The protamine family of sperm nuclear proteins

An overview of the vertebrate members of a diverse family of basic DNA-binding proteins that are synthesized in the late-stage spermatids of many animals and plants and condense the spermatid genome into a genetically inactive state

Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells

<p>Abstract</p> <p>Background</p> <p>A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)₂LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma.</p> <p>Results</p> <p>An analog of the SHAL (DvLPBaPPP)₂LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more ¹¹¹In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the ¹¹¹In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus.</p> <p>Conclusion</p> <p>The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)₂LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)₂LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.</p

Analysis of Synthetic DNAs and DNA-Protamine Complexes with the Scanning Tunneling Microscope

Three duplex DNAs 22, 47, and 100 base-pairs in length have been imaged with the scanning tunneling microscope (STM) after deposition on highly oriented pyrolytic graphite (HOPG). Images of the 47 base-pair (bp) molecules are resolved sufficiently to identify the two phosphodiester strands, the direction of helical coiling (this molecule contains three turns of left-handed helix), and single-stranded ends. Length measurements indicate that all three DNA sequences have adopted an A-like conformation. DNA-protamine complexes were also prepared and imaged under similar conditions. Length measurements of the complexes demonstrate that the binding of bull protamine 1 to the 47-mer stabilizes the DNA in a B conformation and prevents the B to A transition that has been shown to occur as the DNA molecules dehydrate on the surface. Measurements of the diameter of the complex (3 nm) were also obtained and were found to be only slightly larger than the DNA molecule. This observation is consistent with the binding of the protamine molecule inside one of the grooves

Identification of a Novel Drug Lead That Inhibits HCV Infection and Cell-to-Cell Transmission by Targeting the HCV E2 Glycoprotein

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment