Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells.

Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Müller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Müller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo. Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Müller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs. Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Müller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.

SAP102 regulates synaptic AMPAR function through a CNIH-2-dependent mechanism.

The postsynaptic density (PSD)-95-like, disk-large (DLG) membrane-associated guanylate kinase (PSD/DLG-MAGUK) family of proteins scaffold α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complexes to the postsynaptic compartment and are postulated to orchestrate activity-dependent modulation of synaptic AMPAR functions. SAP102 is a key member of this family, present from early development, before PSD-95 and PSD-93, and throughout life. Here we investigate the role of SAP102 in synaptic transmission using a cell-restricted molecular replacement strategy, where SAP102 is expressed against the background of acute knockdown of endogenous PSD-95. We show that SAP102 rescues the decrease of AMPAR-mediated evoked excitatory postsynaptic currents (AMPAR eEPSCs) and AMPAR miniature EPSC (AMPAR mEPSC) frequency caused by acute knockdown of PSD-95. Further analysis of the mini events revealed that PSD-95-to-SAP102 replacement but not direct manipulation of PSD-95 increases the AMPAR mEPSC decay time. SAP102-mediated rescue of AMPAR eEPSCs requires AMPAR auxiliary subunit cornichon-2, whereas cornichon-2 knockdown did not affect PSD-95-mediated regulation of AMPAR eEPSC. Combining these observations, our data elucidate that PSD-95 and SAP102 differentially influence basic synaptic properties and synaptic current kinetics potentially via different AMPAR auxiliary subunits. NEW & NOTEWORTHY Synaptic scaffold proteins postsynaptic density (PSD)-95-like, disk-large (DLG) membrane-associated guanylate kinase (PSD-MAGUKs) regulate synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function. However, the functional diversity among different PSD-MAGUKs remains to be categorized. We show that distinct from PSD-95, SAP102 increase the AMPAR synaptic current decay time, and the effect of SAP102 on synaptic AMPAR function requires the AMPAR auxiliary subunit cornichon-2. Our data suggest that PSD-MAGUKs target and modulate different AMPAR complexes to exert specific experience-dependent modification of the excitatory circuit.

Liquid chromatography with tandem mass spectrometry quantification of urinary proanthocyanin A2 dimer and its potential use as a biomarker of cranberry intake.

The lack of a biomarker for the consumption of cranberries has confounded the interpretation of several studies investigating the effect of cranberry products, especially juices, on health outcomes. The objectives of this pilot study were to develop a liquid chromatography tandem mass spectrometric method for the quantification of the proanthocyanin dimer A-2 in human urine and validate urinary proanthocyanin dimer A-2 as a biomarker of cranberry intake. Five healthy, nonsmoking, premenopausal women (20-30 years of age, body mass index: 18.5-25 kg/m(2) ) were assigned to consume a cranberry beverage containing 140 mg proanthocyanin and 35 kilocalories at 237 mL/day, according to a weekly dosing schedule for 7 weeks. Eleven 24 h and morning spot urine samples each were collected from each subject. A reliable, sensitive method for the detection of proanthocyanin dimer A-2 in urine using liquid chromatography with tandem mass spectrometry was developed with a limit of quantitation of 0.25 ng/mL and a relative standard deviation of 7.26%, precision of 5.7%, and accuracy of 91.7%. While proanthocyanin dimer A-2 was quantifiable in urine, it did not appear to be excreted in a concentration that corresponded to the dosing schedule and intake of cranberry juice.

Cyclophilin A (CyPA) induces chemotaxis independent of its peptidylprolyl cis-trans isomerase activity: direct binding between CyPA and the ectodomain of CD147.

Cyclophilin A (CyPA) is a ubiquitously distributed peptidylprolyl cis-trans isomerase (PPIase) that possesses diverse biological functions. Extracellular CyPA is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. Although it has been identified that the chemotaxis activity of CyPA is mediated through its cell surface signaling receptor CD147, the role of CyPA PPIase activity in this process is disputable, and the underlying molecular mechanism is still poorly understood. In this study, we present the first evidence that CyPA induces leukocyte chemotaxis through a direct binding with the ectodomain of CD147 (CD147(ECT)), independent of its PPIase activity. Although NMR study indicates that the CD147(ECT) binding site on CyPA overlaps with the PPIase active site, the PPIase inactive mutant CyPA(R55A) exhibits similar CD147(ECT) binding ability and chemotaxis activity to those of CyPA(WT). Furthermore, we have identified three key residues of CyPA involved in CD147(ECT) binding and found that mutations H70A, T107A, and R69A result in similar levels of reduction in CD147(ECT) binding ability and chemotaxis activity for CyPA, without affecting the PPIase activity. Our findings indicate that there exists a novel mechanism for CyPA to regulate cellular signaling processes, shedding new light on its applications in drug development and providing a new targeting site for drug design.

An efficient method for refolding the extracellular portion of CD147 from the total bacterial lysate.

CD147 is a widely expressed transmembrane protein that mediates signal transduction, and it plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. The extracellular portion of CD147 (CD147(EC)) is responsible for its functional interactions with different signaling molecules. Due to the existence of two disulfide bonds, CD147(EC) is mainly expressed as an inclusion body in Escherichia coli. Here, we report a convenient rapid-dilution refolding protocol that enables the refolding of CD147(EC) efficiently from total bacterial lysate instead of pure inclusion bodies. Using this method, over 25 mg of CD147(EC) can be purified from 1 l of bacterial culture in M9 medium. The refolded CD147(EC) is well folded as characterized by nuclear magnetic resonance (NMR), and it can induce the expression of matrix metalloproteinase-9 in fibroblast cells. The described protocol is also applicable to the refolding of two immunoglobulin domains of CD147(EC) individually. Interestingly, we noticed that little protein was produced for the C-terminal immunoglobulin (Ig) domain of CD147(EC) by bacteria in M9 medium, even though it was overexpressed in Luria-Bertani (LB) medium. However, when the pH of the bacterial culture in M9 medium was adjusted in accordance with that in LB medium during growth, comparable expression level could be achieved.

Neurogranin, Encoded by the Schizophrenia Risk Gene NRGN, Bidirectionally Modulates Synaptic Plasticity via Calmodulin-Dependent Regulation of the Neuronal Phosphoproteome.

BACKGROUND: Neurogranin (Ng), encoded by the schizophrenia risk gene NRGN, is a calmodulin-binding protein enriched in the postsynaptic compartments, and its expression is reduced in the postmortem brains of patients with schizophrenia. Experience-dependent translation of Ng is critical for encoding contextual memory, and Ng regulates developmental plasticity in the primary visual cortex during the critical period. However, the overall impact of Ng on the neuronal signaling that regulates synaptic plasticity is unknown. METHODS: Altered Ng expression was achieved via virus-mediated gene manipulation in mice. The effect on long-term potentiation (LTP) was accessed using spike timing-dependent plasticity protocols. Quantitative phosphoproteomics analyses led to discoveries in significant phosphorylated targets. An identified candidate was examined with high-throughput planar patch clamp and was validated with pharmacological manipulation. RESULTS: Ng bidirectionally modulated LTP in the hippocampus. Decreasing Ng levels significantly affected the phosphorylation pattern of postsynaptic density proteins, including glutamate receptors, GTPases, kinases, RNA binding proteins, selective ion channels, and ionic transporters, some of which highlighted clusters of schizophrenia- and autism-related genes. Hypophosphorylation of NMDA receptor subunit Grin2A, one significant phosphorylated target, resulted in accelerated decay of NMDA receptor currents. Blocking protein phosphatase PP2B activity rescued the accelerated NMDA receptor current decay and the impairment of LTP mediated by Ng knockdown, implicating the requirement of synaptic PP2B activity for the deficits. CONCLUSIONS: Altered Ng levels affect the phosphorylation landscape of neuronal proteins. PP2B activity is required for mediating the deficit in synaptic plasticity caused by decreasing Ng levels, revealing a novel mechanistic link of a schizophrenia risk gene to cognitive deficits.

Shank Proteins Differentially Regulate Synaptic Transmission.

Shank proteins, one of the principal scaffolds in the postsynaptic density (PSD) of the glutamatergic synapses, have been associated with autism spectrum disorders and neuropsychiatric diseases. However, it is not known whether different Shank family proteins have distinct functions in regulating synaptic transmission, and how they differ from other scaffold proteins in this aspect. Here, we investigate the role of Shanks in regulating glutamatergic synaptic transmission at rat hippocampal SC-CA1 synapses, using lentivirus-mediated knockdown and molecular replacement combined with dual whole-cell patch clamp in hippocampal slice culture. In line with previous findings regarding PSD-MAGUK scaffold manipulation, we found that loss of scaffold proteins via knockdown of Shank1 or Shank2, but not Shank3, led to a reduction of the number but not the unitary response of AMPAR-containing synapses. Only when both Shank1 and Shank2 were knocked down, were both the number and the unitary response of active synapses reduced. This reduction was accompanied by a decrease in NMDAR-mediated synaptic response, indicating more profound deficits in synaptic transmission. Molecular replacement with Shank2 and Shank3c rescued the synaptic transmission to the basal level, and the intact sterile α-motif (SAM) of Shank proteins is required for maintaining glutamatergic synaptic transmission. We also found that altered neural activity did not influence the effect of Shank1 or Shank2 knockdown on AMPAR synaptic transmission, in direct contrast to the activity dependence of the effect of PSD-95 knockdown, revealing differential interaction between activity-dependent signaling and scaffold protein families in regulating synaptic AMPAR function.

Structural and functional characterization of tumor suppressors TIG3 and H-REV107.

H-REV107-like family proteins TIG3 and H-REV107 are class II tumor suppressors. Here we report that the C-terminal domains (CTDs) of TIG3 and H-REV107 can induce HeLa cell death independently. The N-terminal domain (NTD) of TIG3 enhances the cell death inducing ability of CTD, while NTD of H-REV107 plays an inhibitory role. The solution structure of TIG3 NTD is very similar to that of H-REV107 in overall fold. However, the CTD binding regions on NTD are different between TIG3 and H-REV107, which may explain their functional difference. As a result, the flexible main loop of H-REV107, but not that of TIG3, is critical for its NTD to modulate its CTD in inducing cell death.

1H, 13C, and 15N resonance assignments of the N-terminal domain of human TIG3.

Human TIG3 protein is a member of H-REV107 protein family which belongs to the type II tumor suppressor family. TIG3 can induce apoptosis in cancer cells, and it also possesses Ca(2+)-independent phospholipase A(1/2) activity. The NMR assignments of the N-terminal domain of TIG3 are essential for its solution structure determination.

1H, 13C and 15N resonance assignments of SARS-CoV main protease N-terminal domain.

The main protease (M(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. SARS-CoV M(pro) is composed of a catalytic N-terminal domain and an α-helical C-terminal domain linked by a long loop. Even though the N-terminal domain of SARS-CoV M(pro) adopts a similar chymotrypsin-like fold as that of piconavirus 3C protease, the extra C-terminal domain is required for SARS-CoV M(pro) to be enzymatically active. Here, we reported the NMR assignments of the SARS-CoV M(pro) N-terminal domain alone, which are essential for its solution structure determination.

1H, 13C, and 15N resonance assignments of reduced GrxS14 from Populus tremula × tremuloides.

GrxS14 is a monothiol Glutaredoxin (Grx) from Populus tremula × tremuloides, which has a CGFS active site. GrxS14 is located in the chloroplasts and has been found to occur ether as an apo form or as a holo form with a [2Fe-2S] cluster. The holo form contains two monomers of apo GrxS14 bridged by the iron sulphur center, in the presence of two external glutathione molecules (Bandyopadhyay et al. 2008). The NMR assignments of the GrxS14 are essential for its solution structure determination.

Solution structure of the N-terminal catalytic domain of human H-REV107--a novel circular permutated NlpC/P60 domain.

H-REV107 is a Ca(2+)-independent phospholipase A(1/2), and it is also a pro-apoptosis protein belonging to the novel class II tumor suppressor family, H-REV107-like family. Here we report the solution structure of the N-terminal catalytic domain of human H-REV107, which has a similar architecture to classical NlpC/P60 domains, even though their fold topologies are different due to circular permutation in the primary sequence. The phospholipase active site possesses a structurally conserved Cys-His-His catalytic triad as found in NlpC/P60 peptidases, indicating H-REV107 should adopt a similar catalytic mechanism towards phospholipid substrates to that of NlpC/P60 peptidases towards peptides. As H-REV107 is highly similar to lecithin retinol acyltransferase, our study also provides structural insight to this essential enzyme in retinol metabolism.

1H, 13C and 15N resonance assignments of human H-REV107 N-terminal domain.

Human H-REV107 protein is the representative of a novel class II tumor suppressor family, which is lost in tumor cells and can induce cell death after restoration. The NMR assignments of the H-REV107 N-terminal domain are essential for its solution structure determination.

Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease.

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (M(pro)) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. M(pro) is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, M(pro) can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature M(pro) self-assembles into a novel super-active octamer (AO-M(pro)). The crystal structure of AO-M(pro) adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported M(pro) active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of M(pro), in equilibrium between inactive monomer and active dimer, the stable AO-M(pro) exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-M(pro) and its polyprotein substrates with multiple cleavage sites, would make AO-M(pro) functionally much more superior than the M(pro) active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AOM(pro) should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AOM(pro) provides new insights about the functional mechanism of M(pro) and its maturation process.

Beclin 1-independent autophagy induced by a Bcl-XL/Bcl-2 targeting compound, Z18.

Inhibitors of Bcl-XL/Bcl-2 can induce autophagy by releasing the autophagic protein Beclin 1 from its complexes with these proteins. Here we report a novel compound targeting the BH3 binding groove of Bcl-XL/Bcl-2, Z18, which efficiently induces autophagy-associated cell death in HeLa cells, without apparent apoptosis. Unexpectedly, the inhibition of Beclin 1 and phosphatidylinositol 3-kinase have no obvious effect on Z18-induced autophagy in HeLa cells, implying that it is a non-canonical Beclin 1-independent autophagy. Meanwhile, the accumulation of autophagosomes is positively correlated with Z18-induced cell death and the full flux of autophagy is not necessary.

A novel Bcl-XL inhibitor Z36 that induces autophagic cell death in Hela cells.

Inhibition of Bcl2 family proteins Bcl-X(L) and Bcl-2 represents a promising drug development strategy for cancer treatment by triggering apoptosis in cancer cells. Here we report a novel Bcl-X(L) inhibitor, Z36, which unexpectedly induces only autophagic cell death, but not apoptosis. This special property distinguishes Z36 from other previously reported Bcl-X(L) and Bcl-2 inhibitors that induce cancer cell death mainly through apoptosis, and makes Z36 an attractive molecular tool for studying the cellular regulation of autophagic cell death and apoptosis.