Allison R. Sherratt

Strain-promoted cycloadditions involving nitrones and alkynes — rapid tunable reactions for bioorthogonal labeling

The development and applications of strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions have brought about new tools for rapid and specific functionalization of biomolecules in different settings. While a number of strain-promoted reactions have been successfully developed, SPANC reactions offer high reactivity with bimolecular rate constants of k2 that are as fast as 60M(-1)s(-1). SPANC reactions also offer stability of starting materials, particularly in the case of endocyclic nitrones, as well as stereoelectronic tunability of the nitrone moiety to optimize reactivity towards different alkyne reaction partners. Herein we discuss recent advances in the development of SPANC reactions and their applications in bioorthogonal labeling.

Activity-Based Protein Profiling of the Escherichia coli GlpG Rhomboid Protein Delineates the Catalytic Core

Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site. Here we show that a highly reactive and sterically unencumbered fluorophosphonate activity-based protein profiling probe can be used to report on the catalytic integrity of active site residues in the Escherichia coli GlpG protein. We used results obtained with this probe on GlpG in proteomic samples, in combination with a conventional assay of proteolytic function on purified samples, to identify residues that are located on the cytoplasmic side of the lipid bilayer that are required for maximal proteolytic activity. Regions tested include the 90-residue aqueous-exposed N-terminus that encompasses a globular structure that we have determined by solution nuclear magnetic resonance, along with residues on the cytoplasmic side of the transmembrane domain core. While in most cases mutation or elimination of these residues did not significantly alter the catalytic status of the GlpG active site, the lipid-facing residue Arg227 was found to be important for maintaining a catalytically competent active site. In addition, we found a functionally critical region outside the transmembrane domain (TMD) core that is required for maximal protease activity. This region encompasses an additional 8-10 residues on the N-terminal side of the TMD core that precedes the first transmembrane segment and was not previously known to play a role in rhomboid function. These findings highlight the utility of the activity-based protein profiling approach for the characterization of rhomboid function.

Kinugasa Reactions in Water: From Green Chemistry to Bioorthogonal Labelling

The Kinugasa reaction has become an efficient method for the direct synthesis of β-lactams from substituted nitrones and copper(I) acetylides. In recent years, the reaction scope has been expanded to include the use of water as the solvent, and with micelle-promoted [3+2] cycloadditions followed by rearrangement furnishing high yields of β-lactams. The high yields of stable products under aqueous conditions render the modified Kinugasa reaction amenable to metabolic labelling and bioorthogonal applications. Herein, the development of methods for use of the Kinugasa reaction in aqueous media is reviewed, with emphasis on its potential use as a bioorthogonal coupling strategy.

Insights into the effect of detergents on the full-length rhomboid protease from Pseudomonas aeruginosa and its cytosolic domain.

Rhomboids comprise a family of intramembrane serine proteases that catalyze the cleavage of transmembrane segments within the lipid membrane to achieve a wide range of biological functions. A subset of bacterial rhomboids possesses an N-terminal cytosolic domain that appears to enhance proteolytic activity via an unknown mechanism. Structural analysis of a full-length rhomboid would provide new insights into this mechanism, an objective that solution NMR has the potential to realize. For this purpose we purified the rhomboid from Pseudomonas aeruginosa in a range of membrane-mimetic media, evaluated its functional status in vitro and investigated the NMR spectroscopic properties of these samples. In general, NMR signals could only be observed from the cytosolic domain, and only in detergents that did not support rhomboid activity. In contrast, media that supported rhomboid function did not show these resonances, suggesting an association between the cytosolic domain and the protein-detergent complex. Investigations into the ability of the isolated cytosolic domain to bind detergent micelles revealed a denaturing interaction, whereas no interaction occurred with micelles that supported rhomboid activity. The cytosolic domain also did not show any tendency to interact with lipid bilayers found in small bicelles or vesicles made from Escherichia coli phospholipid extracts. Based on these data we propose that the cytosolic domain does not interact with the lipid membrane, but instead enhances rhomboid activity through interactions with some other part of the rhomboid, such as the catalytic core domain.

A New Chemical Probe for Phosphatidylinositol Kinase Activity

Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive “clickable” probe, PIK‐BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK‐BPyne to a wortmannin activity‐based probe also known to target PIKs. We found that PIK‐BPyne can act as an effective in situ activity‐based probe, and for the first time, report changes in PI4K‐IIIβ activity induced by the hepatitis C virus. These results establish the utility of PIK‐BPyne for activity‐based protein profiling studies of PIK function in native biological systems.

Copper-catalysed cycloaddition reactions of nitrones and alkynes for bioorthogonal labelling of living cells

An adapted biocompatible version of the Kinugasa reaction, the copper-catalysed alkyne-nitrone cycloaddition followed by rearrangement (CuANCR), was developed for live-cell labelling. CuANCR labelling was demonstrated for both mammalian and bacterial cells. A method for metabolic incorporation of the nitrone group is also described.

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.

Dual Strain-Promoted Alkyne–Nitrone Cycloadditions for Simultaneous Labeling of Bacterial Peptidoglycans

Bioorthogonal chemistry has been applied to study a multitude of biological processes in complex environments through incorporation and detection of small functional groups. However, few reactions are known to be compatible with each other to allow for studies of more than one biomolecule simultaneously. Here we describe a dual labeling method wherein two stereoelectronically contrasting nitrone tags are incorporated into bacteria peptidoglycan and detected via strain-promoted alkyne-nitrone cycloaddition (SPANC) simultaneously. Furthermore, we show orthogonality with the azide functionality broadening the potential for simultaneous biomolecular target labeling in less accommodating metabolic pathways. We also demonstrate the simultaneous labeling of two different food-associated bacteria, L. innocua (a model for the food-born pathogen L. monocytogenes) and L. lactis (a fermentation bacterium). The ability to monitor multiple processes and even multiple organisms concurrently through nitrone/nitrone or nitrone/azide incorporation strengthens the current bioorthogonal toolbox and gives rise to robust duplex labeling of organisms to potentiate the studies of rapid biological phenomena.

Systems biology methods help develop a better understanding of hepatitis C virus–induced liver injury

R ecently, systems biology methods have risen to the forefront of techniques for elucidating the molecular details of disease progression. There has been a transition from an emphasis on individual genes to more comprehensive analyses, as technologies continue to evolve for quantitative high-throughput molecular detection. This has led to a variety of studies that have integrated different ‘‘omics’’ approaches to identify key factors in complex host-pathogen molecular networks that are potentially clinically relevant targets for therapy. These multidisciplinary strategies continue to enhance our understanding of disease progression and identify prognostic markers; specifically, the identification of key factors linked to disease progression susceptibility and therapy response. To this end, several studies have been performed aiming to link hepatitis C virus (HCV) disease progression and/ or therapy outcome to both genetic and proteomic markers. Genome-wide association and profiling studies have identified human single nucleotide polymorphisms and several interferon stimulated genes, respectively, with varying predictive values. In the current issue of HEPATOLOGY, Katze and colleagues have published seminal studies using systems biology methods to gain a better understanding of how HCV reinfection in liver transplant patients can lead to the rapid development of liver disease. HCV infection remains the leading cause of orthotopic liver transplantation (OLT). For those suffering from chronic HCV infection, OLT remains one of the last recourses. Viremia recurrence is essentially universal in the graft posttransplant; however, the rate of disease progression varies, with 5%-30% of patients developing cirrhosis 5 years posttransplant due to accelerated rates of fibrosis. The accelerated requirement for retransplantation (RT) in this subset poses an additional economic burden, with patient and graft survival rates post-RT being lower than after primary OLT. Serial liver biopsy remains the best way of monitoring disease progression. However, this technique is invasive and risks the possibility of misdiagnosis. Reliable and noninvasive prognostic markers of HCV-associated disease progression are currently being evaluated, with the goal to improve allograft survival. Thus far, significant factors influencing disease progression that have been identified include HCV RNA levels preand post-OLT, viral genetics, donor age, and donor/recipient genetics. There has been limited work, however, investigating molecular signatures that predict clinical disease progression prior to histological evidence of fibrosis. One study, examining gene expression of recurrent HCV-infected biopsies 1 year post-OLT, reported an increase in myofibroblast (MF) and MF-like cell markers and a decrease in retinoidrelated proteins. These observations suggest decreased hepatic stellate cell (HSC) quiescence is correlated with rapid fibrosis progression. Another proteomics study linked an up-regulation of genes associated with oxidative stress and mitochondrial dysfunction to later stages of fibrosis (Batts-Ludwig stage 3-4). These studies emphasized a correlation between oxidative stress, HSC activation, and fibrosis. A more recent study by Mas et al. examined gene expression in biopsies at the time of HCV recurrence to develop a prognostic signature, which, based on nine differentially expressed genes, was capable of distinguishing mild and severe fibrosis at 3 years post-OLT. This report highlighted the potential for identifying predictors of fibrosis in gene expression early after OLT. In this issue of HEPATOLOGY, alternate avenues for diagnosing liver fibrosis, along with characterizing prognostic signatures indicative of rapid disease progression, are explored. These two companion studies utilized systems biology approaches to characterize early molecular signatures that correlated with rapid progression in both liver disease and fibrosis in HCVinfected transplant patients. These reports make use of the novel singular value decomposition initialized multidimensional scaling (SVD-MDS) analysis to separate Abbreviations: DAA, directed anti-viral agent; DEG, differentially expressed gene; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDAC, histone deacetylase; HSC, hepatic stellate cells; MF, myofibroblast; OLT, orthotopic liver transplantation; RT, retransplantation; SVD-MDS, singular value decomposition initialized multidimensional scaling. Address reprint requests to: John Paul Pezacki, Ph.D., Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6. E-mail: John.Pezacki@nrc.ca; fax: 613-941-8447. CopyrightVC 2012 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.25727 Potential conflict of interest: Nothing to report.

Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe

To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells. We utilized an ABPP probe, wortmannin-yne, based on the kinase inhibitor wortmannin, which contains a pendant alkyne group for bioconjugation using bioorthogonal chemistry. We observed changes in the activity of kinases involved in the mitogen-activated protein kinase pathway, apoptosis pathways, and cell cycle control. These results establish changes to the active kinome, as reported by wortmannin-yne, in the proteome of human hepatoma cells actively replicating HCV. The observed changes include kinase activity that affect viral entry, replication, assembly, and secretion, implying that HCV is regulating the pathways that it uses for its life cycle through modulation of the active kinome.