The rockefeller university

Accumulation of neurotoxic amyloid-beta is a major hallmark of Alzheimer's disease(1). Formation of amyloid-beta is catalysed by gamma-secretase, a protease with numerous substrates(2,3). Little is known about the molecular mechanisms that confer substrate specificity on this potentially promiscuous enzyme. Knowledge of the mechanisms underlying its selectivity is critical for the development of clinically effective gamma-secretase inhibitors that can reduce amyloid-beta formation without impairing cleavage of other gamma-secretase substrates, especially Notch, which is essential for normal biological functions(3,4). Here we report the discovery of a novel gamma-secretase activating protein (GSAP) that drastically and selectively increases amyloid-beta production through a mechanism involving its interactions with both gamma-secretase and its substrate, the amyloid precursor protein carboxy-terminal fragment (APP-CTF). GSAP does not interact with Notch, nor does it affect its cleavage. Recombinant GSAP stimulates amyloid-beta production in vitro. Reducing GSAP concentrations in cell lines decreases amyloid-beta concentrations. Knockdown of GSAP in a mouse model of Alzheimer's disease reduces levels of amyloid-beta and plaque development. GSAP represents a type of gamma-secretase regulator that directs enzyme specificity by interacting with a specific substrate. We demonstrate that imatinib, an anticancer drug previously found to inhibit amyloid-beta formation without affecting Notch cleavage 5, achieves its amyloid-beta-lowering effect by preventing GSAP interaction with the gamma-secretase substrate, APP-CTF. Thus, GSAP can serve as an amyloid-beta-lowering therapeutic target without affecting other key functions of gamma-secretase.

Genetic defects in the IL-12-IL-23/IFN-gamma circuit confer Mendelian susceptibility to mycobacteria and salmonella. The IL-12/IFN-gamma axis is essential for anti-tumoral immunity in mice. Cancer susceptibility has not been recognised in these patients so far. We report three relatives with IL-12R beta 1 deficiency. At the age of 25 years old, one patient presented with oesophageal squamous cell carcinoma (OSCC). The patient had no previous risk factors for OSCC. He died at the age of 29 years. OSCC is exceedingly rare in individuals under 30 years and frecuently relates to alcohol intake and smoking. Disorders of the IL-12-IL-23/IFN-gamma axis may predispose to cancer.

The inner ear achieves a wide dynamic range of responsiveness by mechanically amplifying weak sounds. The enormous mechanical gain reported for the mammalian cochlea, which exceeds a factor of 4000, poses a challenge for theory. Here we show how such a large gain can result from an interaction between amplification by low-gain hair bundles and a pressure wave: hair bundles can amplify both their displacement per locally applied pressure and the pressure wave itself. A recently proposed ratchet mechanism, in which hair-bundle forces do not feed back on the pressure wave, delineates the two effects. Our analytical calculations with a WKB approximation agree with numerical solutions.

TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TIEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs.

Hearing depends on reliable and temporally precise neurotransmission by cochlear hair cells. The wide dynamic range and high sensitivity with which these cells encode acoustic stimuli are associated with a presynaptic specialization termed the presynaptic dense body or synaptic ribbon. Apposed to the presynaptic density, this spherical or flattened structure tethers a layer of synaptic vesicles and is thought to facilitate their exocytotic fusion. Although defining the molecular constituents of the hair cell's synaptic ribbon should contribute to our understanding of neurotransmitter release at this synapse, accomplishing this task has been slowed by the difficulty of obtaining sufficient amounts of starting material for protein analysis from hair cells. We isolated synaptic material from chicken cochleas, purified synaptic ribbons with specific immunological reagents, and identified the associated proteins by tandem mass spectrometry. Purification of the ribbons revealed a predominant composition of C-terminal-binding proteins, especially ribeye, in association with the small GTPase Rab3, which is possibly involved in attaching vesicles to the ribbon. In comparison with the components of conventional synapses and of retinal ribbon synapses, we observed that certain regulatory proteins are excluded from the hair cell's synapse. Using antisera against several of the novel proteins and membrane-trafficking components that we had identified, we documented their localization in isolated hair cells. Our results indicate that the ribbon synapses of hair cells display modifications to the presynaptic machinery that are associated with the high-fidelity transmission of acoustic signals to the brain.

A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has the potential to provide access to the chemical diversity encoded in the genomes of uncultured bacteria. One of the challenges facing this approach has been that many natural product biosynthetic gene clusters are too large to be readily captured on a single fragment of cloned eDNA. The reassembly of large eDNA-derived natural product gene clusters from collections of smaller overlapping clones represents one potential solution to this problem. Unfortunately, traditional methods for the assembly of large DNA sequences from multiple overlapping clones can be technically challenging. Here we present a general experimental framework that permits the recovery of large natural product biosynthetic gene clusters on overlapping soil-derived eDNA cosmid clones and the reassembly of these large gene clusters using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. The development of practical methods for the rapid assembly of biosynthetic gene clusters from collections of overlapping eDNA clones is an important step toward being able to functionally study larger natural product gene clusters from uncultured bacteria. (C) 2010 Wiley Periodicals, Inc. Biopolymers 93: 833-844, 2010.

An examination of the micro-organization of visual cortex using two-photon calcium imaging provides a new level of insight into retinotopic maps, finding that retinotopy is scrambled on fine scales in mouse primary visual cortex.

BAFF and APRIL are innate immune mediators that trigger immunoglobulin G (IgG) and IgA class-switch recombination (CSR) in B cells by engaging the receptor TACI. The mechanism that underlies CSR signaling by TACI remains unknown. Here we found that the cytoplasmic domain of TACI encompasses a conserved motif that bound MyD88, an adaptor that activates transcription factor NF-kappa B signaling pathways via a Toll-interleukin 1 (IL-1) receptor (TIR) domain. TACI lacks a TIR domain, yet triggered CSR via the DNA-editing enzyme AID by activating NF-kappa B through a Toll-like receptor (TLR)-like MyD88-IRAK1-IRAK4-TRAF6-TAK1 pathway. TACI-induced CSR was impaired in mice and humans lacking MyD88 or the kinase IRAK4, which indicates that MyD88 controls a B cell-intrinsic, TIR-independent, TACI-dependent pathway for immunoglobulin diversification.

The study of gene regulation in cells has recently begun to shift from a period dominated by the study of transcription factor-DNA interactions to a new focus on RNA regulation. This was sparked by the still-emerging recognition of the central role for RNA in cellular complexity emanating from the RNA World hypothesis, and has been facilitated by technologic advances, in particular high throughput RNA sequencing and crosslinking methods (RNA-Seq, CLIP, and HITS-CLIP). This study will place these advances in context, and, focusing on CLIP, will explain the method, what it can be used for, and how to approach using it. Examples of the successes, limitations, and future of the technique will be discussed. (C) 2010 John Wiley & Sons, Ltd. WIREs RNA 2010 1 266-286

We reported previously that the stability of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs) depends on their interaction with Tel2, the ortholog of yeast Tel2 and Caenorhabditis elegans Clk-2. Here we provide evidence that Tel2 acts with Hsp90 in the maturation of PIKK complexes. Quantitative immuno-blotting showed that the abundance of Tel2 is low compared with the PIKKs, and Tel2 preferentially bound newly synthesized ATM, ATR, mTOR, and DNA-PKcs. Tel2 complexes contained, in addition to Tti1-Tti2, the Hsp90 chaperone, and inhibition of Hsp90 interfered with the interaction of Tel2 with the PIKKs. Analysis of in vivo labeled nascent protein complexes showed that Tel2 and Hsp90 mediate the formation of the mTOR TORC1 and TORC2 complexes and the association of ATR with ATRIP. The structure of yeast Tel2, reported here, shows that Tel2 consists of HEAT-like helical repeats that assemble into two separate a-solenoids. Through mutagenesis, we identify a surface patch of conserved residues involved in binding to the Tti1-Tti2 complex in vitro. In vivo, mutation of this conserved patch affects cell growth, levels of PIKKs, and ATM/ATR-mediated checkpoint signaling, highlighting the importance of Tti1-Tti2 binding to the function of Tel2. Taken together, our data suggest that the Tel2-Tti1-Tti2 complex is a PIKK-specific cochaperone for Hsp90.