The rockefeller university

Protein lipidation and lipid trafficking control many key biological functions in all kingdoms of life. The discovery of diverse lipid species and their covalent attachment to many proteins has revealed a complex and regulated network of membranes and lipidated proteins that are central to fundamental aspects of physiology and human disease. Given the complexity of lipid trafficking and the protein targeting mechanisms involved with membrane lipids, precise and sensitive methods are needed to monitor and identify these hydrophobic molecules in bacteria, yeast, and higher eukaryotes. Although many analytical methods have been developed for characterizing membrane lipids and covalently modified proteins, traditional reagents and approaches have limited sensitivity, do not faithfully report on the lipids of interest, or are not readily accessible. The invention of bloorthogonal ligation reactions, such as the Staudinger ligation and azide-alkyne cycloadditions, has provided new tools to address these limitations, and their use has begun to yield fresh insight into the biology of protein lipidation and lipid trafficking. In this Account, we discuss how these new bioorthogonal ligation reactions and lipid chemical reporters afford new opportunities for exploring the biology of lipid-modified proteins and lipid trafficking. Lipid chemical reporters from our laboratory and several other research groups have enabled improved detection and large-scale proteomic analysis of fatty-acylated and prenylated proteins. For example, fatty acid and isoprenoid chemical reporters in conjunction with bioorthogonal ligation methods have circumvented the limited sensitivity and hazards of radioactive analogues, allowing rapid and robust fluorescent detection of lipidated proteins in all organisms tested. These chemical tools have revealed alterations in protein lipidation in different cellular states and are beginning to provide unique insights in mechanisms of regulation. Notably, the purification of proteins labeled with lipid chemical reporters has allowed both the large-scale analysis of lipidated proteins as well as the discovery of new lipidated proteins involved in metabolism, gene expression, and innate Immunity. Specific lipid reporters have also been developed to monitor the trafficking of soluble lipids; these species are enabling bioorthogonal imaging of membranes in cells and tissues. Future advances in bioorthogonal chemistry, specific lipid reporters, and spectroscopy should provide important new insight into the functional roles of lipidated proteins and membranes in biology.

Transmission of Plasmodium species from a mammalian host to the mosquito vector requires the uptake, during an infected blood meal, of gametocytes, the precursor cells of the gametes. Relatively little is known about the molecular mechanisms involved in the developmental switch from asexual development to sexual differentiation or the maturation and survival of gametocytes. Here, we show that a gene coding for a novel putative transporter, NPT1, plays a crucial role in the development of Plasmodium berghei gametocytes. Parasites lacking NPT1 are severely compromised in the production of gametocytes and the rare gametocytes produced are unable to differentiate into fertile gametes. This is the earliest block in gametocytogenesis obtained by reverse genetics and the first to demonstrate the role of a protein with a putative transport function in sexual development. These results and the high degree of conservation of NPT1 in Plasmodium species suggest that this protein could be an attractive target for the development of novel drugs to block the spread of malaria.

We present a study of B-s(0) decays to the CP-odd final state J/psi f(0)(980) with J/psi -> mu(+)mu(-) and f(0)(980) -> pi(+)pi(-). Using p(p) over bar $ collision data with an integrated luminosity of 3.8 fb(-1) collected by the CDF II detector at the Tevatron we measure a B-s(0) lifetime of tau(B-s(0) -> J/psi f(0)(980)) = 1.70(-0.11)(+0.12)(stat) +/- 0.03(syst)ps. This is the first measurement of the B-s(0) lifetime in a decay to a CP eigenstate and corresponds in the standard model to the lifetime of the heavy B-s(0) eigenstate. We also measure the product of branching fractions of B-s(0) -> J/psi f(0)(980) and f(0)(980) -> pi(+)pi(-) relative to the product of branching fractions of B-s(0) -> J/psi phi and phi -> K+K- to be R-f0/phi = 0.257 +/- 0: 020(stat) +/- 0.014(syst), which is the most precise determination of this quantity to date.

Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.

Pluripotency of embryonic stem cells (ESCs) is defined by their ability to differentiate into three germ layers and derivative cell types' and is established by an interactive network of proteins including OCT4 (also known as POU5F1; ref. 4), NANOG (refs 5,6), SOX2 (ref. 7) and their binding partners. The forkhead box 0 (FoxO) transcription factors are evolutionarily conserved regulators of longevity and stress response whose function is inhibited by AKT protein kinase. Fox() proteins are required for the maintenance of somatic and cancer stem cells(8-13); however, their function in ESCs is unknown. We show that FOXO1 is essential for the maintenance of human ESC pluripotency, and that an orthologue of FOXO1 (Foxo1) exerts a similar function in mouse ESCs. This function is probably mediated through direct control by FOXO1 of OCT4 and SOX2 gene expression through occupation and activation of their respective promoters. Finally, AKT is not the predominant regulator of FOXO1 in human ESCs. Together these results indicate that FOXO1 is a component of the circuitry of human ESC pluripotency. These findings have critical implications for stem cell biology, development, longevity and reprogramming, with potentially important ramifications for therapy.

The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg(2+) was a more potent mediator of attachment than was Ca(2+). The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing.

Cortical areas that directly receive sensory inputs from the thalamus were long thought to be exclusively dedicated to a single modality, originating separate labeled lines. In the past decade, however, several independent lines of research have demonstrated cross-modal responses in primary sensory areas. To investigate whether these responses represent behaviorally relevant information, we carried out neuronal recordings in the primary somatosensory cortex (S1) and primary visual cortex (V1) of rats as they performed whisker-based tasks in the dark. During the free exploration of novel objects, V1 and S1 responses carried comparable amounts of information about object identity. During execution of an aperture tactile discrimination task, tactile recruitment was slower and less robust in V1 than in S1. However, V1 tactile responses correlated significantly with performance across sessions. Altogether, the results support the notion that primary sensory areas have a preference for a given modality but can engage in meaningful cross-modal processing depending on task demand.

G protein-gated K+ channels (Kir3.1-Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here, we present the first crystal structures of a G protein-gated K+ channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G proteins could open a G loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP2 suggest that G proteins open only the G loop gate in the absence of PIP2, but in the presence of PIP2 the G loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na+ ion-binding site, which would allow intracellular Na+ to modulate GIRK channel activity. These data provide a structural basis for understanding multiligand regulation of GIRK channel gating.

This article reports a measurement of the production cross section of prompt isolated photon pairs in proton-antiproton collisions at root s = 1.96 TeV using the CDF II detector at the Fermilab Tevatron collider. The data correspond to an integrated luminosity of 5.36 fb(-1). The cross section is presented as a function of kinematic variables sensitive to the reaction mechanisms. The results are compared with three perturbative QCD calculations: (1) a leading-order parton shower Monte Carlo, (2) a fixed next-to-leading-order calculation and (3) a next-to-leading-order/next-to-next-to-leading-log resummed calculation. The comparisons show that, within their known limitations, all calculations predict the main features of the data, but no calculation adequately describes all aspects of the data.

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.