Information

What is a functional screen?

What is a functional screen?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I was going through this paper, but did not understand a term.
What is the meaning of functional screen?

(I am not a biology student, I don't understand much, and a small simple explanation would be enough)


From the abstract of the linked article (Guttman, 2002):

We used an in vivo genetic screen to identify 13 effectors [… ]. Although sharing little overall homology, the amino-terminal regions of these effectors had strikingly similar amino acid compositions.

And from the body:

The screen relied on the type III secretion signal and the endogenous promoter of the hop gene and was thus highly specific.

Hence, the authors search and identify proteins with a secretory signal under the regulation of a hop promoter. A functional screen in this paper refers to the analysis of protein samples to detect the presence of proteins with a particular function.


Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell.

Results

Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes.

Conclusions

Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.


A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence

Primary fibroblasts respond to activated H-RAS V12 by undergoing premature arrest, which resembles replicative senescence 1 . This irreversible 'fail-safe mechanism' requires p19 ARF , p53 and the Retinoblastoma (Rb) family: upon their disruption, RAS V12 -expressing cells fail to undergo senescence and continue to proliferate 1,2,3,4,5,6,7 . Similarly, co-expression of oncogenes such as c-MYC or E1A rescues RAS V12 -induced senescence. To identify novel genes that allow escape from RAS V12 -induced senescence, we designed an unbiased, retroviral complementary DNA library screen. We report on the identification of DRIL1, the human orthologue of the mouse Bright and Drosophila dead ringer transcriptional regulators. DRIL1 renders primary murine fibroblasts unresponsive to RAS V12 -induced anti-proliferative signalling by p19 ARF /p53/p21 CIP1 , as well as by p16 INK4a . In this way, DRIL1 not only rescues RAS V12 -induced senescence but also causes these fibroblasts to become highly oncogenic. Furthermore, DRIL1 immortalizes mouse fibroblasts, in the presence of high levels of p16 INK4a . Immortalization by DRIL1, whose product binds the pRB-controlled transcription factor E2F1 (ref. 8), is correlated with induction of E2F1 activity. Correspondingly, DRIL1 induces the E2F1 target Cyclin E1, overexpression of which is sufficient to trigger escape from senescence. Thus, DRIL1 disrupts cellular protection against RAS V12 -induced proliferation downstream of the p19 ARF /p53 pathway.


References

Hering, H. & Sheng, M. Dendritic spines: structure, dynamics and regulation. Nature Rev. Neurosci. 2, 880–888 (2001).

Nimchinsky, E. A., Sabatini, B. L. & Svoboda, K. Structure and function of dendritic spines. Annu. Rev. Physiol. 64, 313–353 (2002).

Matsuzaki, M., Honkura, N., Ellis-Davies, G. C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004).

Nagerl, U. V, Eberhorn, N., Cambridge, S. B. & Bonhoeffer, T. Bidirectional activity-dependent morphological plasticity in hippocampal neurons. Neuron 44, 759–767 (2004).

Bagni, C. & Greenough, W. T. From mRNP trafficking to spine dysmorphogenesis: the roots of fragile X syndrome. Nature Rev. Neurosci. 6, 376–387 (2005).

Okamoto, K., Nagai, T., Miyawaki, A. & Hayashi, Y. Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nature Neurosci. 7, 1104–1112 (2004).

Yi, J. J. & Ehlers, M. D. Ubiquitin and protein turnover in synapse function. Neuron 47, 629–632 (2005).

Shi, S. H et al. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284, 1811–1816 (1999).

Zhou, Z. et al. Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation. Neuron 52, 255–269 (2006).

Bingol, B. & Schuman, E. M. Synaptic protein degradation by the ubiquitin proteasome system. Curr. Opin. Neurobiol. 15, 536–541 (2005).

Steward, O. & Schuman, E. M. Protein synthesis at synaptic sites on dendrites. Annu. Rev. Neurosci. 24, 299–325 (2001).

Martin, K. C., Barad, M. & Kandel, E. R. Local protein synthesis and its role in synapse-specific plasticity. Curr. Opin. Neurobiol. 10, 587–592 (2000).

Eberwine, J., Miyashiro, K., Kacharmina, J. E. & Job, C. Local translation of classes of mRNAs that are targeted to neuronal dendrites. Proc. Natl Acad. Sci. USA 98, 7080–7085 (2001).

Ostroff, L. E., Fiala, J. C., Allwardt, B. & Harris, K. M. Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during LTP in developing rat hippocampal slices. Neuron 35, 535–545 (2002).

Wells, D. G., Richter, J. D. & Fallon, J. R. Molecular mechanisms for activity-regulated protein synthesis in the synapto-dendritic compartment. Curr. Opin. Neurobiol. 10, 132–137 (2000).

Kiebler, M. A. & Bassell, G. J. Neuronal RNA granules: movers and makers. Neuron 51, 685–690 (2006).

Krichevsky, A. M. & Kosik, K. S. Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation. Neuron 32, 683–696 (2001).

Ashraf, S. I., McLoon, A. L., Sclarsic, S. M. & Kunes, S. Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila. Cell 124, 191–205 (2006).

Schratt, G. M. et al. A brain-specific microRNA regulates dendritic spine development. Nature 439, 283–289 (2006).

Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004).

Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

Filipowicz, W., Bhattacharyya, S. N. & Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nature Rev. Genet. 9, 102–114 (2008).

Kosik, K. S. The neuronal microRNA system. Nature Rev. Neurosci. 7, 911–920 (2006).

Miska, E. A. et al. Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol. 5, R68 (2004).

Sempere, L. F. et al. Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol. 5, R13 (2004).

Kim, J. et al. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc. Natl Acad. Sci. USA 101, 360–365 (2004).

Yeh, D. C., Duncan, J. A., Yamashita, S. & Michel, T. Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca 2+ -calmodulin. J. Biol. Chem. 274, 33148–33154 (1999).

Rao, A. & Steward, O. Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes. J. Neurosci. 11, 2881–2895 (1991).

Meister, G., Landthaler, M., Dorsett, Y. & Tuschl, T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10, 544–550 (2004).

Zito, K., Knott, G., Shepherd, G. M., Shenolikar, S. & Svoboda, K. Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton. Neuron 44, 321–334 (2004).

Mansfield, J. H. et al. MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nature Genet. 36, 1079–1083 (2004).

Vo, N. et al. A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc. Natl Acad. Sci. USA 102, 16426–16431 (2005).

Rehmsmeier, M., Steffen, P., Hochsmann, M. & Giegerich, R. Fast and effective prediction of microRNA/target duplexes. RNA 10, 1507–1517 (2004).

Kruger, J. & Rehmsmeier, M. RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res. 34, W451–W454 (2006).

Rodenas-Ruano, A., Perez-Pinzon, M. A., Green, E. J., Henkemeyer, M. & Liebl, D. J. Distinct roles for ephrinB3 in the formation and function of hippocampal synapses. Dev. Biol. 292, 34–45 (2006).

Etournay, R. et al. PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa. J. Cell Sci. 118, 2891–2899 (2005).

Ozaki, N. et al. cAMP-GEFII is a direct target of cAMP in regulated exocytosis. Nature Cell Biol. 2, 805–811 (2000).

Husseini Ael, D. et al. Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108, 849–863 (2002).

Linder, M. E. & Deschenes, R. J. Palmitoylation: policing protein stability and traffic. Nature Rev. Mol. Cell Biol. 8, 74–84 (2007).

Kang, R. et al. Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation. Nature 456, 904–909 (2008).

Bhattacharyya, R. & Wedegaertner, P. B. Gα13 requires palmitoylation for plasma membrane localization, Rho-dependent signaling, and promotion of p115-RhoGEF membrane binding. J. Biol. Chem. 275, 14992–14999 (2000).

Kye, M. J. et al. Somatodendritic microRNAs identified by laser capture and multiplex RT-PCR. RNA 13, 1224–1234 (2007).

Lugli, G., Torvik, V. I., Larson, J. & Smalheiser, N. R. Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain. J. Neurochem. 106, 650–661 (2008).

Bernard, O. Lim kinases, regulators of actin dynamics. Int. J. Biochem. Cell Biol. 39, 1071–1076 (2007).

Kurose, H. Gα12 and Gα13 as key regulatory mediator in signal transduction. Life Sci. 74, 155–161 (2003).

Tada, T. & Sheng, M. Molecular mechanisms of dendritic spine morphogenesis. Curr. Opin. Neurobiol. 16, 95–101 (2006).

Obernosterer, G., Leuschner, P. J., Alenius, M. & Martinez, J. Post-transcriptional regulation of microRNA expression. RNA 12, 1161–1167 (2006).

Schratt, G. M., Nigh, E. A., Chen, W. G., Hu, L. & Greenberg, M. E. BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway during neuronal development. J. Neurosci. 24, 9366–9377 (2004).

Paradis, S. et al. An RNAi-based approach identifies molecules required for glutamatergic and GABAergic synapse development. Neuron 53, 217–232 (2007).

Obernosterer, G., Martinez, J. & Alenius, M. Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nature Protocols 2, 1508–1514 (2007).


Abstract

The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein–DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein–DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency.


Chromatin Immunoprecipitation-Based Screen To Identify Functional Genomic Binding Sites for Sequence-Specific Transactivators

FIG. 1 . Analysis of p53, MDM2, and p21 protein levels in MCF-10A cells and HMEC after ADR treatment. Shown is Western blot analysis of p53, MDM2, and p21 protein harvested from MCF-10A and primary HMEC that were not treated (−) or treated (+) with ADR (350 nM) for 8 h. FIG. 2 . Yeast selection system. The candidate upstream activating sequences (UAS) recovered from ChIP were cloned into the pBM947 reporter vector containing the HIS3 gene under the control of a basal GAL1 promoter and a URA1 marker. The pBM947-based library was transformed into an auxotrophic His-deficient yeast strain containing the pRS314SN vector, which expresses a galactose-inducible human wild-type p53 and a TRP1 marker. Yeasts containing both the vectors were grown on galactose-containing, histidine-deficient media (SG-Trp-Ura-His) to assay for the ability of p53 to bind to the potential UAS in the pBM947 vector and activate transcription of the HIS3 gene. Replica plating of all clones on glucose-containing, histidine-deficient media (SD-Trp-Ura-His) was performed to rule out false-positive clones. The clones that grew in the presence of glucose were considered false positive, and only the clones that grew on galactose, and presumably in a p53-dependent manner, were analyzed further. A clone containing a fragment of the p21 promoter encompassing site 1 is indicated as an example of a positive result. FIG. 3 . Analysis of p53 in vivo binding to consensus binding sites. Three sets each of MCF-10A cells, HMEC, and HK cells were identically processed: one set was treated with ADR (350 nM for 5 h) and formaldehyde cross-linked (ADR +, X-L +), another set was not treated with ADR and was formaldehyde cross-linked (ADR −, X-L +), and a final set was treated with ADR and not formaldehyde cross-linked (ADR +, X-L −). The DNA for PCRs was derived from p53-specific and cyclin B1-specific immunoprecipitations (IP) and amplified using primers flanking the p53 response elements in genes encoding the indicated proteins. PCRs were resolved with polyacrylamide gel electrophoresis, and the gels were stained with ethidium bromide. The cyclin B1-specific IPs were included to assess any DNA fragments purified from cross-linked lysates nonspecifically. Input +, genomic input input −, water control. PCR results with primers directed to the coding region of GAPDH serve as a control for nonspecific DNA IP by p53-specific antibodies. FIG. 4 . Comparative analysis of p53 binding to sites in promoter regions of known and candidate target genes. In the left panels, four sets of HMEC were processed as follows: one set was treated with ADR (350 nM for 5 h), formaldehyde cross-linked, and immunoprecipitated with a p53 antibody (solid bars) another set was treated with ADR, formaldehyde cross-linked, and immunoprecipitated with a cyclin B1 antibody (open bars) a third set was treated with ADR, not formaldehyde cross-linked, and immunoprecipitated with a p53 antibody (dotted bars) and a final set was formaldehyde cross-linked but not treated with ADR and then immunoprecipitated with a p53 antibody (gray bars). Quantitative real-time PCR was performed, and each sample was normalized to the same genomic DNA that was isolated from cells that were cross-linked and processed the same with the exception that the immunoprecipitation step was not performed. The binding sites shown are those that were recovered from the library screen (LS), those that were previously reported in the literature (RS), and those that were potential binding sites found by gene analysis using the p53MH algorithm (PS). The base pair match of the binding site to the p53 consensus is shown in parentheses. The results, shown as percentages of input DNA, are from at least three independent experiments, with the error bars representing standard deviations. The right panels show schematics of the genomic structure and localization of known and putative p53 binding sites analyzed. The bar shading indicates species conservation as indicated. Exons are indicated with an E followed by the exon number in either an open box or a shaded box (representing the terminal exon). The sequences of the binding sites present in the regions analyzed are shown, and in parentheses the distances of the binding sites from the start of exon 1 are given. FIG. 5 . p53-dependent regulation of representative candidate target gene expression. The isogenic pair of HCT116 p53 +/+ and p53 −/− cells were treated with ADR (350 nM) for 0, 6, 12, and 24 h the HIp53 cells (p53) and corresponding vector control cell line (Ø) were treated with ponasterone A (10 μM) for 24 h, and the HK cells were infected with a GFP- or p53-expressing adenovirus for 30 h. Total RNA from HCT116 cells and mRNA from HIp53 and HK cells was purified and reverse transcribed and quantitative real-time PCR performed. The samples were normalized to GAPDH, and the results are presented as changes relative to either the 0-h HCT116 p53 +/+ sample (left panel), the HIp53 vector control cells treated with ponasterone (middle panel), or the HK cells infected with a GFP-expressing adenovirus (right panel). The results are the means of three independent experiments (MCF-10A cells and HMEC) or duplicate experiments (HK cells), with error bars representing the standard deviations. Note that the y axes are set to 7.0 with the exceptions of the left and middle panels for the CDKN1A gene and the left and right panels for the EDN-2 gene.

Contents

A functional specification does not define the inner workings of the proposed system it does not include the specification of how the system function will be implemented. Instead, it focuses on what various outside agents (people using the program, computer peripherals, or other computers, for example) might "observe" when interacting with the system.

A functional requirement in a functional specification might state as follows:

When the user clicks the OK button, the dialog is closed and the focus is returned to the main window in the state it was in before this dialog was displayed.

Such a requirement describes an interaction between an external agent (the user) and the software system. When the user provides input to the system by clicking the OK button, the program responds (or should respond) by closing the dialog window containing the OK button.

Purpose Edit

There are many purposes for functional specifications. One of the primary purposes on team projects is to achieve some form of team consensus on what the program is to achieve before making the more time-consuming effort of writing source code and test cases, followed by a period of debugging. Typically, such consensus is reached after one or more reviews by the stakeholders on the project at hand after having negotiated a cost-effective way to achieve the requirements the software needs to fulfill.

  1. To let the developers know what to build.
  2. To let the testers know what tests to run.
  3. To let stakeholders know what they are getting.

Process Edit

In the ordered industrial software engineering life-cycle (waterfall model), functional specification describes what has to be implemented. The next, Systems architecture document describes how the functions will be realized using a chosen software environment. In non industrial, prototypical systems development, functional specifications are typically written after or as part of requirements analysis.

When the team agrees that functional specification consensus is reached, the functional spec is typically declared "complete" or "signed off". After this, typically the software development and testing team write source code and test cases using the functional specification as the reference. While testing is performed, the behavior of the program is compared against the expected behavior as defined in the functional specification.

Methods Edit

One popular method of writing a functional specification document involves drawing or rendering either simple wire frames or accurate, graphically designed UI screenshots. After this has been completed, and the screen examples are approved by all stakeholders, graphical elements can be numbered and written instructions can be added for each number on the screen example. For example, a login screen can have the username field labeled '1' and password field labeled '2,' and then each number can be declared in writing, for use by software engineers and later for beta testing purposes to ensure that functionality is as intended. The benefit of this method is that countless additional details can be attached to the screen examples.


Acknowledgements

We thank Hans Teunissen and Elzo de Wit for their help on the 3C experiment and all members of the Agami laboratory for their technical help and discussions. We are grateful to the NKI Genomics Core Facility for deep-sequencing our samples.

Funding

This work was supported by the ERC-AdG enhReg (322493 to RA), ERC-ITN RNA TRAIN (607720 to RA), China Scholarship Council (CSC) (to LL), The Human Frontier Science Program LT000640/2013 (to APU), and The Dutch Organization for Research NWO-TOP 91216002 (to RA). RE is supported by the Israeli Cancer Association (ICA), with the generous assistance of the ICA Netherlands friends, and by the Marguerite Stolz Research Fellowship Fund. ZM was supported in part by the Gad, Nava, and Shye Shtacher fellowship. RE is a Faculty Fellow of the Edmond J. Safra Center for Bioinformatics at Tel Aviv University.

Availability of data and materials

RNA-seq data are available from the GEO DB accession number GSE112458 [50]. GRO-seq data are available from the GEO DB accession number GSE109290 [51].


References

Anderson, P. & Kedersha, N. RNA granules. J. Cell Biol. 172, 803–808 (2006).

Anderson, P. & Kedersha, N. Stress granules: the Tao of RNA triage. Trends. Biochem. Sci. 33, 141–150 (2008).

Kedersha, N. & Anderson, P. Mammalian stress granules and processing bodies. Methods Enzymol. 431, 61–81 (2007).

Parker, R. & Sheth, U. P bodies and the control of mRNA translation and degradation. Mol. Cell 25, 635–646 (2007).

Eulalio, A., Behm-Ansmant, I. & Izaurralde, E. P bodies: at the crossroads of post-transcriptional pathways. Nature Rev. Mol. Cell Biol. 8, 9–22 (2007).

Kedersha, N. et al. Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol. Biol. Cell 13, 195–210 (2002).

Kimball, S. R., Horetsky, R. L., Ron, D., Jefferson, L. S. & Harding, H. P. Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. Am. J. Physiol. Cell Physiol. 284, C273–C284 (2003).

Kwon, S., Zhang, Y. & Matthias, P. The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response. Genes Dev. 21, 3381–3394 (2007).

Eulalio, A., Behm-Ansmant, I., Schweizer, D. & Izaurralde, E. P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol. Cell. Biol. 27, 3970–3981 (2007).

Sheth, U. & Parker, R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808 (2003).

Lykke-Andersen, J. & Wagner, E. Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. Genes Dev. 19, 351–361 (2005).

Franks, T. M. & Lykke-Andersen, J. TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements. Genes Dev. 21, 719–735 (2007).

Kedersha, N. et al. Stress granules and processing bodies are dynamically liked sites of mRNP remodeling. J. Cell Biol. 169, 871–884 (2005).

Kedersha, N., Tisdale, S., Hickman, T. & Anderson, P. Methods Enzymol. (in the press).

Cougot, N., Babajko, S. & Seraphin, B. Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol. 165, 31–40 (2004).

Kedersha, N. et al. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J. Cell Biol. 151, 1257–1268 (2000).

Hou, J. C. & Pessin, J. E. Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking. Curr. Opin. Cell Biol. 19, 466–473 (2007).

Love, D. C. & Hanover, J. A. The hexosamine signaling pathway: deciphering the 'O-GlcNAc code'. Sci. STKE 2005, re13 (2005).

Marshall, S. Role of insulin, adipocyte hormones, and nutrient-sensing pathways in regulating fuel metabolism and energy homeostasis: a nutritional perspective of diabetes, obesity, and cancer. Sci. STKE 2006, re7 (2006).

Slawson, C., Housley, M. P. & Hart, G. W. O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks. J. Cell. Biochem. 97, 71–83 (2006).

Zachara, N. E. & Hart, G. W. Cell signaling, the essential role of O-GlcNAc! Biochim. Biophys. Acta 1761, 599–617 (2006).

Zachara, N. E. et al. Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J. Biol. Chem. 279, 30133–30142 (2004).

Jones, S. P. et al. Cardioprotection by N-acetylglucosamine linkage to cellular proteins. Circulation 117, 1172–1182 (2008).

Zachara, N. E. The sweet nature of cardioprotection. Am. J. Physiol. Heart Circ. Physiol. 293, H1324–H1326 (2007).

Zachara, N. E. & Hart, G. W. O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim. Biophys. Acta 1673, 13–28 (2004).

Cheung, W. D. & Hart, G. W. AMP-activated protein kinase and p38 MAPK activate O-GlcNAcylation of neuronal proteins during glucose deprivation. J. Biol. Chem. 283, 13009–13020 (2008).

Taylor, R. P. et al. Glucose deprivation stimulates O-GlcNAc modification of proteins through up-regulation of O-linked N-acetylglucosaminyltransferase. J. Biol. Chem. 283, 6050–6057 (2008).

Morris, N. J. et al. Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes. J. Biol. Chem. 273, 3582–3587 (1998).

Nielsen, M. S. et al. The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein. EMBO J. 20, 2180–2190 (2001).

Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol. Cell Proteomics 1, 791–804 (2002).

Dai, M. S. & Lu, H. Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5. J. Biol. Chem. 279, 44475–44482 (2004).

Mazroui, R., Di Marco, S., Kaufman, R. J. & Gallouzi, I. E. Inhibition of the ubiquitin–proteasome system induces stress granule formation. Mol. Biol. Cell 18, 2603–2618 (2007).

Gilks, N. et al. Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol. Biol. Cell 15, 5383–5398 (2004).

Brengues, M. & Parker, R. Accumulation of polyadenylated mRNA, Pab1p, eIF4E, and eIF4G with P-bodies in Saccharomyces cerevisiae. Mol. Biol. Cell 18, 2592–2602 (2007).

Hoyle, N. P., Castelli, L. M., Campbell, S. G., Holmes, L. E. & Ashe, M. P. Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies. J. Cell Biol. 179, 65–74 (2007).

Stoecklin, G., Mayo, T. & Anderson, P. ARE-mRNA degradation requires the 5′-3′ decay pathway. EMBO Rep. 7, 72–77 (2006).


Anatomy of a molecule: What makes remdesivir unique?

The World Health Organization in late January convened experts to discuss experimental therapeutics for patients with the emerging coronavirus with no name, no vaccine and no treatment. The panel reported that &ldquoamong the different therapeutic options, remdesivir was considered the most promising candidate.&rdquo

Within weeks, a clinical trial of the compound was underway in China. Results are expected in April in the meantime, the outbreak of SARS-nCoV-2, the virus that causes COVID-19, has become a global pandemic.

Remdesivir is a nucleoside analog, one of the oldest classes of antiviral drugs. It works by blocking the RNA polymerase that coronaviruses and related RNA viruses need to replicate their genomes and proliferate in the host body.

The molecule originally was synthesized as part of a screen for inhibitors of the hepatitis C virus RNA polymerase. Its inventors at Gilead Sciences decided to move forward with a different nucleoside analog compound to treat hepatitis C. But RNA-dependent RNA polymerases are conserved between many viruses. Experiments in vitro, in cell culture and in animal models have shown that remdesivir has broad-spectrum activity against RNA viruses, including filoviruses (like the one that causes Ebola) and coronaviruses.

Remdesivir resembles the RNA base adenosine, shown here as a monophosphate.

The compound and ATP have some important differences, but some features are very similar. ASBMB Today spoke to medicinal chemist Katherine Seley&ndashRadtke at the University of Maryland, Baltimore County, and structural virologist Craig Cameron at the University of North Carolina, Chapel Hill about what makes the molecule interesting. Click on a feature marked in blue to read their remarks.

3&rsquo hydroxy group

Different classes of nucleoside/nucleotide analogs have different effects on polymerases. Remdesivir is in a class called nonobligate chain terminators, because it should, in theory, be possible to add more nucleotides to a strand of RNA after remdesivir has been added due to the presence of the hydroxyl group at carbon 3 in the sugar.

&ldquoThat hydroxy group is what is required for continued synthesis of nucleic acid, whether it be RNA or DNA,&rdquo said virologist Craig Cameron, a professor at the University of North Carolina at Chapel Hill who studies the interactions between nucleoside analogs and viral polymerases.

Recent research suggests that when mixed with RNA polymerases from coronaviruses or flaviviruses in vitro, remdesivir doesn&rsquot terminate the synthesis of a new RNA strand right away. Instead, Cameron said, &ldquoIt takes a few cycles of nucleotide addition before you can see the termination effect.&rdquo

Those additional nucleotides may help shield remdesivir from coronavirus proofreading enzymes that are known to remove unnatural nucleotide analogs.

Base pairing to uracil

In adenosine in double-stranded RNA, this face of the molecule is involved in base-pairing with uracil. The two nitrogens act as proton donor and acceptor, respectively for hydrogen bonds to atoms in the uracil base.

Chemists think that remdesivir, by presenting a very similar binding face, gets incorporated into a growing RNA strand by viral polymerases.

C-nucleoside bond

The link between ribose and the base is called the glycosidic bond. Usually, it connects the 1&rsquo carbon in the ribose ring to a nitrogen in the base. But in remdesivir (and some other nucleotide analogs) the sugar and the nucleobase are connected by a bond between two carbons.

&ldquoIt definitely provides much greater stability (against) nucleases and other enzymes that can cleave the nucleobase from the sugar,&rdquo said Katherine Seley-Radtke, a medicinal chemist at the University of Maryland, Baltimore County who works on the design and synthesis of antiviral nucleoside analogues. With a C-nucleoside, &ldquoyou&rsquod have to break a carbon-carbon bond, whereas in a normal nucleoside you&rsquore breaking a hemi-aminal bond, which is actually fairly unstable. So having that carbon-carbon bond is a great advantage.&rdquo

1&rsquo cyano group

Ask a group of chemists what jumps out at them about remdesivir, and most will start with this dramatic feature. Substitution at this carbon is unusual, and probably only possible because of the strength of the C-nucleoside bond.

According to an article in the Journal of Medicinal Chemistry, the cyano group was initially added because a precursor molecule, a very effective inhibitor of viral RNA polymerases, also blocked the mitochondrial RNA polymerase in mice. To make a molecule without those toxic side effects, chemists at Gilead tried a series of substitutions at the 1&rsquo carbon. The compound with the cyano group worked best: it still blocked the hepatitis C polymerase, but was no longer incorporated by host cell polymerases.

&ldquoYou can&rsquot predict activity. You have to make it and test it,&rdquo Seley-Radtke said. &ldquoBut even small changes can have amazing consequences.&rdquo

Phosphate

&ldquoYou see all that flotsam and jetsam coming off at the 5&rsquo hydroxyl?&rdquo said Katherine Seley-Radtke. Among medicinal chemists, this type of protecting group is casually known as &ldquoa McGuigan protide.&rdquo Designed by medicinal chemist Chris McGuigan in the 1990s, this type of protecting group and its variations are widely used to deliver nucleotide analogs into cells.

&ldquoIt is a brilliant system, because it accomplishes two things,&rdquo Seley-Radtke said. &ldquoNo. 1, an issue with nucleosides is that they&rsquore polar and their phosphates are even more polar.&rdquo Masking the highly negative phosphate groups with esters or amides reduces the molecule&rsquos overall polarity, letting it cross the plasma membrane into cells.

Second, in order to be recognized by polymerases, the analog needs to resemble a normal nucleotide triphosphate&mdashwhich means it needs to be phosphorylated.

&ldquoThe first phosphorylation, either by cellular or viral kinases, is oftentimes very difficult,&rdquo Seley-Radtke said. &ldquoA lot of those kinases are very, very picky in terms of recognition.&rdquo By arriving in the cell with its first phosphate already in tow, remdesivir and related nucleotide analogs skip that rate-limiting step. After the protecting groups are cleaved, the nucleotide analog is a reasonable substrate for later nucletodie kinases.


Watch the video: What is a Functional Movement Screen? (December 2022).