Studying the RNA silencing pathway with the p19 protein

The origins of the RNA silencing pathway are in defense against invading viruses and in response, viruses have evolved counter‐measures to interfere with the host pathway. The p19 protein is expressed by tombusviruses as a suppressor of RNA silencing and functions to sequester small RNA duplexes, thereby preventing induction of the pathway. p19 exhibits size‐specific and sequence‐independent binding of its small RNA ligands, binding with high affinity to duplexes 20–22 nucleotides long. p19's binding specificity and its ability to sequester small RNAs has made it a unique protein‐based tool for probing the molecular mechanisms of the highly complex RNA silencing pathway in a variety of systems. Furthermore, protein engineering of this 'molecular caliper' promises novel applications in biotechnology and medicine where small RNA molecules are of remarkable interest given their potent gene regulatory abilities

Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) poses a growing threat to global health as it often leads to serious liver diseases and is one of the primary causes for liver transplantation. Currently, no vaccines are available to prevent HCV infection and clinical treatments have limited success. Since HCV has a small proteome, it relies on many host cell proteins to complete its life cycle. In this study, we used a non-directed phenyl sulfonate ester probe (PS4≡) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells.</p> <p>Results</p> <p>The PS4≡ probe successfully targeted 19 active proteins in nine distinct protein families, some that were predominantly labeled <it>in situ </it>compared to the <it>in vitro </it>labeled cell homogenate. Nine proteins revealed altered activity levels during HCV replication. Some candidates identified, such as heat shock 70 kDa protein 8 (or HSP70 cognate), have been shown to influence viral release and abundance of cellular lipid droplets. Other differentially active PS4≡ targets, such as electron transfer flavoprotein alpha, protein disulfide isomerase A5, and nuclear distribution gene C homolog, constitute novel proteins that potentially mediate HCV propagation.</p> <p>Conclusions</p> <p>These findings demonstrate the practicality and versatility of non-directed activity-based protein profiling (ABPP) to complement directed methods and accelerate the discovery of altered protein activities associated with pathological states such as HCV replication. Collectively, these results highlight the ability of <it>in situ </it>ABPP approaches to facilitate the identification of enzymes that are either predominantly or exclusively labeled in living cells. Several of these differentially active enzymes represent possible HCV-host interactions that could be targeted for diagnostic or therapeutic purposes.</p

Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees.</p> <p>Results</p> <p>Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol.</p> <p>Conclusion</p> <p>Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.</p

Studying the RNA silencing pathway with the p19 protein

The origins of the RNA silencing pathway are in defense against invading viruses and in response, viruses have evolved counter-measures to interfere with the host pathway. The p19 protein is expressed by tombusviruses as a suppressor of RNA silencing and functions to sequester small RNA duplexes, thereby preventing induction of the pathway. p19 exhibits size-specific and sequence-independent binding of its small RNA ligands, binding with high affinity to duplexes 20-22 nucleotides long. p19's binding specificity and its ability to sequester small RNAs has made it a unique protein-based tool for probing the molecular mechanisms of the highly complex RNA silencing pathway in a variety of systems. Furthermore, protein engineering of this 'molecular caliper' promises novel applications in biotechnology and medicine where small RNA molecules are of remarkable interest given their potent gene regulatory abilities.Peer reviewed: YesNRC publication: Ye

An Affinity-based Probe for Methyltransferase Enzymes Based on Sinefungin

Epigenetics controls numerous cellular processes, such as gene transcription, signal transduction, and protein stabilization. An understanding of epigenetic mechanisms can lead to the development of therapeutic agents for various diseases. Herein, we report the design and synthesize a sinefungin activity-probe (BpyneSF) that targets methyltranferase enzymes and proteins involved in recognition of methylation. This probe contains a bioorthogonal alkyne residue for conjugation using the copper catalyzed azide-alkyne cycloaddition and a photoactivatable crosslinker group for covalent attachment of the probe to its proteomic targets. We investigate the efficiency and selectivity of the probe to inhibit and label methyltransferase enzymes and we demonstrate, through in-gel fluorescence, on-bead digestion and tandem mass spectrometry, that BpyneSF can label methyltransferase SETD2 and reader proteins in vitro. These results establish the utility of BpyneSF as a tool for activity-based protein profiling in complex biological environments.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Studies of multicomponent Kinugasa reactions in aqueous media

Micelle-promoted, copper-catalyzed multicomponent Kinugasa reactions were studied in aqueous media. Reactions were performed in a \u2018single pot\u2019 for a series of in situ generated C,N-diphenylnitrones with Cu(I) phenylacetylide providing \u3b2-lactams in yields of 45\u201385%. Substituents affect the reaction by either accelerating cycloaddition or minimizing side reactions.Peer reviewed: YesNRC publication: Ye

Host\u2013virus interactions during hepatitis C virus infection : a complex and dynamic molecular biosystem

The hepatitis C virus (HCV) is a global health issue with no vaccine available and limited clinical treatment options. Like other obligate parasites, HCV requires host cellular components of an infected individual to propagate. These host\u2013virus interactions during HCV infection are complex and dynamic and involve the hijacking of host cell environments, enzymes and pathways. Understanding this unique molecular biosystem has the potential to yield new and exciting strategies for therapeutic intervention. Advances in genomics and proteomics have opened up new possibilities for the rapid measurement of global changes at the transcriptional and translational levels during infection. However, these techniques only yield snapshots of host\u2013virus interactions during HCV infection. Other new methods that involve the imaging of biomolecular interactions during HCV infection are required to identify key interactions that may be transient and dynamic. Herein we highlight systems biology based strategies that have helped to identify key host\u2013virus interactions during HCV replication and infection. Novel biophysical tools are also highlighted for identification and visualization of activities and interactions between HCV and its host hepatocyte. As some of these methods mature, we expect them to pave the way forward for further exploration of this complex biosystem and elucidation of mechanisms for HCV pathogenesis and carcinogenesis.Peer reviewed: YesNRC publication: Ye