Modulation of nucleobindin-1 and nucleobindin-2 by caspases

Nucleobindin-1 (NUCB1) and nucleobindin-2 (NUCB2) are multifunctional proteins that interact with Ca(2+), nucleic acids and various regulatory proteins in different signaling pathways. So far, our understanding of the regulation of the biological functions of nucleobindins remains limited. In our proteome-wide selection for downstream caspase substrates, both NUCB1 and NUCB2 are found to be the downstream substrates of caspases. We report here the detailed analyses of the cleavage of nucleobindins by caspases. Significantly, the caspase cleavage sites are located exactly at one of the Ca(2+)-binding EF-hand motifs. Our results suggest that the functions of nucleobindins could be modulated by caspase-mediated cleavage in apoptosis.     

Organic anion transporter OAT1 undergoes constitutive and protein kinase C-regulated trafficking through a dynamin- and clathrin-dependent pathway

Organic anion transporter 1 (OAT1) mediates the body disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We previously demonstrate that OAT1 activity was down-regulated by activation of protein kinase C (PKC), kinetically revealed as a decrease in the maximum transport velocity V(max) without significant change in the substrate affinity K(m) of the transporter. In the current study, we showed that OAT1 constitutively internalized from and recycled back to the plasma membrane, and PKC activation accelerated OAT1 internalization without affecting OAT1 recycling. We further showed that treatment of OAT1-expressing cells with concanavalin A, depletion of K(+) from the cells, or transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block the clathrin-dependent endocytotic pathway, significantly blocked constitutive and PKC-regulated OAT1 internalization. We finally showed that OAT1 colocalized with transferrin, a marker for clathrin-dependent endocytosis, at the cell surface and in the EEA1-positive early endosomes. Together, our findings demonstrated for the first time that (i) OAT1 constitutively traffics between plasma membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 trafficking, and (iii) OAT1 internalization occurs partly through a dynamin- and clathrin-dependent pathway.     

Fungistatic Intensity of Agricultural Soil Against Fungal Agents and Phylogenetic Analysis on the Actinobacteria Involved

A total of 287 agricultural soil samples collected from 26 provinces or autonomous regions of China were tested on their ability to suppress the conidial germination of nine biocontrol fungal agents. These soil samples showed great differences in the degree to inhibit the germination of conidia (22.8% < mean inhibition rate < 97.5%), but all exhibited fungistatic activities above the moderate levels (mean inhibition rate > 50%) to most of tested fungi. Ten soil samples that have stronger fungistatic intensity (germination inhibition rate > 68.3%) to the target fungi, Trichoderma viride and Paecilomyces lilacinus, were selected to evaluate their soil actinobacteria involved fungistasis in soil. Of the 1,000 isolates from those soil samples, 345 actinobacteria exhibited fungistatic activity to conidial germination of T. viride and P. lilacinus with germination inhibition rates higher than 10%. Sequences encoding 16S rRNA gene of the 345 actinobacteria were analyzed by ARDRA and resulted 44 different ARDRA types. Fifty-six isolates, at least one from each unique ARDRA type, were selected for 16S rDNA sequencing and phylogenetic analysis. Results indicated that the actinobacteria involved in the soil fungistasis had close phylogenetic relationship with the members of Sterptomycetaceae, Microbacteriaceae, Micrococcaceae, and Nocardiacea.     

The ensemble folding kinetics of the FBP28 WW domain revealed by an all-atom Monte Carlo simulation in a knowledge-based potential

In this work, we apply a detailed all‐atom model with a transferable knowledge‐based potential to study the folding kinetics of Formin‐Binding protein, FBP28, which is a canonical three‐stranded β‐sheet WW domain. Replica exchange Monte Carlo simulations starting from random coils find native‐like (Cα RMSD of 2.68 Å) lowest energy structure. We also study the folding kinetics of FBP28 WW domain by performing a large number of ab initio Monte Carlo folding simulations. Using these trajectories, we examine the order of formation of two β‐hairpins, the folding mechanism of each individual β‐hairpin, and transition state ensemble (TSE) of FBP28 WW domain and compare our results with experimental data and previous computational studies. To obtain detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Further, a rigorous P_fold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated Φ values. Our analysis shows that the turn structure between first and second β strands is a partially stable structural motif that gets formed before entering the TSE in FBP28 WW domain and there exist two major pathways for the folding of FBP28 WW domain, which differ in the order and mechanism of hairpin formation. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Detection of local polarity of alpha-lactalbumin by N-terminal specific labeling with a new tailor-made fluorescent probe

To detect the local polarity such as the N-terminal domain of a protein molecule, 3-(4-chloro-6-hydrazino-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine has been designed and synthesized as a polarity-sensitive fluorescent probe by using an s-triazine ring as a backbone, neutral red and hydrazine as a polarity-sensitive fluorophore, and a labeling group, respectively. The fluorescence properties of the probe have been characterized. The probe has the following features: (1) stable in various solvents; (2) the long-wavelength emission of >550 nm that can avoid the interferences of the background fluorescence shorter than 500 nm from common biomacromolecules; and (3) the maximum emission wavelength (lambda(em)) sensitive to solvent polarity only but not to pH and temperature. The hydrazino group in such a probe reacts readily with an active carbonyl produced by transamination of a protein molecule, leading to N-terminal specific attachment of the fluorophore and thereby allowing the monitoring of local polarity. With this probe, the polarity of the N-terminal domain in both native and heat-denatured alpha-lactalbumin has been first determined, which corresponds to that with a dielectric constant of about 16, and the hydrophobic core near the N-terminus is found to be conservative for heating. The present strategy may provide a general method to study the local environmental changes of a protein molecule under different denaturation conditions.     

Non-hydroxamate 5-phenylpyrimidine-2,4,6-trione derivatives as selective inhibitors of tumor necrosis factor-alpha converting enzyme

New inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered with a pyrimidine-2,4,6-trione in place of the commonly used hydroxamic acid. These non-hydroxamate TACE inhibitors were developed by incorporating a 4-(2-methyl-4-quinolinylmethoxy)phenyl group, an optimized TACE selective P1' group. Several leads were identified with IC50 values around 100 nM in a porcine TACE assay and selective over MMP-1, -2, -9, -13, and aggrecanase.     

Blocking effect and crystal structure of natrin toxin, a cysteine-rich secretory protein from Naja atra venom that targets the BKCa channel

Cysteine-rich secretory proteins (CRISPs) are widespread in snake venoms. Some members of these CRISPs recently have been found to block L-type Ca(2+) channels or cyclic nucleotide-gated ion (CNG) channels. Here, natrin purified from Naja atra venom, a member of the CRISP family, can induce a further contractile response in the endothelium-denuded thoracic aorta of mouse which has been contracted by a high-K(+) solution. Further experiments show it can block the high-conductance calcium-activated potassium (BK(Ca)) channel in a concentration-dependent manner with an IC(50) of 34.4 nM and a Hill coefficient of 1.02, which suggests that only a single natrin molecule is required to bind an ion channel to block BK(Ca) current. The crystal structure of natrin displaying two domains in tandem shows its cysteine-rich domain (CRD) has relatively independent flexibility, especially for the C-terminal long loop (loop I) of CRD to participate in the interface of two domains. On the basis of previous studies of CNG channel and L-Ca(2+) channel blockers, and the sequence and structural comparison of natrin and stecrisp, the deviation of the vital loop I of CRD is suggested to contribute to different effects of some CRISPs in protein-protein interaction.     

Immunodepletion of albumin for two-dimensional gel detection of new mouse acute-phase protein and other plasma proteins

Immunodepletion of albumin to improve the 2-D gel resolution of human plasma proteins has recently been described. With the importance of mouse models in many studies in which serum or plasma is often analyzed, we have adopted this approach to immunoprecipitate mouse albumin and evaluated its effectiveness for 2-D separation of mouse plasma proteins. Purified polyclonal antibodies against mouse albumin were effective depleting intact albumin as well as its numerous fragments from mouse plasma samples. Removal of albumin resulted in better resolution of mouse plasma proteins. Three proteins, alpha2-macroglobulin, coagulation factor XII, and hemopexin, that were previously either undetectable or poorly resolved, were identified from albumin-depleted 2-D gels by peptide mass fingerprinting. Albumin depletion also led to partial loss of several other proteins such as clusterin and gelsolin. This loss can be attributed to the interaction with albumin itself because the specificity of the antibody was demonstrated by Western blot. When applying this method to the 2-D separation of plasma from inflamed mouse induced by cutaneous burn injury with superimposed Pseudomonas aeruginosa infection, the upregulation of inter alpha-trypsin inhibitor heavy chain 4 (ITIH4) and hemopexin was unambiguously detected along with other mouse acute-phase proteins (APP), including haptoglobin and serum amyloid A. Based on the significant increase of ITIH4, we propose that this protein is a new member of mouse APP that are upregulated during the inflammatory response.     

Discovery of N-benzyl-N'-(4-pipyridinyl)urea CCR5 antagonists as anti-HIV-1 agents (II): modification of the acyl portion

Modification of the acyl moiety in the CCR5 lead molecule 2 led to identification of several new classes of CCR5 antagonists. Antiviral activity and pharmacokinetic properties of the synthesized compounds were evaluated. Structure-activity relationship (SAR) derived from these studies further guided the optimization efforts, ultimately leading to the discovery of 36 with an acceptable drug-like profile.