The rockefeller university

All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential similar to 540-kDa AAA+ (AT-Pases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.

Protein-facilitated shape and topology changes of cell membranes are crucial for many biological processes, such as cell division, protein trafficking, and cell signaling. However, the inherently multiscale nature of membrane remodeling presents a considerable challenge for understanding the mechanisms and physics that drive this process. To address this problem, a multiscale approach that makes use of a diverse set of computational and experimental techniques is required. The atomistic simulations provide high-resolution information on protein-membrane interactions. Experimental techniques, like electron microscopy, on the other hand, resolve high-order organization of proteins on the membrane. Coarse-grained (CG) and mesoscale computational techniques provide the intermediate link between the two scales and can give new insights into the underlying mechanisms. In this Review, we present the recent advances in multiscale computational approaches established in our group. We discuss various CG and mesoscale approaches in studying the protein-mediated large-scale membrane remodeling. (C) 2016 Elsevier Inc. All rights reserved.

Normal brain function depends on the interaction between highly specialized neurons that operate within anatomically and functionally distinct brain regions. Neuronal specification is driven by transcriptional programs that are established during early neuronal development and remain in place in the adult brain. The fidelity of neuronal specification depends on the robustness of the transcriptional program that supports the neuron type-specific gene expression patterns. Here we show that polycomb repressive complex 2 (PRC2), which supports neuron specification during differentiation, contributes to the suppression of a transcriptional program that is detrimental to adult neuron function and survival. We show that PRC2 deficiency in striatal neurons leads to the de-repression of selected, predominantly bivalent PRC2 target genes that are dominated by self-regulating transcription factors normally suppressed in these neurons. The transcriptional changes in PRC2-deficient neurons lead to progressive and fatal neurodegeneration in mice. Our results point to a key role of PRC2 in protecting neurons against degeneration.

Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here, we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (ORF; similar to 5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal. These traits were beneficial, had prion-like patterns of inheritance, were common in wild yeasts, and could be transmitted to naive cells with protein alone. Most inducing proteins were not known prions and did not form amyloid. Instead, they are highly enriched in nucleic acid binding proteins with large intrinsically disordered domains that have been widely conserved across evolution. Thus, our data establish a common type of protein-based inheritance through which intrinsically disordered proteins can drive the emergence of new traits and adaptive opportunities.

Organisms from all domains of life are infected by viruses. In eukaryotes, serine/threonine kinases play a central role in antiviral response. Bacteria, however, are not commonly known to use protein phosphorylation as part of their defense against phages. Here we identify Stk2, a staphylococcal serine/threonine kinase that provides efficient immunity against bacteriophages by inducing abortive infection. A phage protein of unknown function activates the Stk2 kinase. This leads to the Stk2-dependent phosphorylation of several proteins involved in translation, global transcription control, cell-cycle control, stress response, DNA topology, DNA repair, and central metabolism. Bacterial host cells die as a consequence of Stk2 activation, thereby preventing propagation of the phage to the rest of the bacterial population. Our work shows thatmechanisms of viral defense that rely on protein phosphorylation constitute a conserved antiviral strategy across multiple domains of life.

Purpose of reviewTo evaluate how the genomic, transcriptomic, and proteomic profiles of allergic skin diseases, like atopic dermatitis and allergic contact dermatitis, contribute to their understanding and promote their therapeutic development.Recent findingsThe -omics' revolution has facilitated the quantification of inflammatory skin diseases at the molecular level, expanding our understanding of disease pathogenesis. It has also greatly expanded once-limited treatment options and improved the ability to define posttreatment improvements, beyond clinical scores. The findings on the genomic/transcriptomic level are also complemented by proteomic data, contributing to the understanding of the later changes taking place in the final stages of protein formation. Atopic dermatitis is defined as a Th2/Th22 polarized disease with some contributions of Th17 and Th1 pathways. In atopic dermatitis, studies of biologics and small molecules, targeting specific pathways upregulated in atopic dermatitis, seem to provide well tolerated alternatives to conventional immunosuppressive therapies (i.e. corticosteroids and cyclosporine A), particularly for severe patients. Allergic contact dermatitis is defined as having Th1/Th17-centered inflammation, especially with nickel-induced disease, but additional pathways, including Th2 and Th22, are upregulated with other allergens (i.e. fragrance).SummarySupplementing studies of allergic skin diseases with -omics' approaches are transforming the pathogenic understanding, diagnosis and, perhaps, also the treatment of these diseases.

Dietary unsaturated fatty acids, such as oleic acid, have been shown to be covalently incorporated into a small subset of proteins, but the generality and diversity of this protein modification has not been studied. We synthesized unsaturated fatty-acid chemical reporters and determined their protein targets in mammalian cells. The reporters can induce the formation of lipid droplets and be incorporated site-specifically onto known fatty-acylated proteins and label many proteins in mammalian cells. Quantitative proteomics analysis revealed that unsaturated fatty acids modify similar protein targets to saturated fatty acids, including several immunity-associated proteins. This demonstrates that unsaturated fatty acids can directly modify many proteins to exert their unique and often beneficial physiological effects in vivo.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen-and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

Axon degeneration is a tightly regulated, self-destructive program that is a critical feature of many neurodegenerative diseases, but the molecular mechanisms regulating this program remain poorly understood. Here, we identify S-phase kinase-associated protein 1A (Skp1a), a core component of a Skp/Cullin/F-box (SCF)-type E3 ubiquitin ligase complex, as a critical regulator of axon degeneration after injury in mammalian neurons. Depletion of Skp1a prolongs survival of injured axons in vitro and in the optic nerve in vivo. We demonstrate that Skp1a regulates the protein level of the nicotinamide adenine dinucleotide (NAD)(+) synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) in axons. Loss of axonal Nmnat2 contributes to a local ATP deficit that triggers axon degeneration. Knockdown of Skp1a elevates basal levels of axonal Nmnat2, thereby delaying axon degeneration through prolonged maintenance of axonal ATP. Consistent with Skp1a functioning through regulation of Nmnat2, Skp1a knockdown fails to protect axons from Nmnat2 knockdown. These results illuminate the molecular mechanism underlying Skp1adependent axonal destruction.

In a channel flow, the velocity fluctuations are inhomogeneous and anisotropic. Yet, the small-scale properties of the flow are expected to behave in an isotropic manner in the very-large-Reynolds-number limit. We consider the statistical properties of small-scale velocity fluctuations in a turbulent channel flow at moderately high Reynolds number (Re-tau approximate to 1000), using the Johns Hopkins University Turbulence Database. Away from the wall, in the logarithmic layer, the skewness of the normal derivative of the streamwise velocity fluctuation is approximately constant, of order 1, while the Reynolds number based on the Taylor scale is R-lambda approximate to 150. This defines a small-scale anisotropy that is stronger than in turbulent homogeneous shear flows at comparable values of R-lambda In contrast, the vorticity-strain correlations that characterize homogeneous isotropic turbulence are nearly unchanged in channel flow even though they do vary with distance from the wall with an exponent that can be inferred from the local dissipation. Our results demonstrate that the statistical properties of the fluctuating velocity gradient in turbulent channel flow are characterized, on one hand, by observables that are insensitive to the anisotropy, and behave as in homogeneous isotropic flows, and on the other hand by quantities that are much more sensitive to the anisotropy. How this seemingly contradictory situation emerges from the simultaneous action of the flux of energy to small scales and the transport of momentum away from the wall remains to be elucidated.