Structural and Sequence Analysis of Imelysin-Like Proteins Implicated in Bacterial Iron Uptake

Imelysin-like proteins define a superfamily of bacterial proteins that are likely involved in iron uptake. Members of this superfamily were previously thought to be peptidases and were included in the MEROPS family M75. We determined the first crystal structures of two remotely related, imelysin-like proteins. The Psychrobacter arcticus structure was determined at 2.15 Å resolution and contains the canonical imelysin fold, while higher resolution structures from the gut bacteria Bacteroides ovatus, in two crystal forms (at 1.25 Å and 1.44 Å resolution), have a circularly permuted topology. Both structures are highly similar to each other despite low sequence similarity and circular permutation. The all-helical structure can be divided into two similar four-helix bundle domains. The overall structure and the GxHxxE motif region differ from known HxxE metallopeptidases, suggesting that imelysin-like proteins are not peptidases. A putative functional site is located at the domain interface. We have now organized the known homologous proteins into a superfamily, which can be separated into four families. These families share a similar functional site, but each has family-specific structural and sequence features. These results indicate that imelysin-like proteins have evolved from a common ancestor, and likely have a conserved function

What does the structure-function relationship of the HIV-1 Tat protein teach us about developing an AIDS vaccine?

The human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription protein Tat is an important factor in viral pathogenesis. In addition to its function as the key trans-activator of viral transcription, Tat is also secreted by the infected cell and taken up by neighboring cells where it has an effect both on infected and uninfected cells. In this review we will focus on the relationship between the structure of the Tat protein and its function as a secreted factor. To this end we will summarize some of the exogenous functions of Tat that have been implicated in HIV-1 pathogenesis and the impact of structural variations and viral subtype variants of Tat on those functions. Finally, since in some patients the presence of Tat-specific antibodies or CTL frequencies are associated with slow or non-progression to AIDS, we will also discuss the role of Tat as a potential vaccine candidate, the advances made in this field, and the importance of using a Tat protein capable of eliciting a protective or therapeutic immune response to viral challenge

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p

Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-κB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway

Spitzer number counts of active galactic nuclei in the goods fields

We present mid-infrared observations of AGN in the GOODS fields, performed with the Spitzer Space Telescope. These are the deepest infrared and X-ray fields to date and cover a total area of ~0.1 square degrees. AGN are selected on the basis of their hard (2-8 keV) X-ray emission. The median AGN infrared luminosity is at least 10 times larger than the median for normal galaxies with the same redshift distribution, suggesting that the infrared emission is dominated by the central nucleus. The X-ray to infrared luminosity ratios of GOODS AGN, most of which are at 0.5<z<1.5, are similar to the values obtained for AGN in the local Universe. The observed infrared flux distribution has an integral slope of ~1.5 and there are 1000 sources per square degree brighter than ~50 uJy at 3-6 microns. The counts approximately match the predictions of models based on AGN unification, in which the majority of AGN are obscured. This agreement confirms that the faintest X-ray sources, which are dominated by the host galaxy light in the optical, are obscured AGN. Using these Spitzer data, the AGN contribution to the extragalactic infrared background light is calculated by correlating the X-ray and infrared catalogues. This is likely to be a lower limit given that the most obscured AGN are missed in X-rays. We estimate the contribution of AGN missed in X-rays, using a population synthesis model, to be ~45% of the observed AGN contribution, making the AGN contribution to the infrared background at most ~2-10% in the 3-24 micron range, depending on wavelength, lower than most previous estimates. The AGN contribution to the infrared background remains roughly constant with source flux in the IRAC bands but decreases with decreasing flux in the MIPS 24 um band, where the galaxy population becomes more important.Comment: ApJ Accepted, 24 pages, 7 figures, author list modifie

Obscured active galactic nuclei and the X-ray, optical, andfar-infrared number counts of active galactic nuclei in the GOODS fields

The deep X-ray, optical, and far-infrared fields that constitute GOODS are sensitive to obscured AGN (N_H>10^{22} cm^{-2}) at the quasar epoch (z~2-3), as well as to unobscured AGN as distant as z~7. Luminous X-ray emission is a sign of accretion onto a supermassive black hole and thus reveals all but the most heavily obscured AGN. We combine X-ray luminosity functions with appropriate spectral energy distributions for AGN to model the X-ray, optical and far-infrared flux distributions of the X-ray sources in the GOODS fields. A simple model based on the unified paradigm for AGN, with ~3 times as many obscured AGN as unobscured, successfully reproduces the z-band flux distributions measured in the deep HST ACS observations on the GOODS North and South fields. This model is also consistent with the observed spectroscopic and photometric redshift distributions once selection effects are considered. The previously reported discrepancy between observed spectroscopic redshift distributions and the predictions of population synthesis models for the X-ray background can be explained by bias against the most heavily obscured AGN generated both by X-ray observations and the identification of sources via optical spectroscopy. We predict the AGN number counts for Spitzer MIPS 24 um and IRAC 3.6-8 um observations in the GOODS fields, which will verify whether most AGN in the early Universe are obscured in the optical. Such AGN should be very bright far-infrared sources and include some obscured AGN missed even by X-ray observations.Comment: Accepted by ApJ; 39 pages, 13 figure

Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12

FKBP12, a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin, is ubiquitously expressed and interacts with proteins in several intracellular signal transduction systems(1). Although FKBP12 interacts with the cytoplasmic domains of type I receptors of the transforming growth factor-beta (TGF-beta) superfamily in vitro, the function of FKBP12 in TGF-beta superfamily signalling is controversial(2-6). FKBP12 also physically interacts stoichiometrically with multiple intracellular calcium release channels including the tetrameric skeletal muscle ryanodine dine receptor (RyR1)(7,8). In contrast, the cardiac ryanodine receptor, RyR2, appears to bind selectively the FKBP12 homologue, FKBP12.6 (refs 9, 10). To define the functions of FKBP12 in vivo, we generated mutant mice deficient in FKBP12 using embryonic stem (ES) cell technology. FKBP12-deficient mice have normal skeletal muscle but have severe dilated cardiomyopathy and ventricular septal defects that mimic a human congenital heart disorder, noncompaction of left ventricular myocardium(11,12). About 9% of the mutants exhibit exencephaly secondary to a defect in neural tube closure. Physiological studies demonstrate that FKBP12 is dispensable for TGF-beta-mediated signalling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62697/1/391489a0.pd