Wnt/ß-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation.

The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/ß-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, we examine the cellular determinants of ISC proliferation, harnessing kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. We find that Wnt signaling within Prospero-positive cells supports ISC proliferation and that kramer regulates Wnt signaling in this context by antagonizing kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/ß-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/ß-catenin signaling.

Imaging Interorganelle Phospholipid Transport by Extended Synaptotagmins Using Bioorthogonally Tagged Lipids.

The proper distribution of lipids within organelle membranes requires rapid interorganelle lipid transport, much of which occurs at membrane contact sites and is mediated by lipid transfer proteins (LTPs). Our current understanding of LTP mechanism and function is based largely on structural studies and in vitro reconstitution. Existing cellular assays for LTP function use indirect readouts, and it remains an open question as to whether substrate specificity and transport kinetics established in vitro are similar in cellular settings. Here, we harness bioorthogonal chemistry to develop tools for direct visualization of interorganelle transport of phospholipids between the plasma membrane (PM) and the endoplasmic reticulum (ER). Unnatural fluorescent phospholipid analogs generated by the transphosphatidylation activity of phospholipase D (PLD) at the PM are rapidly transported to the ER dependent in part upon extended synaptotagmins (E-Syts), a family of LTPs at ER-PM contact sites. Ectopic expression of an artificial E-Syt-based tether at ER-mitochondria contact sites results in fluorescent phospholipid accumulation in mitochondria. Finally, in vitro reconstitution assays demonstrate that the fluorescent lipids are bona fide E-Syt substrates. Thus, fluorescent lipids generated in situ via PLD activity and bioorthogonal chemical tagging can enable direct visualization of the activity of LTPs that mediate bulk phospholipid transport at ER-PM contact sites.

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization.

Tools for acute manipulation of protein localization enable elucidation of spatiotemporally defined functions, but their reliance on exogenous triggers can interfere with cell physiology. This limitation is particularly apparent for studying mitosis, whose highly choreographed events are sensitive to perturbations. Here we exploit the serendipitous discovery of a phosphorylation-controlled, cell cycle-dependent localization change of the adaptor protein PLEKHA5 to develop a system for mitosis-specific protein recruitment to the plasma membrane that requires no exogenous stimulus. Mitosis-enabled Anchor-away/Recruiter System (MARS) comprises an engineered, 15-kDa module derived from PLEKHA5 capable of recruiting functional protein cargoes to the plasma membrane during mitosis, either through direct fusion or via GFP-GFP nanobody interaction. Applications of MARS include both knock sideways to rapidly extract proteins from their native localizations during mitosis and conditional recruitment of lipid-metabolizing enzymes for mitosis-selective editing of plasma membrane lipid content, without the need for exogenous triggers or perturbative synchronization methods.

Imaging and Editing the Phospholipidome.

Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells.In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT.IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid-protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling.PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling.Collectively, the strategies described here summarize our progress to date in tackling the challenge of assigning precise functions to defined pools of phospholipids in cells. They also point to new challenges and directions for future study, including extension of imaging and membrane editing tools to other classes of lipids. We envision that continued application of bioorthogonal chemistry, optogenetics, and directed evolution will yield new tools and discoveries to interrogate the phospholipidome and reveal new mechanisms regulating phospholipid homeostasis and roles for phospholipids in cell signaling.

Organelle-Selective Membrane Labeling through Phospholipase D-Mediated Transphosphatidylation.

The specialized functions of eukaryotic organelles have motivated chemical approaches for their selective tagging and visualization. Here, we develop chemoenzymatic tools using metabolic labeling of abundant membrane lipids for selective visualization of organelle compartments. Synthetic choline analogues with three N-methyl substituents replaced with 2-azidoethyl and additional alkyl groups enabled the generation of corresponding derivatives of phosphatidylcholine (PC), a ubiquitous and abundant membrane phospholipid. Subsequent bioorthogonal tagging via the strain-promoted azide-alkyne cycloaddition (SPAAC) with a single cyclooctyne-fluorophore reagent enabled differential labeling of the endoplasmic reticulum, the Golgi complex, mitochondria, and lysosomes depending upon the substitution pattern at the choline ammonium center. Key to the success of this strategy was the harnessing of both the organic cation transporter OCT1 to enable cytosolic delivery of these cationic metabolic probes and endogenous phospholipase D enzymes for rapid, one-step metabolic conversion of the choline analogues to the desired lipid products. Detailed analysis of the trafficking kinetics of both the SPAAC-tagged fluorescent PC analogues and their non-fluorescent, azide-containing precursors revealed that the latter exhibit time-dependent differences in organelle selectivity, suggesting their use as probes for visualizing intracellular lipid transport pathways. By contrast, the stable localizations of the fluorescent PC analogues will allow applications not only for organelle-selective imaging but also for local modulation of physiological events with organelle-level precision by tethering of bioactive small molecules, via click chemistry, within defined subcellular membrane environments.

Acylguanidine derivatives of zanamivir and oseltamivir: Potential orally available prodrugs against influenza viruses.

Zanamivir (ZA) and guanidino-oseltamivir carboxylic acid (GOC) are very potent inhibitors against influenza neuraminidase (NA). The guanidinium moiety plays an important role in NA binding; however, its polar cationic nature also hinders the use of ZA and GOC from oral administration. In this study, we investigated the use of ZA and GOC acylguanidine derivatives as possible orally available prodrugs. The acylguanidine derivatives were prepared by coupling with either n-octanoic acid or (S)-naproxen. The lipophilic acyl substituents were verified to improve cell permeability, and may also improve the bioavailability of acylguanidine compounds. In comparison, the acylguanidines bearing linear octanoyl chain showed better NA inhibitory activity and higher hydrolysis rate than the corresponding derivatives having bulky branched naproxen moiety. Our molecular docking experiments revealed that the straight octanoyl chain could extend to the 150-cavity and 430-cavity of NA to gain extra hydrophobic interactions. Mice receiving the ZA octanoylguanidine derivative survived from influenza infection better than those treated with ZA, whereas the GOC octanoylguanidine derivative could be orally administrated to treat mice with efficacy equal to oseltamivir. Our present study demonstrates that incorporation of appropriate lipophilic acyl substituents to the polar guanidine group of ZA and GOC is a feasible approach to develop oral drugs for influenza therapy.

Peramivir conjugates as orally available agents against influenza H275Y mutant.

Peramivir is an efficacious neuraminidase (NA) inhibitor for treatment of influenza by intravenous administration. However, the efficacy of peramivir toward the H275Y mutant is appreciably reduced. To address this drawback, conjugation of peramivir with caffeic acid is devised in this study to enhance the binding affinity with neuraminidases. The C2-OH group of peramivir is elaborated to link with caffeate derivatives, giving the desired conjugates 8 and 9 that possess potent NA inhibitory activity against both wild-type and H275Y viruses with the IC50 values in nanomolar range. The molecular modeling reveals that the caffeate moiety of conjugate 9 prefers to reside in the 295-cavity of H275Y neuraminidase, thus providing additional hydrogen bonds and hydrophobic interactions to compensate the reduced binding affinity of the peramivir moiety due to Glu-276 dislocation in H275Y mutant. In comparison with peramivir, the lipophilicity of conjugates 8 and 9 also increases by incorporation of the caffeate moiety. Thus, conjugates 8 and 9 offer better effect to protect MDCK cells from infection of H275Y virus with low EC50 value (∼17 nM). Administration of conjugates 8 or 9 by oral gavage is effective in treatment of mice that are infected by lethal dose of wild-type or H275Y influenza viruses. Considering drug metabolism, since the ester linkage in conjugate 8 is susceptible to hydrolysis in plasma, conjugate 9 with robust amide linkage may be a better candidate for development into orally available anti-influenza drug that is also active to mutant viruses.

Peramivir analogues bearing hydrophilic side chains exhibit higher activities against H275Y mutant than wild-type influenza virus.

Peramivir is an effective anti-influenza drug in the clinical treatment of influenza, but its efficacy toward the H275Y mutant is reduced. The previously reported cocrystal structures of inhibitors in the mutant neuraminidase (NA) suggest that the hydrophobic side chain should be at the origin of reduced binding affinity. In contrast, zanamivir having a hydrophilic glycerol side chain still possesses high affinity toward the H275Y NA. We thus designed five peramivir analogues (5-9) carrying hydrophilic glycol or glycerol side chains, and evaluated their roles in anti-influenza activity, especially for the H275Y mutant. The synthetic sequence involves a key step of (3 + 2) cycloaddition reactions between alkenes and nitrile oxides to construct the scaffold of peramivir carrying the desired hydrophilic side chains and other appropriate functional groups. The molecular docking experiments reveal that the hydrophilic side chain can provide extra hydrogen bonding with the translocated Glu-276 residue in the H275Y NA active site. Thus, the H275Y mutant may be even more sensitive than wild-type virus toward the peramivir analogues bearing hydrophilic side chains. Notably, the peramivir analogue bearing a glycerol side chain inhibits the H275Y mutant with an IC50 value of 35 nM, which is better than the WSN virus by 9 fold.