Azaspiracid: First evidence of protein binding in shellfish

The occurrence of azaspiracid (AZA) toxins in contaminated shellfish has been the focus of much research. The present study investigated the binding properties of these toxins in mussels of the species Mytilus edulis. The work involved extraction of proteins and AZAs from contaminated mussel hepatopancreas and examination of the extracts by isoelectric focusing (IEF), size exclusion chromatography (SEC) and sodium docecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Liquid chromatography coupled with tandem mass spectrometry analysis (LC–MS/MS) was also performed in this study to identify AZAs. Blank mussels were subjected to the same purification and analytical procedures.

AZAs were found to be weakly bound to a protein with a molecular weight of 45 kDa, in samples of contaminated mussels. This protein, which was abundant in contaminated mussels, was also present in blank mussels, albeit at much lower concentrations. It was further noted that a 22 kDa protein was also present only in contaminated mussel samples.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKB alpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKB alpha at the plasma membrane. Binding of CTMP reduces the activity of PKB alpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Protein surface recognition using geometrically pure Ru(II) tris(bipyridine) derivatives

This manuscript illustrates that the geometric arrangement of protein-binding groups around a ruthenium(II) core leads to dramatic differences in cytochrome c (cyt c) binding highlighting that it is possible to define synthetic receptors with shape complementarity to protein surfaces.

Mutations within subdomain II of the extracellular region of epidermal growth factor receptor selectively alter TGF alpha binding

The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.

The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases

UNLABELLED: Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins.

IMPORTANCE: It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.

Mapping protein-DNA interactions using ChIP-sequencing

Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.

Chromatin binding by the androgen receptor in prostate cancer

Alterations in transcriptional programs are fundamental to the development of cancers. The androgen receptor is central to the normal development of the prostate gland and to the development of prostate cancer. To a large extent this is believed to be due to the control of gene expression through the interaction of the androgen receptor with chromatin and subsequently with coregulators and the transcriptional machinery. Unbiased genome-wide studies have recently uncovered the recruitment sites that are gene-distal and intragenic rather than associated with proximal promoter regions. Whilst expression profiles from AR-positive primary prostate tumours and cell lines can directly relate to the AR cistrome in prostate cancer cells, this distribution raises significant challenges in making direct mechanistic connections. Furthermore, extrapolating from datasets assembled in one model to other model systems or clinical samples poses challenges if we are to use the AR-directed transcriptome to guide the development of novel biomarkers or treatment decisions. This review will provide an overview of the androgen receptor before addressing the challenges and opportunities created by whole-genome studies of the interplay between the androgen receptor and chromatin.