Hydrodynamic and thermodynamic analysis of PEGylated human serum albumin.

Covalent labeling of therapeutic drugs and proteins with polyethylene glycol (PEGylation) is an important modification for improving stability, solubility, and half-life. PEGylation alters protein solution behavior through its impact on thermodynamic nonideality by increasing the excluded volume, and on hydrodynamic nonideality by increasing the frictional drag. To understand PEGylation's impact, we investigated the thermodynamic and hydrodynamic properties of a model system consisting of PEGylated human serum albumin derivatives using analytical ultracentrifugation (AUC) and dynamic light scattering (DLS).

Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase.

Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of T(H)17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response (AAR) pathway.

Allosteric interaction of nucleotides and tRNA(ala) with E. coli alanyl-tRNA synthetase.

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution.

The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK.

The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK.

Human platelet 12-lipoxygenase, new findings about its activity, membrane binding and low-resolution structure.

Human platelet 12-lipoxygenase (hp-12LOX, 662 residues+iron nonheme cofactor) and its major metabolite 12S-hydroxyeicosatetraenoic acid have been implicated in cardiovascular and renal diseases, many types of cancer and inflammatory responses. However, drug development is slow due to a lack of structural information. The major hurdle in obtaining a high-resolution X-ray structure is growing crystals, a process that requires the preparation of highly homogenous, reproducible and stable protein samples.

Daunomycin binding to detergent micelles: a model system for evaluating the hydrophobic contribution to drug-DNA interactions.

The interaction of daunomycin with sodium dodecyl sulfate and Triton X-100 micelles was investigated as a model for the hydrophobic contribution to the free energy of DNA intercalation reactions. Measurements of visible absorbance, fluorescence lifetime, steady-state fluorescence emission intensity, and fluorescence anisotropy indicate that the anthraquinone ring partitions into the hydrophobic micelle interior. Fluorescence quenching experiments using both steady-state and lifetime measurements demonstrate reduced accessibility of daunomycin in sodium dodecyl sulfate micelles to the anionic quencher iodide and to the neutral quencher acrylamide.

Activation of the ATPase activity of adeno-associated virus Rep68 and Rep78.

Rep68 and Rep78 DNA helicases, encoded by adeno-associated virus 2 (AAV2), are required for replication of AAV viral DNA in infected cells. They bind to imperfect palindromic elements in the inverted terminal repeat structures at the 3'- and 5'-ends of virion DNA. The ATPase activity of Rep68 and Rep78 is stimulated up to 10-fold by DNA containing the target sequence derived from the inverted terminal repeat; nontarget DNA stimulates ATPase activity at 50-fold higher concentrations.

Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase.

Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions. The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicates that the monomer is inactive and that the active enzyme is at least a dimer.

Adeno-associated virus rep protein-mediated inhibition of transcription of the adenovirus major late promoter in vitro.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication.

Mechanisms of adeno-associated virus genome encapsidation.

The defective parvovirus, adeno-associated virus (AAV), is under close scrutiny as a human gene therapy vector. AAV's non-pathogenic character, reliance on helper virus co-infection for replication and wide tissue tropism, make it an appealing vector system. The virus' simplicity and ability to generate high titer vector preparations have contributed to its wide spread use in the gene therapy community.

Identification of an adeno-associated virus Rep protein binding site in the adenovirus E2a promoter.

Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus.

A biochemical characterization of the adeno-associated virus Rep40 helicase.

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins.

Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase.

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate.