Tuning of the outer hair cell motor by membrane cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.     

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.     

Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery

BACKGROUND: The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety. METHODS: Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently. RESULTS: Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone. CONCLUSIONS: Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes, generated in this study have considerable potential to facilitate DNA delivery and enhance transfection. The domains in these fusion proteins would be promising in the development of non-viral gene delivery vectors particularly in cells that do not divide.     

SecA interacts with ribosomes in order to facilitate posttranslational translocation in bacteria

In Escherichia coli, translocation of exported proteins across the cytoplasmic membrane is dependent on the motor protein SecA and typically begins only after synthesis of the substrate has already been completed (i.e., posttranslationally). Thus, it has generally been assumed that the translocation machinery also recognizes its protein substrates posttranslationally. Here we report a specific interaction between SecA and the ribosome at a site near the polypeptide exit channel. This interaction is mediated by conserved motifs in SecA and ribosomal protein L23, and partial disruption of this interaction in?vivo by introducing mutations into the genes encoding SecA or L23 affects the efficiency of translocation by the posttranslational pathway. Based on these findings, we propose that SecA could interact with its nascent substrates during translation in order to efficiently channel them into the "posttranslational" translocation pathway.     

Effects of arginine on heat-induced aggregation of concentrated protein solutions

Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein.     

A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening

We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.     

Therapeutic efficacy of lipoic acid in combination with dimercaptosuccinic acid against lead-induced renal tubular defects and on isolated brush-border enzyme activities

The combined therapeutic potentials of lipoic acid and dimercaptosuccinic acid were compared against their sole administrations in restoring the altered lead sensitive indices in urine and isolated renal brush-border preparations. Toxicity was induced in male albino rats (Wistar strain) by administering lead acetate (0.2%) in drinking water for 5 weeks, followed by therapy comprising lipoic acid (25 mg/kg body weight) and dimercaptosuccinic acid (20 mg/kg body weight) solely as well as combined during the 6th week. Changes in kidney weights encountered upon lead administration improved after therapy with lipoic acid and dimercaptosuccinic acid. Renal integrity was assessed by measuring the activities of alkaline phosphatase, acid phosphatase, lactate dehydrogenase, leucine aminopeptidase, N-acetyl-beta-D-glucosaminidase, gamma-glutamyl transferase and beta-glucuronidase in urine along with some urinary constituents (urea, uric acid, creatinine, protein and phosphorous). The effects of lead were also studied on isolated brush-border enzymes (alkaline phosphatase, acid phosphatase, gamma-glutamyl transferase and beta-glucuronidase) that showed a decline upon its administration. Increased activities of urinary enzymes were accompanied by increase in the urinary constituents. Increase in renal lead content was paralleled by a drastic fall in the renal delta-aminolevulinic acid dehydratase and a rise in urinary lead levels. Relative to the administration of lead, the combined therapy showed betterment on the renal integrity with respect to the functional parameters assessed, thereby indicating its efficacy over the monotherapies.