DNA methylation, which occurs predominantly at CpG dinucleotides, is a potent epigenetic repressor of transcription. Because DNA methylation is reversible, there is much interest in understanding the mechanisms by which it can be regulated by DNA-binding transcription factors. We discuss several models that, by incorporating sequence motifs, CpG density, and methylation levels, attempt to link the binding of a transcription factor with the acquisition or loss of DNA methylation at promoters and distal regulatory elements. Additional in vivo genome-wide characterization of transcription factor binding patterns and high-resolution DNA methylation analyses are clearly required for stronger support of each model.
Immunosurveillance of tumor cells depends on NKp30, a major activating receptor of human natural killer (NK) cells. The human BCL2-associated athanogene 6 (BAG-6, also known as BAT3; 1126 amino acids) is a cellular ligand of NKp30. To date, little is known about the molecular details of this receptor ligand system. Within the current study, we have located the binding site of NKp30 to a sequence stretch of 250 amino acids in the C-terminal region of BAG-6 (BAG-6686–936). BAG-6686–936 forms a noncovalent dimer of 57–59 kDa, which is sufficient for high affinity interaction with NKp30 (KD < 100 nm). As our most important finding, BAG-6686–936 inhibits NKp30-dependent signaling, interferon-γ release, and degranulation of NK cells in the presence of malignantly transformed target cells. Based on these data, we show for the first time that BAG-6686–936 comprises a subdomain of BAG-6, which is sufficient for receptor docking and inhibition of NKp30-dependent NK cell cytotoxicity as part of a tumor immune escape mechanism. These molecular insights provide an access point to restore tumor immunosurveillance by NK cells and to increase the efficacy of cellular therapies.
The cornea is the clear, outermost portion of the eye composed of three layers: an epithelium that provides a protective barrier while allowing transmission of light into the eye, a collagen-rich stroma, and an endothelium monolayer. How cornea development and aging is controlled is poorly understood. Here we characterize the mouse cornea transcriptome from early embryogenesis through aging and compare it with transcriptomes of other epithelial tissues, identifying cornea-enriched genes, pathways, and transcriptional regulators. Additionally, we profiled cornea epithelium and stroma, defining genes enriched in these layers. Over 10,000 genes are differentially regulated in the mouse cornea across the time course, showing dynamic expression during development and modest expression changes in fewer genes during aging. A striking transition time point for gene expression between postnatal days 14 and 28 corresponds with completion of cornea development at the transcriptional level. Clustering classifies co-expressed, and potentially co-regulated, genes into biologically informative categories, including groups that exhibit epithelial or stromal enriched expression. Based on these findings, and through loss of function studies and ChIP-seq, we show that the Ets transcription factor EHF promotes cornea epithelial fate through complementary gene activating and repressing activities. Furthermore, we identify potential interactions between EHF, KLF4, and KLF5 in promoting cornea epithelial differentiation. These data provide insights into the mechanisms underlying epithelial development and aging, identifying EHF as a regulator of cornea epithelial identity and pointing to interactions between Ets and KLF factors in promoting epithelial fate. Furthermore, this comprehensive gene expression data set for the cornea is a powerful tool for discovery of novel cornea regulators and pathways.
Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of d-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible β-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide.
Smooth muscle (SM) development consists of several processes, including cell fate determination, differentiation, and maturation. The molecular mechanisms controlling SM early differentiation have been studied extensively. However, little is known about the mechanism underlying SM maturation. Cell division cycle 7 (Cdc7) has been shown to regulate cell fate determination in the initial phase of transforming growth factor-β (TGF-β)-induced SM differentiation. Our present study indicates that Cdc7 also regulates SM maturation. Knockdown of Cdc7 suppresses TGF-β-induced expression of SM myosin heavy chain, a late marker of SM differentiation. Cdc7 overexpression, on the other hand, enhances SM myosin heavy chain expression. Interestingly, Cdc7 activates the mRNA expression and promoter activity of myocardin (Myocd), a master regulator of SM differentiation, whose transcription is blocked in the initial phase of the differentiation because TGF-β does not induce Myocd mRNA until after the early SM markers are induced. These data suggest that Cdc7 mediates TGF-β-induced SM maturation via activation of Myocd transcription. Mechanistically, Cdc7 physically and functionally interacts with Nkx2.5 to regulate Myocd promoter activity. Cdc7 appears to enhance Nkx2.5 binding to Myocd promoter, leading to Myocd activation. Taken together, our studies demonstrate that Cdc7 regulates the initial and late phase of SM differentiation through distinct mechanisms.
Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.
Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.
Dopamine neurotoxicity is associated with several neurodegenerative diseases, and neurons utilize several mechanisms, including uptake and metabolism, to protect them from injury. Metabolism of dopamine involves three enzymes: monoamine oxidase, catechol O-methyltransferase, and sulfotransferase. In primates but not lower order animals, a sulfotransferase (SULT1A3) is present that can rapidly metabolize dopamine to dopamine sulfate. Here, we show that SULT1A3 and a closely related protein SULT1A1 are highly inducible by dopamine. This involves activation of the D1 and NMDA receptors. Both ERK1/2 phosphorylation and calcineurin activation are required for induction. Pharmacological agents that inhibited induction or siRNA targeting SULT1A3 significantly increased the susceptibility of cells to dopamine toxicity. Taken together, these results show that dopamine can induce its own metabolism and protect neuron-like cells from damage, suggesting that SULT1A3 activity may be a risk factor for dopamine-dependent neurodegenerative diseases.
Acid-sensing ion channels (ASICs) are a group of trimeric cation permeable channels gated by extracellular protons that are mainly expressed in the nervous system. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. In this report, we employed the substituted cysteine accessibility method and site-directed mutagenesis to examine the mechanism of activation of ASIC1a by extracellular protons. We found that the modification of E238C and D345C channels by MTSET reduced proton apparent affinity for activation. Furthermore, the introduction of positively charged residues at position 345 rendered shifted biphasic proton activation curves. Likewise, channels bearing mutations at positions 79 and 416 in the palm domain of the channel showed reduced proton apparent affinity and biphasic proton activation curves. Of significance, the effect of the mutations at positions 79 and 345 on channel activation was additive. E79K-D345K required a change to a pH lower than 2 for maximal activation. In summary, this study provides direct evidence for the presence of two distinct proton coordination sites in the extracellular region of ASIC1a, which jointly facilitate pore opening in response to extracellular acidification.
During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.
Prohibitin (PHB) has been reported to play a crucial role in adipocyte differentiation and mitochondrial function. However, the regulative mechanism of PHB during adipogenesis remains unclear. In this study, we determined that the levels of both microRNA (miR)-27a and miR-27b were down-regulated following adipogenic induction of human adipose-derived stem cells, whereas the mRNA level of PHB was up-regulated. Overexpression of miR-27a or miR-27b inhibited PHB expression and adipocyte differentiation. Using PHB 3′-UTR luciferase reporter assay, we observed that miR-27a and miR-27b directly targeted PHB in human adipose-derived stem cells. A compensation of PHB partially restored the adipogenesis inhibited by miR-27. Moreover, we demonstrated the novel finding that ectopic expression of miR-27a or miR-27b impaired mitochondrial biogenesis, structure integrity, and complex I activity accompanied by excessive reactive oxygen species production. Our data suggest that miR-27 is an anti-adipogenic microRNA partly by targeting PHB and impairing mitochondrial function. Pharmacological modulation of miR-27 function may provide a new therapeutic strategy for the treatment of obesity.
Hec1 and Nuf2, core components of the NDC80 complex, are essential for kinetochore-microtubule attachment and chromosome segregation. It has been shown that both Hec1 and Nuf2 utilize their coiled-coil domains to form a functional dimer; however, details of the consequential significance and structural requirements to form the dimerization interface have yet to be elucidated. Here, we showed that Hec1 required three contiguous heptad repeats from Leu-324 to Leu-352, but not the entire first coiled-coil domain, to ensure overall stability of the NDC80 complex through direct interaction with Nuf2. Substituting the hydrophobic core residues, Leu-331, Val-338, and Ile-345, of Hec1 with alanine completely eliminated Nuf2 binding and blocked mitotic progression. Moreover, unlike most coiled-coil proteins, where the buried positions are composed of hydrophobic residues, Hec1 possessed an unusual distribution of glutamic acid residues, Glu-334, Glu-341, and Glu-348, buried within the interior dimerization interface, which complement with three Nuf2 lysine residues: Lys-227, Lys-234, and Lys-241. Substituting these corresponding residues with alanine diminished the binding affinity between Hec1 and Nuf2, compromised NDC80 complex formation, and adversely affected mitotic progression. Taken together, these findings demonstrated that three buried glutamic acid-lysine pairs, in concert with hydrophobic interactions of core residues, provide the major specificity and stability requirements for Hec1-Nuf2 dimerization and NDC80 complex formation.
ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation.
♦ See referenced article, J. Biol. Chem. 2013, 288, 34414–34426
Mutations in a subfamily of the ATP-binding cassette (ABC) transporters called the A-subtype are implicated in a number of diseases. These ABCA transporters are involved in lipid transport, but not much is known about the lipids they transport and in which direction. In this Paper of the Week, Faraz Quazi and Robert Molday at the University of British Columbia in Canada purified and reconstituted three ABCA transporters (ABCA1, ABCA7, and ABCA4) into liposomes and carried out fluorescent lipid transport studies. The investigators demonstrated that ABCA1, which is involved in Tangier disease, and ABCA7 export phosphatidylcholine and phosphatidylserine across membranes; ABCA4, which is implicated in Stargardt disease, imports phosphatidylethanolamine. By analyzing mutants of the transporters, the investigators showed that nine ABCA1 Tangier mutants and ABCA4 Stargardt mutants had significantly lower phospholipid transport activity and notable subcellular mislocalization. They concluded, “ABCA transporters actively flip phospholipids across membranes, a function severely reduced for disease-causing mutants.”
Relative fluorescent phospholipid transport activity of ABCA1 and ABCA4 disease-associated mutants. A, purified WT ABCA1 and disease-causing mutants. B, purified WT ABCA4 and mutants.
α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3β4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the β4-Trp-59 and β4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe-9 interacts with the β4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively “avoid” nAChR receptors while targeting receptors involved with nociception.
Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe3+-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe3+-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe3+-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe3+-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.
♦ See referenced article, J. Biol. Chem. 2013, 288, 34443–34458
Heme oxygenase catalyzes the breakdown of heme to biliverdin, iron, and carbon monoxide. It is not a heme protein in itself but uses heme as both its active center and substrate. In this Paper of the Week, a team led by Masao Ikeda-Saito at Tohoku University in Japan studied the structure of HmuO, the heme oxygenase from the pathogenic bacterium Corynebacterium diphtheria, by a combination of x-ray crystallography and molecular dynamics. They analyzed the enzyme without its substrate, as well as the enzyme bound to biliverdin and Fe3+. Their data suggest that the reaction center of HmuO undergoes significant conformational changes when it binds to heme. By analyzing two intermediates of the enzyme, one bound to Fe3+-biliverdin and another to only biliverdin, the authors suggest that the structural changes in the enzyme upon heme binding produce an exit for Fe2+, so it can be released without the enzyme having to undergo more structural changes.
Schematic representation of the iron release from the Fe3+-biliverdin complex.
Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability.
The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1–17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx. Minimally biased docking of the tested ligands into the antagonist-bound KOR structure generated distinct binding modes, which were then evaluated biochemically and pharmacologically. Our analysis identified two types of mutations: those that affect receptor function primarily via ligand binding and those that primarily affect function. The shared and differential mechanisms of agonist binding and activation in KOR are further discussed. Usually, mutations affecting function more than binding were located at the periphery of the binding site and did not interact strongly with the various ligands. Analysis of the crystal structure along with the present results provide fundamental insights into the activation mechanism of the KOR and suggest that “functional” residues, along with water molecules detected in the crystal structure, may be directly involved in transduction of the agonist binding event into structural changes at the conserved rotamer switches, thus leading to receptor activation.
Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat−/− and Rpe65−/− mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat−/− and Rpe65−/− mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases.
Predicting enzymatic behavior in silico is an integral part of our efforts to understand biology. Hundreds of millions of compounds lie in targeted in silico libraries waiting for their metabolic potential to be discovered. In silico “enzymes” capable of accurately determining whether compounds can inhibit or react is often the missing piece in this endeavor. This problem has now been solved for the cytosolic sulfotransferases (SULTs). SULTs regulate the bioactivities of thousands of compounds—endogenous metabolites, drugs and other xenobiotics—by transferring the sulfuryl moiety (SO3) from 3′-phosphoadenosine 5′-phosphosulfate to the hydroxyls and primary amines of these acceptors. SULT1A1 and 2A1 catalyze the majority of sulfation that occurs during human Phase II metabolism. Here, recent insights into the structure and dynamics of SULT binding and reactivity are incorporated into in silico models of 1A1 and 2A1 that are used to identify substrates and inhibitors in a structurally diverse set of 1,455 high value compounds: the FDA-approved small molecule drugs. The SULT1A1 models predict 76 substrates. Of these, 53 were known substrates. Of the remaining 23, 21 were tested, and all were sulfated. The SULT2A1 models predict 22 substrates, 14 of which are known substrates. Of the remaining 8, 4 were tested, and all are substrates. The models proved to be 100% accurate in identifying substrates and made no false predictions at Kd thresholds of 100 μm. In total, 23 “new” drug substrates were identified, and new linkages to drug inhibitors are predicted. It now appears to be possible to accurately predict Phase II sulfonation in silico.
Lipins are evolutionarily conserved Mg2+-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.
WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.
T cell immunoglobulin and mucin domain 3 (Tim-3) dampens the response of CD4+ and CD8+ effector T cells via induction of cell death and/or T cell exhaustion and enhances the ability of macrophages to clear pathogens via binding to galectin 9. Here we provide evidence that human Tim-3 is a target of A disintegrin and metalloprotease (ADAM)-mediated ectodomain shedding resulting in a soluble form of Tim-3. We identified ADAM10 and ADAM17 as major sheddases of Tim-3 as shown by ADAM-specific inhibitors and the ADAM10 pro-domain in HEK293 cells and ADAM10/ADAM17-deficient murine embryonic fibroblasts. PMA-induced shedding of Tim-3 was abrogated by deletion of amino acids Glu181–Asp190 of the stalk region and Tim-3 lacking the intracellular domain was not efficiently cleaved after PMA stimulation. Surprisingly, a single lysine residue within the intracellular domain rescues shedding of Tim-3. Shedding of endogenous Tim-3 was found in primary human CD14+ monocytes after PMA and ionomycin stimulation. Importantly, the recently described down-regulation of Tim-3 from Toll-like receptor-activated CD14+ monocytes was caused by ADAM10- and ADAM17-mediated shedding. Inhibition of Tim-3 shedding from lipopolysaccharide-induced monocytes did not influence lipopolysaccharide-induced TNFα and IL-6 but increases IL-12 expression. In summary, we describe Tim-3 as novel target for ADAM-mediated ectodomain shedding and suggest a role of Tim-3 shedding in TLR-mediated immune responses of CD14+ monocytes.
PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.
Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease.
Enterohemorrhagic Escherichia coli and other attaching/effacing bacterial pathogens cause diarrhea in humans. These pathogens use a type III secretion system to inject virulence proteins (effectors) into host cells, some of which inhibit the innate immune system. The enterohemorrhagic E. coli NleH1 effector prevents the nuclear translocation of RPS3 (ribosomal protein S3) to inhibit its participation as a nuclear “specifier” of NF-κB binding to target gene promoters. NleH1 binds to RPS3 and inhibits its phosphorylation on Ser-209 by IκB kinase-β (IKKβ). However, the precise mechanism of this inhibition is unclear. NleH1 possesses a Ser/Thr protein kinase activity that is essential both for its ability to inhibit the RPS3/NF-κB pathway and for full virulence of the attaching/effacing mouse pathogen Citrobacter rodentium. However, neither RPS3 nor IKKβ is a substrate of NleH1 kinase activity. We therefore screened ∼9,000 human proteins to identify NleH1 kinase substrates and identified CRKL (v-Crk sarcoma virus CT10 oncogene-like protein), a substrate of the BCR/ABL kinase. Knockdown of CRKL abundance prevented NleH1 from inhibiting RPS3 nuclear translocation and NF-κB activity. CRKL residues Tyr-198 and Tyr-207 were required for interaction with NleH1. Lys-159, the kinase-active site of NleH1, was necessary for its interaction with CRKL. We also identified CRKL as an IKKβ interaction partner, mediated by CRKL Tyr-198. We propose that the CRKL interaction with IKKβ recruits NleH1 to the IKKβ complex, where NleH1 then inhibits the RPS3/NF-κB pathway.
To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.
Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.
Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.
Allergic asthma is conventionally considered as a Th2 immune response characterized by eosinophilic inflammation. Recent investigations revealed that Th17 cells play an important role in the pathogenesis of non-eosinophilic asthma (NEA), resulting in steroid-resistant neutrophilic airway inflammation. Heme oxygenase-1 (HO-1) has anti-inflammation, anti-oxidation, and anti-apoptosis functions. However, its role in NEA is still unclear. Here, we explore the role of HO-1 in a mouse model of NEA. HO-1 inducer hemin or HO-1 inhibitor tin protoporphyrin IX was injected intraperitoneally into ovalbumin-challenged DO11.10 mice. Small interfering RNA (siRNA) was delivered into mice to knock down HO-1 expression. The results show that induction of HO-1 by hemin attenuated airway inflammation and decreased neutrophil infiltration in bronchial alveolar lavage fluid and was accompanied by a lower proportion of Th17 cells in mediastinal lymph nodes and spleen. More importantly, induction of HO-1 down-regulated Th17-related transcription factor retinoic acid-related orphan receptor γt (RORγt) expression and decreased IL-17A levels, all of which correlated with a decrease in phosphorylated STAT3 (p-STAT3) level and inhibition of Th17 cell differentiation. Consistently, the above events could be reversed by tin protoporphyrin IX. Also, HO-1 siRNA transfection abolished the effect of hemin induced HO-1 in vivo. Meanwhile, the hemin treatment promoted the level of Foxp3 expression and enhanced the proportion of regulatory T cells (Tregs). Collectively, our findings indicate that HO-1 exhibits anti-inflammatory activity in the mouse model of NEA via inhibition of the p-STAT3-RORγt pathway, regulating kinetics of RORγt and Foxp3 expression, thus providing a possible novel therapeutic target in asthmatic patients.
Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.
VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca2+ levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca2+ levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.
The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.
Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.
β-Thalassemia (β-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in β-globin gene (HBB). It is estimated that 4.5% of the population in the world carry β-Thal mutants (1), posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However, homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here, we describe a robust process combining efficient generation of integration-free β-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly, the gene-corrected β-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal β-globin. Our studies provide an efficient and universal strategy to correct different types of β-globin mutations in β-Thal iPSCs for disease modeling and applications.
Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.
Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.
A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.
The stem cell protein SALL4 plays a critical role in hematopoiesis by regulating the cell fate. In primitive hematopoietic precursors, it activates or represses important genes via recruitment of various epigenetic factors such as DNA methyltransferases, and histone deacylases. Here, we demonstrate that LSD1, a histone lysine demethylase, also participates in the trans-repressive effects of SALL4. Based on luciferase assays, the amine oxidase domain of LSD1 is important in suppressing SALL4-mediated reporter transcription. In freshly isolated adult mouse bone marrows, both SALL4 and LSD1 proteins are preferentially expressed in undifferentiated progenitor cells and co-localize in the nuclei. Further sequential chromatin immunoprecipitation assay confirmed that these two factors share the same binding sites at the promoter regions of important hematopoietic regulatory genes including EBF1, GATA1, and TNF. In addition, studies from both gain- and loss-of-function models revealed that SALL4 dynamically controls the binding levels of LSD1, which is accompanied by a reversely changed histone 3 dimethylated lysine 4 at the same promoter regions. Finally, shRNA-mediated knockdown of LSD1 in hematopoietic precursor cells resulted in altered SALL4 downstream gene expression and increased cellular activity. Thus, our data revealed that histone demethylase LSD1 may negatively regulate SALL4-mediated transcription, and the dynamic regulation of SALL4-associated epigenetic factors cooperatively modulates early hematopoietic precursor proliferation.
ATP-dependent proteases are responsible for most energy-dependent protein degradation across all species. Proteases initially bind an unstructured region on a substrate and then translocate along the polypeptide chain, unfolding and degrading protein domains as they are encountered. Although this process is normally processive, resulting in the complete degradation of substrate proteins to small peptides, some substrates are released prematurely. Regions of low sequence complexity within the substrate such as the glycine-rich region (GRR) from p105 or glycine-alanine repeats (GAr) from the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, can trigger partial degradation and fragment release. Loss of processivity could be due to inability to hold on to the substrate (faster release) or inability to unfold and degrade a substrate domain (slower unfolding). I previously showed that the GRR slows domain unfolding by the proteasome (Kraut, D. A., Israeli, E., Schrader, E. K., Patil, A., Nakai, K., Nanavati, D., Inobe, T., and Matouschek, A. (2012) ACS Chem. Biol. 7, 1444–1453). In contrast, a recently published study concluded that GArs increase the rate of substrate release from ClpXP, a bacterial ATP-dependent protease (Too, P. H., Erales, J., Simen, J. D., Marjanovic, A., and Coffino, P. (2013) J. Biol. Chem. 288, 13243–13257). Here, I show that these apparently contradictory results can be reconciled through a reanalysis of the ClpXP GAr data. This reanalysis shows that, as with the proteasome, low complexity sequences in substrates slow their unfolding and degradation by ClpXP, with little effect on release rates. Thus, despite their evolutionary distance and limited sequence identity, both ClpXP and the proteasome share a common mechanism by which substrate sequences regulate the processivity of degradation.
Tryptophanyl-tRNA Synthetase (TrpRS) Urzyme (fragments A and C), a 130-residue construct containing only secondary structures positioning the HIGH and KMSKS active site signatures and the specificity helix, accelerates tRNATrp aminoacylation with ∼10-fold specificity toward tryptophan, relative to structurally related tyrosine. We proposed that including the 76-residue connecting peptide 1 insertion (Fragment B) might enhance tryptophan affinity and hence amino acid specificity, because that subdomain constrains the orientation of the specificity helix. We test that hypothesis by characterizing two new constructs: the catalytic domain (fragments A–C) and the Urzyme supplemented with the anticodon-binding domain (fragments A, C, and D). The three constructs, together with the full-length enzyme (fragments A–D), comprise a factorial experiment from which we deduce individual and combined contributions of the two modules to the steady-state kinetics parameters for tryptophan-dependent 32PPi exchange, specificity for tryptophan versus tyrosine, and aminoacylation of tRNATrp. Factorial design directly measures the energetic coupling between the two more recent modules in the contemporary enzyme and demonstrates its functionality. Combining the TrpRS Urzyme individually in cis with each module affords an analysis of long term evolution of amino acid specificity and tRNA aminoacylation, both essential for expanding the genetic code. Either module significantly enhances tryptophan activation but unexpectedly eliminates amino acid specificity for tryptophan, relative to tyrosine, and significantly reduces tRNA aminoacylation. Exclusive dependence of both enhanced functionalities of full-length TrpRS on interdomain coupling energies between the two new modules argues that independent recruitment of connecting peptide 1 and the anticodon-binding domain during evolutionary development of Urzymes would have entailed significant losses of fitness.
The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nm. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5′-phosphate bound to OPRTs with Kd values near 40 nm. Analogues designed with a C5-pyrimidine carbon–carbon bond to ribocation mimics gave Kd values in the range of 80–500 nm. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5′-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum.
VOLUME 282 (2007) PAGES 21542–21550
The original Fig. 3C had errors. The “nuclear” panel contained the same Western blot images as the “cytosolic” panel and the cytosolic panel did not indicate that separate lanes from the original blot had been spliced together. We have now replaced the nuclear panel with the correct images. A line in the new cytosolic panel indicates the point of splicing. This correction does not change the interpretation of the results or the conclusions.
VOLUME 286 (2011) PAGES 29748–29757
The micro-batch method used for crystallization was first described in the following reference, which we failed to cite in the paper (Chayen, N. E., Stewart, P. D., Maeder, D. L., and Blow, D. M. (1990) An automated system for micro-batch protein crystallization and screening. J. Appl. Cryst. 23, 297–302).
Inflammatory immune responses play an important role in mucosal homeostasis and gut diseases. Nuclear factor κB (NF-κB), central to the proinflammatory cascade, is activated in necrotizing enterocolitis (NEC), a devastating condition of intestinal injury with extensive inflammation in premature infants. TGF-β is a strong immune suppressor and a factor in breast milk, which has been shown to be protective against NEC. In an NEC animal model, oral administration of the isoform TGF-β1 activated the downstream effector Smad2 in intestine and significantly reduced NEC incidence. In addition, TGF-β1 suppressed NF-κB activation, maintained levels of the NF-κB inhibitor IκBα in the intestinal epithelium, and systemically decreased serum levels of IL-6 and IFN-γ. The immature human fetal intestinal epithelial cell line H4 was used as a reductionistic model of the immature enterocyte to investigate mechanism. TGF-β1 pretreatment inhibited the TNF-α-induced IκBα phosphorylation that targets the IκBα protein for degradation and inhibited NF-κB activation. Chromatin immunoprecipitation (ChIP) assays demonstrated decreased NF-κB binding to the promoters of IL-6, IL-8, and IκBα in response to TNF-α with TGF-β1 pretreatment. These TGF-β1 effects appear to be mediated through the canonical Smad pathway as silencing of the TGF-β central mediator Smad4 resulted in loss of the TGF-β1 effects. Thus, TGF-β1 is capable of eliciting anti-inflammatory effects by inhibiting NF-κB specifically in the intestinal epithelium as well as by decreasing systemic IL-6 and IFN-γ levels. Oral administration of TGF-β1 therefore can potentially be used to protect against gastrointestinal diseases.
Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 220.127.116.11). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.
Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584–28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.
Ribonucleases play an important role in RNA metabolism. Yet, they are also potentially destructive enzymes whose activity must be controlled. Here we describe a novel regulatory mechanism affecting RNase R, a 3′ to 5′ exoribonuclease able to act on essentially all RNAs including those with extensive secondary structure. Most RNase R is sequestered on ribosomes in growing cells where it is stable and participates in trans-translation. In contrast, the free form of the enzyme, which is deleterious to cells, is extremely unstable, turning over with a half-life of 2 min. RNase R binding to ribosomes is dependent on transfer-messenger RNA (tmRNA)-SmpB, nonstop mRNA, and the modified form of ribosomal protein S12. Degradation of the free form of RNase R also requires tmRNA-SmpB, but this process is independent of ribosomes, indicating two distinct roles for tmRNA-SmpB. Inhibition of RNase R binding to ribosomes leads to slower growth and a massive increase in RNA degradation. These studies indicate a previously unknown role for ribosomes in cellular homeostasis.
NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of the NQO2 gene in mice leads to γ radiation-induced myeloproliferative diseases. In this report, we showed that the 20 S proteasome and NQO2 both interact with myeloid differentiation factor CCAAT-enhancer-binding protein α (C/EBPα). The interaction of the 20 S proteasome with C/EBPα led to the degradation of C/EBPα. NQO2, in the presence of its cofactor NRH, protected C/EBPα against 20 S degradation. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20 S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBPα. Exposure of mice and HL-60 cells to γ radiation enhanced the levels of NQO2, which led to an increased NQO2 interaction with C/EBPα and decreased 20 S interaction with C/EBPα. NQO2 stabilization of C/EBPα was independent of NQO1, even though both interacted with the same C/EBPα domain. NQO2−/− mice, deficient in NQO2, failed to stabilize C/EBPα. This contributed to the development of γ radiation-induced myeloproliferative disease in NQO2−/− mice.
Defects in cellular metabolism have been widely implicated in causing male infertility, but there has been little progress in understanding the underlying mechanism. Here we report that several key metabolism genes are regulated in the testis by Rhox5, the founding member of a large X-linked homeobox gene cluster. Among these Rhox5-regulated genes are insulin 2 (Ins2), resistin (Retn), and adiponectin (Adipoq), all of which encode secreted proteins that have profound and wide-ranging effects on cellular metabolism. The ability of Rhox5 to regulate their levels in the testis has the potential to dictate metabolism locally in this organ, given the existence of the blood-testes barrier. We demonstrate that Ins2 is a direct target of Rhox5 in Sertoli cells, and we show that this regulation is physiologically significant, because Rhox5-null mice fail to up-regulate Ins2 expression during the first wave of spermatogenesis and have insulin-signaling defects. We identify other Rhox family members that induce Ins2 transcription, define protein domains and homeodomain amino acid residues crucial for this property, and demonstrate that this regulation is conserved. Rhox5-null mice also exhibit altered expression of other metabolism genes, including those encoding the master transcriptional regulators of metabolism, PPARG and PPARGC1A, as well as SCD1, the rate-limiting enzyme for fatty acid metabolism. These results, coupled with the known roles of RHOX5 and its target metabolism genes in spermatogenesis in vivo, lead us to propose a model in which RHOX5 is a central transcription factor that promotes the survival of male germ cells via its effects on cellular metabolism.
The proform of the WF146 protease, an extracellular subtilase produced by thermophilic Bacillus sp. WF146, matures efficiently at high temperatures. Here we report that the proform, which contains an N-terminal propeptide composed of a core domain (N*) and a linker peptide, is intrinsically able to mature via multiple pathways. One autocatalytic pathway is initiated by cis-processing of N* to generate an autoprocessed complex N*-IWT, and this step is followed by truncation of the linker peptide and degradation of N*. Another autocatalytic pathway is initiated by trans-processing of the linker peptide followed by degradation of N*. Unlike most reported subtilases, the maturation of the WF146 protease occurs not only autocatalytically but also hetero-catalytically whereby heterogeneous proteases accelerate the maturation of the WF146 protease via trans-processing of the proform and N*-IWT. Although N* acts as an intramolecular chaperone and an inhibitor of the mature enzyme, the linker peptide is susceptible to proteolysis, allowing the trans-processing reaction to occur auto- and hetero-catalytically. These studies also demonstrate that the WF146 protease undergoes subtle structural adjustments during the maturation process and that the binding of Ca2+ is required for routing the proform to mature properly at high temperatures. Interestingly, under Ca2+-free conditions, the proform is cis-processed into a unique propeptide-intermediate complex (N*-IE) capable of re-synthesis of the proform. Based on the basic catalytic principle of serine proteases and these experimental results, a mechanism for the cis-processing/re-synthesis equilibrium of the proform and the role of the linker peptide in regulation of this equilibrium has been proposed.
Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the “static” images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.
DNA polymerase β (pol β) is responsible for gap filling synthesis during repair of damaged DNA as part of the base excision repair pathway. Human pol β mutations were recently identified in a high percentage (∼30%) of tumors. Characterization of specific cancer variants is particularly useful to further the understanding of the general mechanism of pol β while providing context to disease contribution. We showed that expression of the carcinoma variant E295K induces cellular transformation. The poor polymerase activity exhibited by the variant was hypothesized to be caused by the destabilization of proper active site assembly by the glutamate to lysine mutation. Here, we show that this variant exhibits an unusual preference for binding dCTP opposite a templating adenine over the cognate dTTP. Biochemical studies indicate that the noncognate competes with the cognate nucleotide for binding to the polymerase active site with the noncognate incorporation a function of higher affinity and not increased activity. In the crystal structure of the variant bound to dA:dCTP, the fingers domain closes around the mismatched base pair. Nucleotide incorporation is hindered because key residues in the polymerase active site are not properly positioned for nucleotidyl transfer. In contrast to the noncognate dCTP, neither the cognate dTTP nor its nonhydrolyzable analog induced fingers closure, as isomorphous difference Fourier maps show that the cognate nucleotides are bound to the open state of the polymerase. Comparison with published structures provides insight into the structural rearrangements within pol β that occur during the process of nucleotide discrimination.
The activation of a high affinity Ca2+ influx system (HACS) in the plasma membrane is required for survival of yeast cells exposed to natural or synthetic inhibitors of essential processes (secretory protein folding or sterol biosynthesis) in the endoplasmic reticulum (ER). The mechanisms linking ER stress to HACS activation are not known. Here we show that Kch1, a recently identified low affinity K+ transporter in the plasma membrane of Saccharomyces cerevisiae, is up-regulated in response to several ER stressors and necessary for HACS activation. The activation of HACS required extracellular K+ and was also dependent on the high affinity K+ transporters Trk1 and Trk2. However, a paralog of Kch1 termed Kch2 was not expressed and not necessary for HACS activation in these conditions. The pathogenic yeast Candida albicans carries only one homolog of Kch1/Kch2, and homozygous knock-out mutants were similarly deficient in the activation of HACS during the responses to tunicamycin. However, the Kch1 homolog was not necessary for HACS activation or cell survival in response to several clinical antifungals (azoles, allylamines, echinocandins) that target the ER or cell wall. Thus, Kch1 family proteins represent a conserved linkage between HACS and only certain classes of ER stress in these yeasts.
Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions.
Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pKa of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.
Metabotropic GABAB receptors are crucial for controlling the excitability of neurons by mediating slow inhibition in the CNS. The strength of receptor signaling depends on the number of cell surface receptors, which is thought to be regulated by trafficking and degradation mechanisms. Although the mechanisms of GABAB receptor trafficking are studied to some extent, it is currently unclear whether receptor degradation actively controls the number of GABAB receptors available for signaling. Here we tested the hypothesis that proteasomal degradation contributes to the regulation of GABAB receptor expression levels. Blocking proteasomal activity in cultured cortical neurons considerably enhanced total and cell surface expression of GABAB receptors, indicating the constitutive degradation of the receptors by proteasomes. Proteasomal degradation required Lys48-linked polyubiquitination of lysines 767/771 in the C-terminal domain of the GABAB2 subunit. Inactivation of these ubiquitination sites increased receptor levels and GABAB receptor signaling in neurons. Proteasomal degradation was mediated by endoplasmic reticulum-associated degradation (ERAD) as shown by the accumulation of receptors in the endoplasmic reticulum upon inhibition of proteasomes, by the increase of receptor levels, as well as receptor signaling upon blocking ERAD function, and by the interaction of GABAB receptors with the essential ERAD components Hrd1 and p97. In conclusion, the data support a model in which the fraction of GABAB receptors available for plasma membrane trafficking is regulated by degradation via the ERAD machinery. Thus, modulation of ERAD activity by changes in physiological conditions may represent a mechanism to adjust receptor numbers and thereby signaling strength.
Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.
The S1A serine proteases function in many key biological processes such as development, immunity, and blood coagulation. S1A proteases contain a highly conserved disulfide bond (Cys191–Cys220 in chymotrypsin numbering) that links two β-loop structures that define the rim of the active site pocket. Mast cell βII-tryptase is a S1A protease that is associated with pathological inflammation. In this study, we have found that the conserved disulfide bond (Cys220–Cys248 in βII-tryptase) exists in oxidized and reduced states in the enzyme stored and secreted by mast cells. The disulfide bond has a standard redox potential of −301 mV and is stoichiometrically reduced by the inflammatory mediator, thioredoxin, with a rate constant of 350 m−1 s−1. The oxidized and reduced enzymes have different substrate specificity and catalytic efficiency for hydrolysis of both small and macromolecular substrates. These observations indicate that βII-tryptase activity is post-translationally regulated by an allosteric disulfide bond. It is likely that other S1A serine proteases are similarly regulated.
Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.
Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ϵ (PKCϵ) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3–5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCϵ, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.
Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.
At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence.