Removal of cholesterol from peripheral tissues to the bloodstream via reverse cholesterol transport (RCT) is a process of major biological importance. Here we demonstrate that lymphatic drainage is required for RCT. We have previously shown that hypercholesterolemia in mice is associated with impaired lymphatic drainage and increased lipid accumulation in peripheral tissues. We now show that restoration of lymphatic drainage in these mice significantly improves cholesterol clearance. Conversely, obstruction of lymphatic vessels in wild-type mice significantly impairs RCT. Finally, we demonstrate using silencing RNA interference, neutralizing antibody, and transgenic mice that removal of cholesterol by lymphatic vessels is dependent on the uptake and transcytosis of HDL by scavenger receptor class B type I expressed on lymphatic endothelium. Collectively, this study challenges the current view that lymphatic endothelium is a passive exchange barrier for cholesterol transport and provides further evidence for its interplay with lipid biology in health and disease.

Post-translationally isoprenylated proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these protein probes, we have demonstrated that RabGDI and the related Rab escort protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab-GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.

Bind and shine: An approach for the selective detection of both enzymes and non‐enzymatic proteins using an environment‐sensitive fluorescent turn‐on probe is described (see scheme). This approach targets the hydrophobic ligand‐binding domain of the target protein to trigger the fluorescence turn‐on and was shown to be specific for the targeted protein.

Lymphangiogenesis is an important physiological response to inflammatory insult, acting to limit inflammation. Macrophages, dendritic cells, and lymphocytes are known to drive lymphangiogenesis. In this study, we show that neutrophils recruited to sites of inflammation can also coordinate lymphangiogenesis. In the absence of B cells, intranodal lymphangiogenesis induced during prolonged inflammation as a consequence of immunization is dependent on the accumulation of neutrophils. When neutrophils are depleted in wild-type mice developing skin inflammation in response to immunization or contact hypersensitization, lymphangiogenesis is decreased and local inflammation is increased. We demonstrate that neutrophils contribute to lymphangiogenesis primarily by modulating vascular endothelial growth factor (VEGF)-A bioavailability and bioactivity and, to a lesser extent, secreting VEGF-D. We further show that neutrophils increased VEGF-A bioavailability and bioactivity via the secretion of matrix metalloproteinases 9 and heparanase. Together, these findings uncover a novel function for neutrophils as organizers of lymphangiogenesis during inflammation.

Genetically encoded fluorescent sensor proteins offer the possibility to probe the concentration of key metabolites in living cells. The approaches currently used to generate such fluorescent sensor proteins lack generality, as they require a protein that undergoes a conformational change upon metabolite binding. Here we present an approach that overcomes this limitation. Our biosensors consist of SNAP-tag, a fluorescent protein and a metabolite-binding protein. SNAP-tag is specifically labeled with a synthetic molecule containing a ligand of the metabolite-binding protein and a fluorophore. In the labeled sensor, the metabolite of interest displaces the intramolecular ligand from the binding protein, thereby shifting the sensor protein from a closed to an open conformation. The readout is a concomitant ratiometric change in the fluorescence intensities of the fluorescent protein and the tethered fluorophore. The observed ratiometric changes compare favorably with those achieved in genetically encoded fluorescent sensor proteins. Furthermore, the modular design of our sensors permits the facile generation of ratiometric fluorescent sensors at wavelengths not covered by autofluorescent proteins. These features should allow semisynthetic fluorescent sensor proteins based on SNAP-tag to become important tools for probing previously inaccessible metabolites.

Prenylated Rab GTPases regulate intracellular vesicle trafficking in eukaryotic cells by associating with specific membranes and recruiting a multitude of Rab-specific effector proteins. Prenylation, membrane delivery, and recycling of all 60 members of the Rab GTPase family are regulated by two related molecules, Rab escort protein (REP) and GDP dissociation inhibitor (GDI). Biophysical analysis of the interaction of prenylated proteins is complicated by their low solubility in aqueous solutions. Here, we used expressed protein ligation to construct a semisynthetic fluorescent analogue of prenylated Rab7, Rab7-NBD-farnesyl. This molecule is soluble in the absence of detergent but is otherwise similar in its behavior to naturally prenylated Rab7 GTPase. To obtain information on the interaction of natively mono-and diprenylated Rab7 GTPases with REP and GDI molecules, we stabilized the former molecules in solution by using the ␤-subunit of Rab geranylgeranyl transferase, which we demonstrate to function as an unspecific chaperone of prenylated proteins. Using competitive titrations of mixtures of natively prenylated and fluorescent Rab, we demonstrate that monogeranylgeranylated Rab7 binds to the REP protein with a Kd value of Ϸ70 pM. The affinity of doubly prenylated Rab7 is Ϸ20-fold weaker. In contrast, GDI binds both prenylated forms of Rab7 with comparable affinities (Kd ‫؍‬ 1-5 nM) but has extremely low affinity to unprenylated Rab molecules. The obtained data allow us to formulate a thermodynamic model for the interaction of RabGTPases with their regulators and membranes and to explain the need for both REP and GDI in Rab function.geranylgeranyl ͉ protein prenylation

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ABSTRACT:We report the semisynthesis of a fluorescent glutamate sensor protein on cell surfaces. Sensor excitation at 547 nm yields a glutamate-dependent emission spectrum between 550 and 700 nm that can be exploited for ratiometric sensing. On cells, the sensor displays a ratiometric change of 1.56. The high sensitivity toward glutamate concentration changes of the sensor and its exclusive extracellular localization make it an attractive tool for glutamate sensing in neurobiology.T he amino acid glutamate is the prevalent neurotransmitter in the vertebrate nervous system. It is used at well over 90% of the synapses in the human brain and influences essentially all forms of behavior, including consciousness, sensory perception, motor control, and mood. 1 Further, glutamate is involved at most synapses that are modifiable, that is, that are capable of adapting to changing patterns of stimuli by enhancing or reducing the efficiency of synaptic transmission. 2 These processes are thought to be responsible for high-order brain functions, such as learning and memory. Three fluorescent sensor proteins for investigating the role of glutamate in neurobiology have been developed so far. 3−5 However, the modest performance of these sensor proteins has limited their use. Here we present a semisynthetic fluorescent sensor protein for glutamate which shows higher sensitivity toward glutamate concentration changes and operates at longer wavelengths than the previously reported sensors.Semisynthetic fluorescent sensor proteins (Snifits), 6,7 are fusion proteins consisting of SNAP-tag, 8 CLIP-tag, 9 and an analyte-binding protein ( Figure 1A). SNAP-and CLIP-tag are labeled with a synthetic fluorescent ligand and a second synthetic fluorophore, respectively. The ligand binds to the binding protein in an intramolecular fashion and thereby keeps the sensor protein in a closed conformation. Free analyte can compete for binding to the binding protein and can shift the equilibrium to the open conformation. This shift can be detected by a change in the Forster resonance energy transfer (FRET) efficiency between the two fluorophores.For the construction of a glutamate sensor protein based on the Snifit sensor concept, we have chosen the ionotropic glutamate receptor 5 (iGluR5) as the binding protein for two reasons. First, due to the modular construction of ionotropic glutamate receptors, it is possible to express its glutamate binding domain S1S2 as a soluble protein in bacteria while conserving both the high affinity and specificity toward glutamate. 10,11 The possibility to characterize the soluble binding protein in vitro before its use on cell surfaces facilitates sensor development. Second, it is known that the stereoselective functionalization of the γ-carbon of the glutamate side chain does not significantly perturb its affinity toward iGluR5, 12,13 suggesting an attachment point for the required synthetic tether. We therefore prepared the tethered glutamate analogue 1 (Figure 1B, Scheme S1−S3) that contains a Cy5 fluorophore a...