Bradley P. Kane

Design of sensitive fluorogenic substrates for human cathepsin D

Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimers disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E‐NH2, where X=cysteine, methylcysteine, ethylcysteine, tert‐butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k cat/K m ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure‐based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.

Fluorescence and nucleic acid binding properties of the human T-cell leukemia virus-type 1 nucleocapsid protein

We used intrinsic tryptophan fluorescence to study the nucleocapsid protein from human T-cell leukemia virus-type one, HTLV-1 p15, an 85-amino-acid protein with two Trp-containing zinc-finger motifs. Fluorescence spectra suggested an interaction between the two zinc fingers and another interaction involving the C-terminal tail and the zinc fingers. Titrations with nucleic acid revealed similar, sub-micromolar affinity for poly(dT) and poly(U) in 1 mM sodium phosphate, pH 7. Double-stranded DNA bound an order of magnitude weaker, suggesting helix-destabilizing activity. Base preference of p15 was T approximately U>I approximately C approximately G>A; affinity spanned about one order of magnitude. HTLV-1 p15 bound weaker and with less variation than reported values for either human or simian immunodeficiency virus homologues. The low affinity of p15 for nonspecific nucleic acids distinguishes it from other nucleocapsid proteins, and may suggest its involvement in additional steps of the virus life cycle other than RNA packaging.

Fluorescence and nucleic acid binding properties of bovine leukemia virus nucleocapsid protein.

We used the intrinsic fluorescence of bovine leukemia virus p12, a nucleocapsid protein with two tryptophan-containing zinc fingers (ZFs), to study its conformation and binding to single-stranded nucleic acids. Spectral emission maxima suggested solvent-exposed tryptophans. A peptide derived from ZF1 had a higher quantum yield and longer average lifetime (tau) than ZF2. BLV p12 tau and rotational correlation time were greater than ZF values, but all de-metallated sequences gave similar results. Apo p12 showed reduced fluorescence intensity, tau and loss of secondary structure. DNA-binding affinity of p12 was in the nanomolar range, and decreased 14-fold after Zn++ ejection. Nucleobase preference of BLV p12 was different from the closely related HTLV-1 but similar to HIV-1 and SIV nucleocapsids, both phylogenetically distant.

Reaction of HIV-1 NC p7 zinc fingers with electrophilic reagents

Publisher Summary Recently, it has been shown that Cys (X)2 Cys (X)4 His (X)4 Cys array (CCHC) zinc finger peptides are susceptible to chemical attack by a wide variety of oxidizing agents. The metal-chelated sulfur thiolates in the CCHC zinc fingers of HIV-1 p7 are known to react with a variety of chemical groups, including maleimides, nitrosos, disulfoxides, thiocarbamoyl-disulfides, and other substituted disulfides as well as oxidizing agents, such as Cu +2 , Fe +3 , and Hg +2 ions. The reaction mechanism for the thiuram disulfide class of oxidizing agents and maleimide class of alkylating agents are examined and presented in this chapter. Thiuram disulfides are examined in detail, as a member of this class of compounds, tetraethylthiuram disulfide (Antabuse) is an FDA-approved drug for alcohol abuse therapy and has very low in vivo toxicity. These compounds have functional groups that can modify zinc fingers in nucleocapsid (NC) protein and have antiviral activity but are not necessarily specific for the virus. To initiate studies leading to the design of reagents with greater specificity for the viral NC protein, it is necessary to determine the mechanism of action for model compounds and in particular to determine the initial site of attack on the NC protein. While the studies of NC protein alone demonstrated little if any cross-linking, the results with HIV-1 virus showed extensive oligomerization. The mature virion contains a compact ribonucleoprotein complex formed by the genomic RNA and ca. 2,500 copies of the NC protein. Therefore, the high concentration of NC in the viral particle the formation of intermolecular disulfide bonds over intramolecular ones is expected to be favored following virus treatment with thiuram disulfides.