the action of trypsin, suggesting that beta-turn formation acts as a deterrent to proteolytic cleavage.     

Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: modulation of the pH-activity profile

Engineering of the PhoN enzyme of Salmonella typhimurium due to its superior characteristics for bioremediation of heavy metals has been advocated by Macaskie and colleagues [Basnakova, G., Stephens, E.R., Thaller, M.C., Rossolini, G.M., Macaskie, L.E., 1998. The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows. Appl. Microbiol. Biotechnol. 50, 266-272]. The native enzyme hydrolyzes disparate organophosphates and exhibits optimal phosphatase activity at pH 5.5, for instance, with substrate p-nitrophenyl phosphate. Structurally guided Ile-78 was mutated using site-directed mutagenesis to Ala, Asp and His residues, with an aim to shift the optimum pH of the PhoN enzyme. Encouragingly, the I78A mutant displays significantly higher (as high as 160%) enzymatic efficiency over a broad pH range of 3.0-9.0, compared to the wild-type PhoN. The higher catalytic efficiency is due to the increase in k(cat), and can be mainly attributed to a deshielding of catalytic His-158 from the bulk-solvent. The I78D mutant possesses nearly twice the specific activity at the optimum pH of 7.0. The alkaline shift of the pH-activity profile agrees well with reasoning based on electrostatics. An increase in K(m), however, lowers the catalytic efficiency of the I78D mutant at the optimum pH. The I78H mutant, counter-intuitively, also exhibits an alkaline shift in the pH-optimum. Nonetheless, the active site scaffold in I78H mutant may not be disturbed, as similar steady-state kinetic parameters are observed for both I78H mutant and wild-type PhoN at their respective pH optima.     

In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron

The in vitro competency of mature cereal embryos (winter, spring and durum wheats, oat, barley and triticale) was assessed for direct multiple shoot production on culture media containing the plant growth regulators, thidiazuron (TDZ) and/or 6–benzylaminopurine (BAP). Mature embryos of CDC Dancer oat showed the best response, with 69 shoots per explant on culture medium containing a combination of 4.5 μM TDZ and 4.4 μM BAP. TDZ alone induced about 16 shoots per explant from the oat. Among the wheat genotypes, durum wheat showed the most number of shoots (35) per explant on culture medium containing 4.5 μM of TDZ and 4.4 μM of BAP. With TDZ alone, shoot regeneration for durum wheat ranged from 27–32 shoots per explant. The regeneration frequency from the three winter wheat genotypes ranged from 11–25 shoots per explant and was highest on culture medium containing 9.1 μM TDZ and 4.4 μM BAP. The latter culture medium was also effective for a triticale genotype, inducing 34 shoots per explant. The regeneration from mature embryos of barley genotypes ranged from 5–9 shoots per explant. The mature embryos of all the cereals tested could be used for in vitro regeneration with TDZ and TDZ+BAP combinations.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.