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

Kinugasa reactions in water: From green chemistry to bioorthogonal labelling

The Kinugasa reaction has become an efficient method for the direct synthesis of \u3b2-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 \u3b2-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.Peer reviewed: YesNRC publication: Ye

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-1s-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.Peer reviewed: YesNRC publication: Ye

Correction: Chigrinova, M., et al. Kinugasa reactions in water: From green chemistry to bioorthogonal labelling. Molecules 2015, 20, 6959-6969

Peer reviewed: YesNRC publication: Ye

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. This journal isPeer reviewed: YesNRC publication: Ye

Activity-Based Protein Profiling of the <i>Escherichia coli</i> 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 <i>Escherichia coli</i> 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

Mean speeds and travel distances of DII-core coated LDs compared to LDs in [mock transfected] Huh-7 cells^a.

a<p>The mean speeds and overall travel distances of LDs are compared in DII-core expressing Huh-7 cells and LDs in mock cells (enclosed in square brackets). The error represents standard error of the mean. The n represents the number of LDs from eight or more cells in different fields of view, assessed by particle tracking for both mutant (enclosed in round brackets) and mock samples (enclosed in square brackets). Live-cell imaging was conducted for duration of four minutes acquiring each frame at rate of 1.65 sec/frame. To minimize variability for LD speeds, all of the experiments that directly compared DII-core coated LDs to LDs in a mock sample were observed in cells of the same biological replicate. The ratios were calculated by dividing the mean speed of DII-core coated LDs by LDs in the mock cells.</p

Tracking LD mobility at distinct locations of the cell.

<p>While all of the mutants were tracked accordingly, Huh-7 cells expressing DII-core^G161A is a representative image acquired from a large data set. Huh-7 cells expressing DII-core^G161A is shown as (A) a merged image of DIC and TPF, and (B) TPF. DII-core^G161A coated LDs are selected, and indicated by the arrows, to demonstrate fluorescence overlap between TPF and DIC. (C) LDs localized at different areas within the transfected cell (green outline) were segregated into regions relative to the center of the nucleus, such as close (orange shading), mid (blue), and far (no shading). Each black arrow represents a DII-core^G161A coated LD for each of the segregated region, and the velocities were measured for each direction in the close (D), mid (E), far (F) regions. The red arrow selects for a region of dense LDs in the perinuclear region with higher levels of DII-core^G161A. (G) The velocity of the LD, identified by the red arrow was measured. All scale bars represent 10 µm.</p

Hydrophobic triaryl-substituted \u3b2-lactams as activity-based probes for profiling eukaryotic enzymes and host-pathogen interactions

ABPP with \u3b2-lactams: We identified the eukaryotic targets of \u3b2-lactam-containing compounds by activity-based protein profiling. Using this method, we demonstrated that \u3b2-lactam-based activity probes can be applied to identify differentially active enzymes in different cell lines and during hepatitis C virus replication.Peer reviewed: YesNRC publication: Ye

GFP-tagged DII-core^wt colocalizes with LDs.

<p>(A) Schematic representation of HCV core protein. Distinct interactions belong to each of the three core protein domains. The mature and immature forms are also shown, and are generated by the two host proteases: signal peptidase (SP, blue), and signal peptide peptidase (SPP, red). (B) GFP-tagged DII-core^wt contains the membrane binding domain consisting of two α-amphipathic helices separated by a hydrophobic loop. (C-D) CARS microscopy imaging of LDs in Huh-7 cells expressing GFP-tagged DII-core^wt. All images were collected approximately 20 hours after Huh-7 cells were transfected with (D) DII-core^wt and (C) without DII-core^wt, which contained only the lipofectamine transfection reagent. (C) Lipid volumes measured by voxel analysis for mock Huh-7 cells are shown in the CARS image. (D) CARS imaging captures DII-core^wt induced LD biogenesis and redistribution towards the perinuclear region. The two values in panel 2 represent the average LD volume for cells expressing DII-core^wt (top value, double asterisks) and non-expressing DII-core cells (single asterisks) within the same field of view (bottom value) as measured by voxel analysis. The error represents standard error of the mean. The n represents the amount of cells quantified for LD density. This experiment was conducted under two biological replicates. Panel 4 is a magnified image selected by a region of interest from the merged image to project a clearer view of colocalization between DII-core^wt and LDs. All scale bars represent 10 µm.</p