Prions are self-replicating protein aggregates and are the primary causative factor in a number of neurological diseases in mammals. The prion protein (PrP) undergoes a conformational transformation leading to aggregation into an infectious cellular pathogen. Prion-like protein spreading and transmission of aggregates between cells have also been demonstrated for other proteins associated with Alzheimer disease and Parkinson disease. This protein-only phenomenon may therefore have broader implications in neurodegenerative disorders. The minireviews in this thematic series highlight the recent advances in prion biology and the roles these unique proteins play in disease.
Prions are self-propagating protein conformers that cause a variety of neurodegenerative disorders in humans and animals. Mouse models have played key roles in deciphering the biology of prions and in assessing candidate therapeutics. The development of transgenic mice that form prions spontaneously in the brain has advanced our understanding of sporadic and genetic prion diseases. Furthermore, the realization that many proteins can become prions has necessitated the development of mouse models for assessing the potential transmissibility of common neurodegenerative diseases. As the universe of prion diseases continues to expand, mouse models will remain crucial for interrogating these devastating illnesses.
Recently, synthetic prions with a high level of specific infectivity have been produced from chemically defined components in vitro. A major insight arising from these studies is that various classes of host-encoded cofactor molecules such as phosphatidylethanolamine and RNA molecules are required to form and maintain the specific conformation of infectious prions. Synthetic mouse prions formed with phosphatidylethanolamine exhibit levels of specific infectivity ∼1 million-fold greater than “protein-only” prions (Deleault, N. R., Walsh, D. J., Piro, J. R., Wang, F., Wang, X., Ma, J., Rees, J. R., and Supattapone, S. (2012) Proc. Natl. Acad. Sci. U.S.A. 109, E1938–E1946). Moreover, cofactor molecules also appear to regulate prion strain properties by limiting the potential conformations of the prion protein (see Deleault et al. above). The production of fully infectious synthetic prions provides new opportunities to study the mechanism of prion infectivity directly by structural and biochemical methods.
Work over the past 4 years indicates that multiple proteins associated with neurodegenerative diseases, especially Tau and α-synuclein, can propagate aggregates between cells in a prion-like manner. This means that once an aggregate is formed it can escape the cell of origin, contact a connected cell, enter the cell, and induce further aggregation via templated conformational change. The prion model predicts a key role for extracellular protein aggregates in mediating progression of disease. This suggests new therapeutic approaches based on blocking neuronal uptake of protein aggregates and promoting their clearance. This will likely include therapeutic antibodies or small molecules, both of which can be developed and optimized in vitro prior to preclinical studies.
Neurodegenerative diseases are characterized by the aggregation of misfolded proteins in the brain. Among these disorders are the prion diseases, which are transmissible, and in which the misfolded proteins (“prions”) are also the infectious agent. Increasingly, it appears that misfolded proteins in Alzheimer and Parkinson diseases and the tauopathies also propagate in a “prion-like” manner. However, the association between prion formation, spread, and neurotoxicity is not clear. Recently, we showed that in prion disease, protein misfolding leads to neurodegeneration through dysregulation of generic proteostatic mechanisms, specifically, the unfolded protein response. Genetic and pharmacological manipulation of the unfolded protein response was neuroprotective despite continuing prion replication, hence dissociating this from neurotoxicity. The data have clear implications for treatment across the spectrum of these disorders, targeting pathogenic processes downstream of protein misfolding.
The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.
Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.
The heat shock response, resulting in the production of heat shock proteins or molecular chaperones, is triggered by elevated temperature and a variety of other stressors. Its master regulator is heat shock transcription factor 1 (HSF1). Heat shock factors generally exist in multiple isoforms. The two known isoforms of HSF1 differ in the inclusion (HSF1α) or exclusion (HSF1β) of exon 11. Although there are some data concerning the differential expression patterns and transcriptional activities of HSF2 isoforms during development, little is known about the distinct properties of the HSF1 isoforms. Here we present evidence for two novel HSF1 isoforms termed HSF1γα and HSF1γβ, and we show that the HSF1 isoform ratio differentially regulates heat shock protein gene transcription. Hsf1γ isoforms are expressed in various mouse tissues and are translated into protein. Furthermore, after heat shock, HSF1γ isoforms are exported from the nucleus more rapidly or degraded more quickly than HSF1α or HSF1β. We also show that each individual HSF1 isoform is sufficient to induce the heat shock response and that expression of combinations of HSF1 isoforms, in particular HSF1α and HSF1β, results in a synergistic enhancement of the transcriptional response. In addition, HSF1γ isoforms potentially suppress the synergistic effect of HSF1α and HSF1β co-expression. Collectively, our observations suggest that the expression of HSF1 isoforms in a specific ratio provides an additional layer in the regulation of heat shock protein gene transcription.
The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is key to Ca2+ homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca2+ uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca2+ studies showed decreased nitric oxide (·NO)-induced 45Ca2+ uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca2+ stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca2+ influx. Adenoviral overexpression of calreticulin, an ER Ca2+ binding protein, increased ionomycin-releasable stores, VEGF-induced Ca2+ influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca2+ homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca2+ stores.
Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with •NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged •NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief •NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief •NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of •NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of •NO.
Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.
ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.
The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion.
The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C9–C17 in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.
The TWEAK-fibroblast growth factor-inducible 14 (Fn14) system is a critical regulator of denervation-induced skeletal muscle atrophy. Although the expression of Fn14 is a rate-limiting step in muscle atrophy on denervation, mechanisms regulating gene expression of Fn14 remain unknown. Methylation of CpG sites within promoter region is an important epigenetic mechanism for gene silencing. Our study demonstrates that Fn14 promoter contains a CpG island close to transcription start site. Fn14 promoter also contains multiple consensus DNA sequence for transcription factors activator protein 1 (AP1) and specificity protein 1 (SP1). Denervation diminishes overall genomic DNA methylation and causes hypomethylation at specific CpG sites in Fn14 promoter leading to the increased gene expression of Fn14 in skeletal muscle. Abundance of DNA methyltransferase 3a (Dnmt3a) and its interaction with Fn14 promoter are repressed in denervated skeletal muscle of mice. Overexpression of Dnmt3a inhibits the gene expression of Fn14 and attenuates skeletal muscle atrophy upon denervation. Denervation also causes the activation of ERK1/2, JNK1/2, and ERK5 MAPKs and AP1 and SP1, which stimulate the expression of Fn14 in skeletal muscle. Collectively, our study provides novel evidence that Dnmt3a and MAPK signaling regulate the levels of Fn14 in skeletal muscle on denervation.
High mobility group nucleosomal binding domain 2 (HMGN2) is a small and unique non-histone protein that has many functions in a variety of cellular processes, including regulation of chromatin structure, transcription, and DNA repair. In addition, it may have other roles in antimicrobial activity, cell homing, and regulating cytokine release. Although the biochemical properties of HMGN2 protein are regulated by acetylation and phosphorylation, it is not yet known whether HMGN2 activity can also be regulated by SUMOylation. In this study, we demonstrated for the first time that HMGN2 is modified by covalent attachment of small ubiquitin-related modifier 1 (SUMO1) by pro-inflammatory signal and identified the major SUMOylated lysine residues that localize to the HMGN2 nucleosome-binding domain at Lys-17 and Lys-35. SENP1 can deSUMOylate SUMOylated HMGN2, and PIAS1 is the E3 ligase responsible for SUMOylation of HMGN2. Finally, using SUMO1-conjugated HMGN2 purified from a basal SUMOylation system in Escherichia coli, we demonstrated that SUMOylated HMGN2 has decreased the binding affinity to nucleosome core particles in comparison to unSUMOylated HMGN2. These observations potentially provide new perspectives for understanding the functions of HMGN2 in inflammatory reaction.
To address whether mitochondrial biogenesis is essential for skeletal myogenesis, C2C12 myogenesis was investigated after knockdown of NADH dehydrogenase (ubiquintone) flavoprotein 1 (NDUFV1), which is an oxidative phosphorylation complex I subunit that is the first subunit to accept electrons from NADH. The NDUFVI knockdown enhanced C2C12 myogenesis by decreasing the NAD+/NADH ratio and subsequently inactivating SIRT1 and SIRT1 activators (pyruvate, SRT1720, and resveratrol) abolished the NDUFV1 knockdown-induced myogenesis enhancement. However, the insulin-elicited activation of insulin receptor β (IRβ) and insulin receptor substrate-1 (IRS-1) was reduced with elevated levels of protein-tyrosine phosphatase 1B after NDUFV1 knockdown in C2C12 myotubes. The NDUFV1 knockdown-induced blockage of insulin signaling was released by protein-tyrosine phosphatase 1B knockdown in C2C12 myotubes, and we found that NDUFV1 or SIRT1 knockdown did not affect mitochondria biogenesis during C2C12 myogenesis. Based on these data, we can conclude that complex I dysfunction-induced SIRT1 inactivation leads to myogenesis enhancement but blocks insulin signaling without affecting mitochondria biogenesis.
A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A–D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A–D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo.
NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na+/H+ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na+/H+ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.
REV-ERBα and REV-ERBβ are members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors that play important roles in the regulation of circadian physiology, metabolism, and immune function. Although the REV-ERBs were originally characterized as orphan receptors, recent studies have demonstrated that they function as receptors for heme. Here, we demonstrate that cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX (ZnPP) are ligands that bind directly to the REV-ERBs. However, instead of mimicking the agonist action of heme, CoPP and ZnPP function as antagonists of REV-ERB function. This was unexpected because the only distinction between these ligands is the metal ion that is coordinated. To understand the structural basis by which REV-ERBβ can differentiate between a porphyrin agonist and antagonist, we characterized the interaction between REV-ERBβ with heme, CoPP, and ZnPP using biochemical and structural approaches, including x-ray crystallography and NMR. The crystal structure of CoPP-bound REV-ERBβ indicates only minor conformational changes induced by CoPP compared with heme, including the porphyrin ring of CoPP, which adopts a planar conformation as opposed to the puckered conformation observed in the heme-bound REV-ERBβ crystal structure. Thus, subtle changes in the porphyrin metal center and ring conformation may influence the agonist versus antagonist action of porphyrins and when considered with other studies suggest that gas binding to the iron metal center heme may drive alterations in REV-ERB activity.
In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs.
NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn8) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.
In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo.
Endoplasmic reticulum (ER) stress and ER stress-associated unfolded protein response (UPR) can promote cancer cell survival, but it remains unclear whether they can influence oncogene-induced senescence. The present study examined the role of ER stress in senescence using oncogene-dependent models. Increased ER stress attenuated senescence in part by up-regulating phosphorylated protein kinase B (p-AKT) and decreasing phosphorylated extracellular signal-regulated kinase (p-ERK). A positive feed forward loop between p-AKT, ER stress, and UPR was discovered whereby a transient increase of ER stress caused reduced senescence and promotion of tumorigenesis. Decreased ER stress was further correlated with increased senescence in both mouse and human tumors. Interestingly, H-RAS-expressing Pparβ/δ null cells and tumors having increased cell proliferation exhibited enhanced ER stress, decreased cellular senescence, and/or enhanced tumorigenicity. Collectively, these results demonstrate a new role for ER stress and UPR that attenuates H-RAS-induced senescence and suggest that PPARβ/δ can repress this oncogene-induced ER stress to promote senescence in accordance with its role as a tumor modifier that suppresses carcinogenesis.
Mesenchymal cells alter and retain their phenotype during skeletal development through activation or suppression of signaling pathways. For example, we have shown that Wnt3a only stimulates osteoblast differentiation in cells with intrinsic osteogenic potential (e.g. MC3T3-E1 pre-osteoblasts) and not in fat cell precursors or fibroblasts (3T3-L1 pre-adipocytes or NIH3T3 fibroblasts, respectively). Wnt3a promotes osteogenesis in part by stimulating autocrine production of the osteoinductive ligand Bmp2. Here, we show that the promoter regions of the genes for Bmp2 and the osteoblast marker Alp are epigenetically locked to prevent their expression in nonosteogenic cells. Both genes have conserved CpG islands that exhibit increased CpG methylation, as well as decreased acetylation and increased methylation of histone H3 lysine 9 (H3-K9) specifically in nonosteogenic cells. Treatment of pre-adipocytes or fibroblasts with the CpG-demethylating agent 5′-aza-2′-deoxycytidine or the histone deacetylase inhibitor trichostatin-A renders Bmp2 and Alp responsive to Wnt3a. Hence, drug-induced epigenetic activation of Bmp2 gene expression contributes to Wnt3a-mediated direct trans-differentiation of pre-adipocytes or fibroblasts into osteoblasts. We propose that direct conversion of nonosteogenic cells into osteoblastic cell types without inducing pluripotency may improve prospects for novel epigenetic therapies to treat skeletal afflictions.
Translation of mRNA plays a critical role in consolidation of long-term memory. Here, we report that markers of initiation of mRNA translation are activated during training for contextual memory and that they undergo diurnal oscillation in the mouse hippocampus with maximal activity observed during the daytime (zeitgeber time 4–8 h). Phosphorylation and activation of eukaryotic translation initiation factor 4E (eIF4E), eIF4E-binding protein 1 (4EBP1), ribosomal protein S6, and eIF4F cap-complex formation, all of which are markers for translation initiation, were higher in the hippocampus during the daytime compared with night. The circadian oscillation in markers of mRNA translation was lost in memory-deficient transgenic mice lacking calmodulin-stimulated adenylyl cyclases. Moreover, disruption of the circadian rhythm blocked diurnal oscillations in eIF4E, 4EBP1, rpS6, Akt, and ERK1/2 phosphorylation and impaired memory consolidation. Furthermore, repeated inhibition of translation in the hippocampus 48 h after contextual training with the protein synthesis inhibitor anisomycin impaired memory persistence. We conclude that repeated activation of markers of translation initiation in hippocampus during the circadian cycle might be critical for memory persistence.
Enzymatic activation of Cu,Zn-superoxide dismutase (SOD1) requires not only binding of a catalytic copper ion but also formation of an intramolecular disulfide bond. Indeed, the disulfide bond is completely conserved among all species possessing SOD1; however, it remains obscure how disulfide formation controls the enzymatic activity of SOD1. Here, we show that disulfide formation is a primary event in the folding process of prokaryotic SOD1 (SodC) localized to the periplasmic space. Escherichia coli SodC was found to attain β-sheet structure upon formation of the disulfide bond, whereas disulfide-reduced SodC assumed little secondary structure even in the presence of copper and zinc ions. Moreover, reduction of the disulfide bond made SodC highly susceptible to proteolytic degradation. We thus propose that the thiol-disulfide status in SodC controls the intracellular stability of this antioxidant enzyme and that the oxidizing environment of the periplasm is required for the enzymatic activation of SodC.
The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.
The distinguishing feature of self-inactivating (SIN) retroviral vectors is the deletion of the enhancer/promoter sequences in the U3 region of the 3′ long terminal repeat. This design is used to overcome transcriptional interference and prevent downstream transcription from the 3′ long terminal repeat. SIN vectors were derived from a number of different retroviruses. Studies of SIN vectors show that extensive U3 deletions in HIV-based vectors do not alter viral titers or the in vitro and in vivo properties of the vectors. However, deletion of the U3 sequences in γ- and α-retroviruses correlates with defects in 3′ RNA processing and reduces viral titers by >10-fold. Here, we studied the steps in the retroviral life cycle that are affected by the deletion of sequences in the 3′ U3 region of Moloney murine leukemia virus-derived retroviral vectors. The results show that the amounts of both full-length and internal RNA transcripts of U3-minus vectors are reduced in the nuclei of transfected cells, an effect that is probably due to a general defect in 3′ RNA processing. Furthermore, full-length RNA transcripts were also defective in terms of nuclear export. This defect was complemented by transferring the U3 region to another position within the retroviral vector, indicating that the U3 region contains position-independent cis-acting sequences that are required for the transport of full-length viral transcripts. The results also suggest that the leader region of Moloney murine leukemia virus contains inhibitory/regulatory sequences, which prevent export and mediate nuclear retention of full-length viral RNA.
Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.
Although soluble species of the amyloid-β peptide Aβ42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-β (Aβ). Here, we show that Aβ peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aβ42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aβ in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aβ in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aβ42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aβ42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aβ42 wild-type peptide. Shorter peptides (Aβ38 or Aβ40) and other longer peptides (nontoxic Aβ42 G33A substitution or Aβ43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aβ42 impacts gene regulation, and deleterious effects of Aβ42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.
♦ See referenced article, J. Biol. Chem. 2014, 289, 20182–20191
Soluble amyloid-β42 peptides eventually precipitate to form insoluble plaques that are a characteristic formation in the brains of Alzheimer patients. However, not much is known about the biological activities of the soluble peptides. In this Paper of the Week, a team led by Gerhard Multhaup at McGill University in Canada demonstrated that soluble amyloid-β42 peptides affect gene transcription in the nucleus. The investigators studied amyloid-β peptides in lengths that ranged from 38 to 43 amino acids in cultured neuroblastoma cells and in mouse models. They used biochemical analysis, optical microscopy, and electron microscopy to show that amyloid-β42 peptides in the nucleus repressed transcription of two specific genes. Importantly, the authors used chromatin immunoprecipitation assays to demonstrate a direct effect of the peptides on gene expression. More notably, the amyloid-β42 peptides increased the transcription of the gene from which the amyloid-β peptides are derived. The authors note that the genes regulated by amyloid-β42 peptides could serve as targets for new therapeutics.
Electron micrographs of hippocampal neuronal nuclei taken from wild-type and mutant mice, which express both mouse and human amyloid precursor protein. The cells were labeled with an antibody against amyloid-β42 peptides.
Alcohol dehydrogenase 5 (ADH5) is a conserved enzyme for alcohol and aldehyde metabolism in mammals. Despite dynamic expression throughout neurogenesis, its role in neuronal development remains unknown. Here we present the first evidence that ADH5 is a negative regulator of neuronal differentiation. Gene expression analyses identify a constant reduction of ADH5 levels throughout neuronal development. Overexpression of ADH5 reduces both development and adult neuronal differentiation of mouse neurons. This effect depends on the catalytic activity of ADH5 and involves ADH5-mediated denitrosation of histone deacetylase 2 (HDAC2). Our results indicate that ADH5 counteracts neuronal differentiation of human neural stem cells and that this effect can be reversed by pharmacological inhibition of ADH5. Based on these observations, we propose that ADH5 is a novel suppressor of neuronal differentiation and maturation. Inhibition of ADH5 may improve adult neurogenesis in a physiological or pathological setting.
Brain metabolism is thought to be maintained by neuronal-glial metabolic coupling. Glia take up glutamate from the synaptic cleft for conversion into glutamine, triggering glial glycolysis and lactate production. This lactate is shuttled into neurons and further metabolized. The origin and role of lactate in severe traumatic brain injury (TBI) remains controversial. Using a modified weight drop model of severe TBI and magnetic resonance (MR) spectroscopy with infusion of 13C-labeled glucose, lactate, and acetate, the present study investigated the possibility that neuronal-glial metabolism is uncoupled following severe TBI. Histopathology of the model showed severe brain injury with subarachnoid and hemorrhage together with glial cell activation and positive staining for Tau at 90 min post-trauma. High resolution MR spectroscopy of brain metabolites revealed significant labeling of lactate at C-3 and C-2 irrespective of the infused substrates. Increased 13C-labeled lactate in all study groups in the absence of ischemia implied activated astrocytic glycolysis and production of lactate with failure of neuronal uptake (i.e. a loss of glial sensing for glutamate). The early increase in extracellular lactate in severe TBI with the injured neurons rendered unable to pick it up probably contributes to a rapid progression toward irreversible injury and pan-necrosis. Hence, a method to detect and scavenge the excess extracellular lactate on site or early following severe TBI may be a potential primary therapeutic measure.
Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.
The HIV-1 protein Rev oligomerizes on viral transcripts and directs their nuclear export. Previously, a Fab against Rev generated by phage display was used to crystallize and solve the structure of the Rev oligomerization domain. Here we have investigated the capability of this Fab to block Rev oligomerization and inhibit HIV-1 replication. The Fab itself did not have antiviral activity, but when a Tat-derived cell-penetrating peptide was appended, the resulting molecule (FabRev1-Tat) was strongly inhibitory of three different CCR5-tropic HIV-1 isolates (IC50 = 0.09–0.44 μg/ml), as assessed by suppression of reverse transcriptase activity in infected peripheral blood mononuclear cells, and had low cell toxicity (TC50 > 100 μg/ml). FabRev1-Tat was taken up by both peripheral blood mononuclear and HEK293T cells, appearing in both the cytoplasm and nucleus, as shown by immunofluorescence confocal laser scanning microscopy. Computational alanine scanning was used to identify key residues in the complementarity-determining regions to guide mutagenesis experiments. Residues in the light chain CDR3 (LCDR3) were assessed to be important. Residues in LCDR3 were mutated, and LCDR3-Tyr92 was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. Peptides corresponding to all six CDR regions were synthesized and tested for Rev binding. None of the linear peptides had significant affinity for Rev, but four of the amide-cyclic forms did. Especially cyclic-LCDR3 (LGGYPAASYRTA) had high affinity for Rev and was able to effectively depolymerize Rev filaments, as shown by both surface plasmon resonance and electron microscopy.
The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfrwa5 mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury.
Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1–5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1–5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1–5 protein inside experimental data-based models showed how two chains of HAMP1–5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1–5 structure mediate its function.
In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)- Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins.
O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase. A recent computational screen identified a possible candidate for the base J-associated glucosyltransferase (JGT) in trypanosomatid genomes. We demonstrate that recombinant JGT utilizes uridine diphosphoglucose to transfer glucose to hmU in the context of dsDNA. Mutation of conserved residues typically involved in glucosyltransferase catalysis impairs DNA glucosylation in vitro. The deletion of both alleles of JGT from the genome of Trypanosoma brucei generates a cell line that completely lacks base J. Reintroduction of JGT in the JGT KO restores J synthesis. Ablation of JGT mRNA levels by RNAi leads to the sequential reduction in base J and increased levels of hmU that dissipate rapidly. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only glucosyltransferase enzyme required for the second step of the pathway. Similar to the activity of the related Ten-Eleven Translocation (TET) family of dioxygenases on 5mC, our studies also suggest the ability of the base J-binding protein enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. Here we discuss the regulation of hmU and base J formation in the trypanosome genome by JGT and base J-binding protein.
Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins β-arrestin (βARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and βARR1/2 terminate plasma membrane Ca2+ signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. βARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from βARRs, to recycle. Thus, both ECE-1 and βARRs mediate the resensitization of NK1R Ca2+ signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or βARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of βARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation.
Heparan sulfate (HS) plays a crucial role in the fibrosis associated with chronic allograft dysfunction by binding and presenting cytokines and growth factors to their receptors. These interactions critically depend on the distribution of 6-O-sulfated glucosamine residues, which is generated by glucosaminyl-6-O-sulfotransferases (HS6STs) and selectively removed by cell surface HS-6-O-endosulfatases (SULFs). Using human renal allografts we found increased expression of 6-O-sulfated HS domains in tubular epithelial cells during chronic rejection as compared with the controls. Stimulation of renal epithelial cells with TGF-β induced SULF2 expression. To examine the role of 6-O-sulfated HS in the development of fibrosis, we generated stable HS6ST1 and SULF2 overexpressing renal epithelial cells. Compared with mock transfectants, the HS6ST1 transfectants showed significantly increased binding of FGF2 (p = 0.0086) and pERK activation. HS6ST1 transfectants displayed a relative increase in mono-6-O-sulfated disaccharides accompanied by a decrease in iduronic acid 2-O-sulfated disaccharide structures. In contrast, SULF2 transfectants showed significantly reduced FGF2 binding and phosphorylation of ERK. Structural analysis of HS showed about 40% down-regulation in 6-O-sulfation with a parallel increase in iduronic acid mono-2-O-sulfated disaccharides. To assess the relevance of these data in vivo we established a murine model of fibrosis (unilateral ureteric obstruction (UUO)). HS-specific phage display antibodies (HS3A8 and RB4EA12) showed significant increase in 6-O-sulfation in fibrotic kidney compared with the control. These results suggest an important role of 6-O-sulfation in the pathogenesis of fibrosis associated with chronic rejection.
Reelin is an extracellular protein that controls many aspects of pre- and postnatal brain development and function. The molecular mechanisms that mediate postnatal activities of Reelin are not well understood. Here, we first set out to express and purify the full length Reelin protein and a biologically active central fragment. Second, we investigated in detail the signal transduction mechanisms elicited by these purified Reelin proteins in cortical neurons. Unexpectedly, we discovered that the full-length Reelin moiety, but not the central fragment, is capable of activating Erk1/2 signaling, leading to increased p90RSK phosphorylation and the induction of immediate-early gene expression. Remarkably, Erk1/2 activation is not mediated by the canonical signal transduction pathway, involving ApoER2/VLDLR and Dab1, that mediates other functions of Reelin in early brain development. The activation of Erk1/2 signaling likely contributes to the modulation of neuronal maturation and synaptic plasticity by Reelin in the postnatal and adult brain.
Several neurodegenerative diseases are characterized by the aggregation and posttranslational modifications of Tau protein. Its “repeat domain” (TauRD) is mainly responsible for the aggregation properties, and oligomeric forms are thought to dominate the toxic effects of Tau. Here we investigated the conformational transitions of this domain during oligomerization and aggregation in different states of β-propensity and pseudo-phosphorylation, using several complementary imaging and spectroscopic methods. Although the repeat domain generally aggregates more readily than full-length Tau, its aggregation was greatly slowed down by phosphorylation or pseudo-phosphorylation at the KXGS motifs, concomitant with an extended phase of oligomerization. Analogous effects were observed with pro-aggregant variants of TauRD. Oligomers became most evident in the case of the pro-aggregant mutant TauRDΔK280, as monitored by atomic force microscopy, and the fluorescence lifetime of Alexa-labeled Tau (time-correlated single photon counting (TCSPC)), consistent with its pronounced toxicity in mouse models. In cell models or primary neurons, neither oligomers nor fibrils of TauRD or TauRDΔK280 had a toxic effect, as seen by assays with lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, respectively. However, oligomers of pro-aggregant TauRDΔK280 specifically caused a loss of spine density in differentiated neurons, indicating a locally restricted impairment of function.
Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[3H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.
The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases.
Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease.
Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.
Mitochondria capture and subsequently release Ca2+ ions, thereby sensing and shaping cellular Ca2+ signals. The Ca2+ uniporter MCU mediates Ca2+ uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca2+ against Na+ or H+, respectively. Here we study the role of these ion exchangers in mitochondrial Ca2+ extrusion and in Ca2+-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca2+ efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca2+ ([Ca2+]mt) elevations. NCLX overexpression enhanced the rates of Ca2+ efflux, whereas increasing LETM1 levels had no impact on Ca2+ extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca2+]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na+/Ca2+ exchanger inhibitor CGP37157. The [Ca2+]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na+/Ca2+ exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca2+ extrusion from mitochondria. By controlling the duration of matrix Ca2+ elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca2+ signals into redox changes.
Second messengers such as phosphoinositides and calcium are known to control diverse processes involved in the development of malaria parasites. However, the underlying molecular mechanisms and pathways need to be unraveled, which may be achieved by understanding the regulation of effectors of these second messengers. Calcium-dependent protein kinase (CDPK) family members regulate diverse parasitic processes. Because CDPKs are absent from the host, these kinases are considered as potential drug targets. We have dissected the function of an atypical CDPK from Plasmodium falciparum, PfCDPK7. The domain architecture of PfCDPK7 is very different from that of other CDPKs; it has a pleckstrin homology domain adjacent to the kinase domain and two calcium-binding EF-hands at its N terminus. We demonstrate that PfCDPK7 interacts with PI(4,5)P2 via its pleckstrin homology domain, which may guide its subcellular localization. Disruption of PfCDPK7 caused a marked reduction in the growth of the blood stage parasites, as maturation of rings to trophozoites was markedly stalled. In addition, parasite proliferation was significantly attenuated. These findings shed light on an important role for PfCDPK7 in the erythrocytic asexual cycle of malaria parasites.
In eukaryotes, there are three major RNA polymerases (Pol) in the nucleus, which are commonly described as transcribing non-overlapping subsets of genes. Structural studies have highlighted a conserved core shared among all three transcription systems. Initiation of human Pol III from TATA box-containing Pol II promoters under conditions with impaired Pol II transcription activity have been described previously. RNA polymerase III and Pol II were found to co-localize at the promoters of the c-myc gene and the RPPH1 sRNA in vivo. Here, I report that Pol III can, like Pol II, initiate transcription from most tested Pol II core promoters when assayed with crude human nuclear extracts (HSK, SNF, or Dignam). Both polymerases often initiate from the same transcription start site, and depend on a TATA box or AT-rich region but not the downstream promoter element (DPE) or the motif ten element (MTE). Moderate (∼2-fold) changes in the ratio of DNA template to nuclear extract were sufficient to change Pol II-mediated transcription to a mixture of Pol II- and Pol III-, or to a solely Pol III-dependent initiation of transcription from Pol II promoters. Polymerase specificity is thus not fixed but a variable that depends on the properties of the promoter and the transcription conditions. These findings provide functional evidence for a close similarity between the Pol II and Pol III transcription complexes, and additionally explain previous controversies in the literature.