Nicotinamide phosphoribosyltransferase prompts bleomycin-induced pulmonary fibrosis by driving macrophage M2 polarization in mice.

Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible, fatal interstitial lung disease lacking specific therapeutics. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the nicotinamide adenine dinucleotide (NAD) salvage biosynthesis pathway and a cytokine, has been previously reported as a biomarker for lung diseases; however, the role of NAMPT in pulmonary fibrosis has not been elucidated. Methods: We identified the NAMPT level changes in pulmonary fibrosis by analyzing public RNA-Seq databases, verified in collected clinical samples and mice pulmonary fibrosis model by Western blotting, qRT-PCR, ELISA and Immunohistochemical staining. We investigated the role and mechanism of NAMPT in lung fibrosis by using pharmacological inhibition on NAMPT and Nampt transgenic mice. In vivo macrophage depletion by clodronate liposomes and reinfusion of IL-4-induced M2 bone marrow-derived macrophages (BMDMs) from wild-type mice, combined with in vitro cell experiments, were performed to further validate the mechanism underlying NAMPT involving lung fibrosis. Results: We found that NAMPT increased in the lungs of patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis. NAMPT inhibitor FK866 alleviated BLM-induced pulmonary fibrosis in mice and significantly reduced NAMPT levels in bronchoalveolar lavage fluid (BALF). The lung single-cell RNA sequencing showed that NAMPT expression in monocytes/macrophages of IPF patients was much higher than in other lung cells. Knocking out NAMPT in mouse monocytes/macrophages (Namptfl/fl;Cx3cr1CreER) significantly alleviated BLM-induced pulmonary fibrosis in mice, decreased NAMPT levels in BALF, reduced the infiltration of M2 macrophages in the lungs and improved mice survival. Depleting monocytes/macrophages in Namptfl/fl;Cx3cr1CreER mice by clodronate liposomes and subsequent pulmonary reinfusion of IL-4-induced M2 BMDMs from wild-type mice, reversed the protective effect of monocyte/macrophage NAMPT-deletion on lung fibrosis. In vitro experiments confirmed that the mechanism of NAMPT engaged in pulmonary fibrosis is related to the released NAMPT by macrophages promoting M2 polarization in a non-enzyme-dependent manner by activating the STAT6 signal pathway. Conclusions: NAMPT prompts bleomycin-induced pulmonary fibrosis by driving macrophage M2 polarization in mice. Targeting the NAMPT of monocytes/macrophages is a promising strategy for treating pulmonary fibrosis.

PARP-1 inhibitor alleviates cerebral ischemia/reperfusion injury by reducing PARylation of HK-1 and LDH in mice.

Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.

The Depletion of NAMPT Disturbs Mitochondrial Homeostasis and Causes Neuronal Degeneration in Mouse Hippocampus.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in the salvaging synthesis pathway of the nicotinamide adenine dinucleotide (NAD). Both NAMPT and NAD progressively decline upon aging and neurodegenerative diseases. The depletion of NAMPT induces mitochondrial dysfunction in motor neurons and causes bioenergetic stress in neurons. However, the roles of NAMPT in hippocampus neurons need to be further studied. Using floxed Nampt (Namptflox/flox) mice, we knocked out Nampt specifically in the hippocampus CA1 neurons by injecting rAAV-hSyn-Cre-APRE-pA. The depletion of NAMPT in hippocampus neurons induced cognitive deficiency in mice. Nevertheless, no morphological change of hippocampus neurons was observed with immunofluorescent imaging. Under the transmission electron microscope, we observed mitochondrial swollen and mitochondrial number decreasing in the cell body and the neurites of hippocampus neurons. In addition, we found the intracellular Aß (6E10) increased in the hippocampus CA1 region. The intensity of Aß42 remained unchanged, but it tended to aggregate. The GFAP level, an astrocyte marker, and the Iba1 level, a microglia marker, significantly increased in the mouse hippocampus. In the primary cultured rat neurons, NAMPT inhibition by FK866 decreased the NAD level of neurons at > 10-9 M. FK866 dropped the mitochondrial membrane potential in the cell body of neurons at > 10-9 M and in the dendrite of neurons at > 10-8 M. FK866 decreased the number and shortened the length of branches of neurons at > 10-7 M. Together, likely due to the injury of mitochondria, the decline of NAMPT level can be a critical risk factor for neurodegeneration.

Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells.

Lung ischemia-reperfusion injury (LIRI) is associated with many diseases, including primary graft dysfunction after lung transplantation, and has no specific and effective therapies. Necroptosis contributes to the pathogenesis of ischemia-reperfusion injury. Necrostatin-1 (Nec-1), the necroptosis inhibitor targeting RIPK1, has been reported to alleviate ischemia-reperfusion injury in various organs. However, the underlying mechanism of Nec-1 in LIRI remains unclear. In this paper, an in vivo LIRI model was built up by left lung hilar clamping in mice, and an in vitro cold ischemia-reperfusion (CI/R) model using BEAS-2B cells was applied to mimic the lung transplantation setting. We found Nec-1 significantly alleviated ischemia-reperfusion-induced lung injury, cytokine releasing, and necroptosis of epithelial cells in mouse lungs. In vitro, Nec-1 also mitigated CI/R-induced cell death and inflammatory responses in BEAS-2B cells, and these protective effects were achieved by simultaneously inhibiting the formation of necrosome and RIPK1-dependent apoptosis. However, Nec-1 decreased the necrosome number but increased the apoptosis level in lung tissues after ischemia reperfusion. We further clarified that Nec-1 could also attenuate lung injury by promoting neutrophil apoptosis from flow cytometry. In conclusion, Nec-1 alleviated lung ischemia-reperfusion injury by inhibiting necroptosis and apoptosis of epithelial cells and promoting the apoptosis of neutrophils. Thus, Nec-1 could be a promising medication against primary graft dysfunction after lung transplantation.

Ischemia-Reperfusion Injury in a Simulated Lung Transplant Setting Differentially Regulates Transcriptomic Profiles between Human Lung Endothelial and Epithelial Cells.

Current understanding of mechanisms of ischemia-reperfusion-induced lung injury during lung preservation and transplantation is mainly based on clinical observations and animal studies. Herein, we used cell and systems biology approaches to explore these mechanisms at transcriptomics levels, especially by focusing on the differences between human lung endothelial and epithelial cells, which are crucial for maintaining essential lung structure and function. Human pulmonary microvascular endothelial cells and human lung epithelial cells were cultured to confluent, subjected to different cold ischemic times (CIT) to mimic static cold storage with preservation solution, and then subjected to warm reperfusion with a serum containing culture medium to simulate lung transplantation. Cell morphology, viability, and transcriptomic profiles were studied. Ischemia-reperfusion injury induced a CIT time-dependent cell death, which was associated with dramatic changes in gene expression. Under normal control conditions, endothelial cells showed gene clusters enriched in the vascular process and inflammation, while epithelial cells showed gene clusters enriched in protein biosynthesis and metabolism. CIT 6 h alone or after reperfusion had little effect on these phenotypic characteristics. After CIT 18 h, protein-biosynthesis-related gene clusters disappeared in epithelial cells; after reperfusion, metabolism-related gene clusters in epithelial cells and multiple gene clusters in the endothelial cells also disappeared. Human pulmonary endothelial and epithelial cells have distinct phenotypic transcriptomic signatures. Severe cellular injury reduces these gene expression signatures in a cell-type-dependent manner. Therapeutics that preserve these transcriptomic signatures may represent new treatment to prevent acute lung injury during lung transplantation.

Nicotinamide phosphoribosyltransferase inhibitor ameliorates mouse aging-induced cognitive impairment.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme for the synthesis of nicotinamide adenine dinucleotide (NAD) in the salvaging pathway. Though NAMPT inhibitors such as FK866 were originally developed as anti-cancer drugs, they also display neuroprotective effects. Here we show that the administration of FK866 at 0.5 mg/kg (ip, qod) for four weeks, i.e., ∼1% of the dose used for the treatment of cancer, significantly alleviates the aging-induced impairment of cognition and locomotor activity. Mechanistically, FK866 enhanced autophagy, reduced protein aggregation, and inhibited neuroinflammation indicated by decreasing TNFα, IL-6, GFAP, and Iba1 levels in the aged mouse brain. Though FK866 did not affect the total NAD and nicotinamide mononucleotide (NMN) levels in the mouse brain at the dose we used, FK866 increased nicotinamide (NAM) level in the young mouse brain and decreased NAM level in the aged mouse brain. On the other hand, FK866 did not affect the serum glucose, cholesterol, and triglyceride of young and aged mice and exhibited no effects on the various indices of young mice. Thus, the NAMPT inhibitor can be repurpose to counteract the cognitive impairment upon aging. We also envision that NAMPT inhibitor can be used for the treatment of age-related neurodegenerative diseases.

[Poly adenosine diphosphate-ribosylation and neurodegenerative diseases].

The morbidity of neurodegenerative diseases are increased in recent years, however, the treatment is limited. Poly ADP-ribosylation (PARylation) is a post-translational modification of protein that catalyzed by poly(ADP-ribose) polymerase (PARP). Studies have shown that PARylation is involved in many neurodegenerative diseases such as stroke, Parkinson's diseases, Alzheimer's disease, amyotrophic lateral sclerosis and so on, by affecting intracellular translocation of protein molecules, protein aggregation, protein activity, and cell death. PARP inhibitors have showed neuroprotective efficacy for neurodegenerative diseases in pre-clinical studies and phase Ⅰ clinical trials. To find new PARP inhibitors with more specific effects and specific pharmacokinetic characteristics will be the new direction for the treatment of neurodegenerative diseases. This paper reviews the recent progress on PARylation in neurodegenerative diseases.

FLIM-FRET-Based Structural Characterization of a Class-A GPCR Dimer in the Cell Membrane.

Class-A G protein-coupled receptors (GPCRs) are known to homo-dimerize in the membrane. Yet, methods to characterize the structure of GPCR dimer in the native environment are lacking. Accordingly, the molecular basis and functional relevance of the class-A GPCR dimerization remain unclear. Here, we present the dimeric structural model of GPR17 in the cell membrane. The dimer mainly involves transmembrane helix 5 (TM5) at the interface, with F229 in TM5, a critical residue. An F229A mutation makes GPR17 monomeric regardless of the expression level of the receptor. Monomeric mutants of GPR17 display impaired ERK1/2 activation and cannot be properly internalized upon agonist treatment. Conversely, the F229C mutant is cross-linked as a dimer and behaves like wild-type. Importantly, the GPR17 dimer structure has been modeled using sparse inter-protomer FRET distance restraints obtained from fluorescence lifetime imaging microscopy. The same approach can be applied to characterizing the interactions of other important membrane proteins in the cell.

Cangrelor alleviates bleomycin-induced pulmonary fibrosis by inhibiting platelet activation in mice.

Pulmonary fibrosis is a progressive chronic inflammatory lung disease whose pathogenesis is complicated. Platelets and neutrophils play important roles in the progression of pulmonary inflammation. We have reported that cangrelor, a non-sepesific GPR17 antagonist, alleviates pulmonary fibrosis partly by inhibiting macrophage inflammation in mice. Cangrelor is also a well-known anti-platelet agent. To test whether cangrelor mitigated pulmonary fibrosis partly through the inhibition of platelets, bleomycin (BLM) was used to induce pulmonary fibrosis in C57BL/6 J mice. We found that cangrelor (10 mg/kg) not only significantly decreased BLM-induced release of inflammatory cytokines (PF4, CD40 L and MPO), but also decreased the increment of platelets, neutrophils and platelet-neutrophil aggregates in the fibrotic lung and in the peripheral blood of BLM-treated mice. In addition, cangrelor decreased the number of CD40 and MPO double positive neutrophils and the expression level of CD40 in BLM-treated mouse lungs. Based on these results we conclude that cangrelor alleviates BLM-induced lung inflammation and pulmonary fibrosis in mice, partly through inhibition of platelet activation, therefore reducing the infiltration of neutrophils due to the adhesion of platelets and neutrophils mediated by CD40 - CD40 L interaction. Cangrelor could be a potential therapeutic medicine for pulmonary fibrosis.

Nicotinamide phosphoribosyltransferase secreted from microglia via exosome during ischemic injury.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the salvage pathway of nicotinamide adenine dinucleotide synthesis. NAMPT can also be secreted and functions as a cytokine. We have previously shown that in the brain, NAMPT expression and secretion can be induced in microglia upon neuroinflammation and injury. Yet the mechanism for NAMPT secretion remains unclear. Here we show that NAMPT can be actively secreted from microglia upon the treatment of ischemia-like injury - oxygen-glucose deprivation and recovery (OGD/R). We confirmed that classical ER-Golgi pathway is not involved in NAMPT secretion. NAMPT secretion was further enhanced by ATP, and the secretion was mediated by P2X7 receptor and by intracellular Ca2+ . Importantly, we found that phospholipase D inhibitor, n-butanol, phospholipase D siRNA, and wortmannin significantly decreased OGD/R-induced and ATP-enhanced release of NAMPT in microglia. After excluding the mechanisms of involving secretory autophagy, endosomes, and secretory lysosome, we have concluded that microglial NAMPT is secreted mainly via exosome. Immune-electron microscopy identifies NAMPT in extracellular vesicles with the size and morphology characteristic of exosome. With the vesicles harvested by ultra-centrifugation, exosomal NAMPT is further confirmed by Western blotting analysis. Intriguingly, the amount of NAMPT relative to exosomal protein markers remains unchanged upon the treatment of OGD/R, suggesting a constant load of exosomal NAMPT in microglia. Taken together, we have identified NAMPT is actively secreted via exosome from microglia during neuroinflammation of ischemic injury.

Physicochemical-property guided design of a highly sensitive probe to image nitrosative stress in the pathology of stroke.

In vivo real-time imaging of nitrosative stress in the pathology of stroke has long been a formidable challenge due to both the presence of the blood-brain barrier (BBB) and the elusive nature of reactive nitrogen species, while this task is also informative to gain a molecular level understanding of neurovascular injury caused by nitrosative stress during the stroke episode. Herein, using a physicochemical property-guided probe design strategy in combination with the reaction-based probe design rationale, we have developed an ultrasensitive probe for imaging nitrosative stress evolved in the pathology of stroke. This probe demonstrates an almost zero background fluorescence signal but a maximum 1000-fold fluorescence enhancement in response to peroxynitrite, the nitrosative stress marker. Due to its good physicochemical properties, the probe readily penetrates the BBB after intravenous administration, and quickly accumulates in mice brain to sense local vascular injuries. After accomplishing its imaging mission, the probe is easily metabolized and therefore won't cause safety concerns. These desirable features make the probe competent for the straightforward visualization of nitrosative stress progression in stroke pathology.

Nicotinamide ribose ameliorates cognitive impairment of aged and Alzheimer's disease model mice.

Nicotinamide adenine dinucleotide (NAD) supplementation to repair the disabled mitochondria is a promising strategy for the treatment of Alzheimer's disease (AD) and other dementia. Nicotinamide ribose (NR) is a safe NAD precursor with high oral bioavailability, and has beneficial effects on aging. Here, we applied NR supplied food (2.5 g/kg food) to APP/PS1 transgenic AD model mice and aged mice for 3 months. Cognitive function, locomotor activity and anxiety level were assessed by standard behavioral tests. The change of body weight, the activation of microglia and astrocytes, the accumulation of Aß and the level of serum nicotinamide phosphoribosyltransferase (NAMPT) were determined for the evaluation of pathological processes. We found that NR supplementation improved the short-term spatial memory of aged mice, and the contextual fear memory of AD mice. Moreover, NR supplementation inhibited the activation of astrocytes and the elevation of serum NAMPT of aged mice. For AD model mice, NR supplementation inhibited the accumulation of Aß and the migration of astrocyte to Aß. In addition, NR supplementation inhibit the body weight gain of aged and APP/PS1 mice. Thus, NR has selective benefits for both AD and aged mice, and the oral uptake of NR can be used to prevent the progression of dementia.

[Effects of FK866 on migration of A549 cells and related mechanism].

OBJECTIVE: : To investigate the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 on the migration of human non-small cell cancer A549 cells and related mechanism. METHODS: : The inhibition effect of FK866 on A549 cells was tested by MTT assay. A549 cells were treated with 1.0 and 10.0 nmol/L FK866, and the cell migration was evaluated by modified wound scratch assay. The mRNA expression of E-cadherin and vimentin was detected by real-time RT-PCR, and the expression of ERK1/2 and pERK1/2 was determined by Western blotting. RESULTS: : FK866 inhibited the proliferation of A549 cells in a time-and concentration-dependent manner; after treatment for 72 h, the IC50 of FK866 was 9.55 nmol/L. When 1.0 nmol/L or 10.0 nmol/L FK866 was continuously applied 48 h before and 48 h after a scratch was made in wound scratch assay, the migration of A549 cells was significantly inhibited. However, when the FK866 was applied only 48 h after the scratch, the migration of A549 cells was inhibited by 10.0 nmol/L but not by 1.0 nmol/L FK866. The mRNA expression of E-cadherin and vimentin, and the activated ERK1/2 were significantly increased after 1.0 nmol/L FK866 treatment for 72 h. The pretreatment with nicotinamide adenine dinucleotide (NAD) precursor nicotinamide mononucleotide(1.0 mmol/L) or ERK1/2 inhibitor U0126 (10.0 µmol/L) reversed the up-regulation of E-cadherin and vimentin expression induced by FK866. CONCLUSIONS: s: Low concentration of FK866 decreases the migration of A549 cells through the inhibition of NAD level, activation of ERK1/2 and up-regulation of E-cadherin expression. However, it also up-regulates the expression of vimentin, indicating that it may have dual effects on the migration of tumor cells.

[Effects of cysteinyl leukotriene receptors on phagocytosis of mouse microglial cells].

OBJECTIVE: : To determine the effects of cysteinyl leukotriene receptors (CysLT1R and CysLT2R) on phagocytosis of mouse BV2 microglial cells. METHODS: : BV2 cells were stimulated with microglial activators lipopolysaccharide (LPS) or CysLT receptor agonists LTD4. The phagocytosis of BV2 cells was observed by immunofluorescence analysis and flow cytometry. The intracellular distributions of CysLT1R and CysLT2R in BV2 cells were examined with immunofluorescence staining. RESULTS: : Both LPS and LTD4 could significantly enhance the phagocytosis of BV2 cells, and such effect could be inhibited by CysLT1R selective antagonist Montelukast and CysLT2R selective antagonist HAMI 3379. The activation of BV2 cells induced by LTD4 or LPS resulted in changes in intracellular distributions of CysLT1R and CysLT2R. CysLT1R and CysLT2R was co-localization with a similar distribution. CONCLUSIONS: : CysLT1R and CysLT2R regulate the phagocytosis of mouse BV2 microglial cells with a synergistic effect.

[Research progress on receptor interacting proteins in inflammation].

Receptor interacting proteins (RIPs) are a group of threonine/serine protein kinases, which have relatively conserved kinase domains and different non-kinase domains, and are involved in physiological and pathological processes including innate immune response and inflammation. In recent years, many studies have shown that RIPs mediate cell necroptosis and triggers inflammatory responses by participating in the formation of necrotic complexes, and RIP1 and RIP3 are particularly closely related to cell necrosis. Cell necroptosis is a well-regulated way of cell death. The death signal that transmit through the TNF signaling pathway and the Toll-like receptor signaling pathway can recruit and phosphorylate mixed lineage kinase domain-like protein (MLKL), and eventually leading to disintegration and death of cells, and the release of cells intercellular material after cell disintegration can trigger an inflammatory reaction. This review mainly focuses on the major signaling pathways and molecular mechanisms that are involved in the mediation of necrosis and inflammation by RIPs. It also highlights the importance of RIPs in the development of inflammatory diseases and their potentials as therapeutic targets for inflammatory diseases.

Cangrelor alleviates pulmonary fibrosis by inhibiting GPR17-mediated inflammation in mice.

Pulmonary fibrosis is a progressive and intractable lung disease. Macrophages play a critical role in the progression of pulmonary fibrosis. Cangrelor, an anti-platelet agent, is also a non-selective Gprotein-coupled receptor 17 (GPR17) antagonist. GPR17 mediates microglial inflammation in the chronic phase of cerebral ischemia and regulates allergic pulmonary inflammation. In this study, we observed the effects of cangrelor on bleomycin (BLM)-induced macrophage cellular inflammation and BLM-induced pulmonary fibrosis in C57BL/6J mice. We found that BLM significantly increased GPR17 expression, the mRNA synthesis and release of inflammatory cytokines including TNF-α, IL-6 and TGF-ß1 in murine RAW 264.7 macrophage cells. Knockdown of GPR17 attenuated the BLM-induced inflammatory responses. Cangrelor (2.5 µM-10 µM) significantly alleviated BLM-induced inflammatory response in RAW 264.7 macrophage cells in concentration-dependent manner. In BLM-induced fibrotic mouse lungs, GPR17 expression and GPR17-positive macrophages were increased. Cangrelor (2.5 mg/kg-10 mg/kg) alleviated pulmonary fibrosis in dose-dependent manner. Cangrelor not only reduced the number of GPR17-positive macrophages, but also decreased BLM-induced mRNA synthesis and release of inflammatory cytokine. As such, we concluded that cangrelor alleviates BLM-induced pulmonary fibrosis by suppressing GPR17-mediated inflammation. Cangrelor could be a potential therapeutic drug for pulmonary fibrosis.

Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques.

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques. CXMS allows the modeling of protein conformational states that are alternatives to the crystal structure. SAXS provides ensemble-averaged low-resolution shape information. Importantly, smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight into protein dynamics and shall be widely implemented in structural biology.

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch.

Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last ß-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pKa value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pH-sensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals.

NAMPT inhibitor protects ischemic neuronal injury in rat brain via anti-neuroinflammation.

Nicotinamide phosphoribosyltransferase (NAMPT) is an important neuroprotective factor in cerebral ischemia, and it has been reported that NAMPT inhibitors can aggravate neuronal injury in the acute phase. However, because it is a cytokine, NAMPT participates in many inflammatory diseases in the peripheral system, and its inhibitors have therapeutic effects. Following cerebral ischemia, the peripheral and resident inflammatory and immune cells produce many pro-inflammatory mediators in the ischemic area, which induce neuroinflammation and impair the brain. However, the effects of NAMPT inhibitors in the neuroinflammation after ischemic brain injury remain unknown. Here, we found that FK866, a potent NAMPT inhibitor, decreased the level of TNF-α, NAMPT and IL-6 in the ischemic brain tissue one day after middle-cerebral-artery occlusion and reperfusion (MCAO/R), improved neurological dysfunction, decreased infarct volume and neuronal loss, and inhibited microgliosis and astrogliosis 14days after MCAO/R. The expression of NAMPT protein was induced in Iba1-positive microglia/macrophages in the ischemia core 14days after MCAO/R. In vitro studies show that oxygen-glucose deprivation and recovery (OGD/R) activate microglia. Activated microglia increased the activity of NF-κB, increased the mRNA synthesis of TNF-α, NAMPT and IL-6, and increased the secretion of TNF-α, NAMPT and IL-6. On the other hand, NAMPT can act synergistically with other cytokines and activate microglia. FK866 strongly inhibited these changes and alleviated OGD/R-induced activation of microglia. As such, NAMPT is a crucial determinant of cellular inflammation after cerebral ischemia. NAMPT inhibitors are novel compounds to protect neuronal injury from ischemia via anti-inflammatory effects.

Specific cell surface labeling of GPCRs using split GFP.

Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two ß-stands (ß-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine ß-strands (ß-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications.