The human Rad9-Rad1-Hus1 checkpoint complex stimulates flap endonuclease 1

The toroidal damage checkpoint complex Rad9-Rad1-Hus1 (9-1-1) has been characterized as a sensor of DNA damage. Flap endonuclease 1 (FEN1) is a structure-specific nuclease involved both in removing initiator RNA from Okazaki fragments and in DNA repair pathways. FEN1 activity is stimulated by proliferating cell nuclear antigen (PCNA), a toroidal sliding clamp that acts as a platform for DNA replication and repair complexes. We show that 9-1-1 also binds and stimulates FEN1. Stimulation is observed on a variety of flap, nick, and gapped substrates simulating repair intermediates. Blocking 9-1-1 entry to the double strands prevents a portion of the stimulation. Like PCNA stimulation, 9-1-1 stimulation cannot circumvent the tracking mechanism by which FEN1 enters the substrate; however, 9-1-1 does not substitute for PCNA in the stimulation of DNA polymerase beta. This suggests that 9-1-1 is a damage-specific activator of FEN1.     

Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange

Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins.     

Cleavage mechanism of human Mus81-Eme1 acting on Holliday-junction structures

Recombination-mediated repair plays a central role in maintaining genomic integrity during DNA replication. The human Mus81-Eme1 endonuclease is involved in recombination repair, but the exact structures it acts on in vivo are not known. Using kinetic and enzymatic analysis of highly purified recombinant enzyme, we find that Mus81-Eme1 catalyzes coordinate bilateral cleavage of model Holliday-junction structures. Using a self-limiting, cruciform-containing substrate, we demonstrate that bilateral cleavage occurs sequentially within the lifetime of the enzyme-substrate complex. Coordinate bilateral cleavage is promoted by the highly cooperative nature of the enzyme and results in symmetrical cleavage of a cruciform structure, thus, Mus81-Eme1 can ensure coordinate, bilateral cleavage of Holliday junction-like structures.     

Human recombinant interleukin-1β stimulates glycosaminoglycan production in human synovial fibroblast cultures

Human recombinant interleukin-1β (rIL-1β) stimulated glycosaminoglycan (GAG) production in human synovial fibroblast cultures. A dose-dependent increase in GAG production was found, to a maximum of 500%. Increase was detected at doses as low as 1 pg/ml of rIL-1β, reached a maximum at 10-100 pg/ml, and was apparent 10 hours after addition of rIL-1β. Stimulation of GAG was always accompanied by increased accumulation of prostaglandin E (PGE) in culture media and by increased collagenase production in approximately one-half the experiments. Indomethacin (5 m̈/ml) completely inhibited PGE stimulation by rIL-1β, but only partially inhibited that of GAG overproduction and had no effect on collagenase production. Hydrocortisone (2 m̈/ml) inhibited stimulation of all 3 parameters. Stimulation of hyaluronate in synovial cultures prevailed over that of sulfated GAG, which occurred to à lesser extent. Our results support earlier suggestions that interleukin-1 is a major active mononuclear cell factor that is capable of inducing profound changes in connective tissue cell function.     

Human Astrovirus 1–8 Seroprevalence Evaluation in a United States Adult Population

Human astroviruses are an important cause of viral gastroenteritis globally, yet few studies have investigated the serostatus of adults to establish rates of previous infection. Here, we applied biolayer interferometry immunosorbent assay (BLI-ISA), a recently developed serosurveillance technique, to measure the presence of blood plasma IgG antibodies directed towards the human astrovirus capsid spikes from serotypes 1–8 in a cross-sectional sample of a United States adult population. The seroprevalence rates of IgG antibodies were 73% for human astrovirus serotype 1, 62% for serotype 3, 52% for serotype 4, 29% for serotype 5, 27% for serotype 8, 22% for serotype 2, 8% for serotype 6, and 8% for serotype 7. Notably, seroprevalence rates for capsid spike antigens correlate with neutralizing antibody rates determined previously. This work is the first seroprevalence study evaluating all eight classical human astrovirus serotypes.     

LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Epilepsy, characterized by unprovoked, recurrent seizures, affects 1 to 2% of the population worldwide. Many genes that cause inherited epilepsy when mutated encode ion channels, and dysregulated synaptic transmission often causes epilepsy (1, 2). Although antiepileptic drugs have mainly targeted ion channels, they are not always effective and have adverse effects. It is therefore important to clarify the detailed processes for synaptic transmission and how they are affected in epilepsy.Recent superresolution imaging of the synapse reveals previously overlooked subsynaptic nano-organizations and pre- and postsynaptic nanodomains (3–6), and mathematical simulation suggests their nanometer-scale coordination in individual synapses for efficient synaptic transmission: presynaptic neurotransmitter release machinery and postsynaptic receptors precisely align across the synaptic cleft to make “transsynaptic nanocolumns” (7, 8).So far, numerous transsynaptic cell-adhesion molecules have been identified (9–12), including presynaptic Neurexins and type IIa receptor protein tyrosine phosphatases (PTPδ, PTPσ, and LAR) and postsynaptic Neuroligins, LRRTMs, NGL-3, IL1RAPL1, Slitrks, and SALMs. Neurexins–Neuroligins have attracted particular attention because of their synaptogenic activities when overexpressed and their genetic association with neuropsychiatric disorders (e.g., autism). Another type of transsynaptic adhesion complex mediated by synaptically secreted Cblns (e.g., Neurexin–Cbln1–GluD2) promotes synapse formation and maintenance (13–15). Genetic studies in Caenorhabditis elegans show that secreted Ce-Punctin, the ortholog of the mammalian ADAMTS-like family, specifies cholinergic versus GABAergic identity of postsynaptic domains and functions as an extracellular synaptic organizer (16). However, the molecular identity and in vivo physiological significance of transsynaptic nanocolumns remain incompletely understood.LGI1, a neuronal secreted protein, and its receptor ADAM22 have recently emerged as major determinants of brain excitability (17) as 1) mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (18); 2) mutations in the ADAM22 gene cause infantile epileptic encephalopathy with intractable seizures and intellectual disability (19, 20); 3) Lgi1 or Adam22 knockout mice display lethal epilepsy (21–24); and 4) autoantibodies against LGI1 cause limbic encephalitis characterized by seizures and amnesia (25–28). Functionally, LGI1–ADAM22 regulates AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated synaptic transmission (17, 22, 29) and Kv₁ channel-mediated neuronal excitability (30, 31). Recent structural analysis shows that LGI1 and ADAM22 form a 2:2 heterotetrameric assembly (ADAM22–LGI1–LGI1–ADAM22) (32), suggesting the transsynaptic configuration.In this study, we identify ADAM22-mediated synaptic protein networks in the brain, including pre- and postsynaptic MAGUKs and their functional bindings to transmembrane proteins (NMDA/AMPA glutamate receptors, voltage-dependent ion channels, cell-adhesion molecules, and vesicle-fusion machinery). ADAM22 knock-in mice lacking the MAGUK-binding motif show lethal epilepsy of hippocampal origin. In this mouse, postsynaptic PSD-95 nano-assembly as well as nano-scale alignment between pre- and postsynaptic proteins are significantly impaired. Importantly, PSD-95 is no longer able to modulate AMPAR-mediated synaptic transmission without binding to ADAM22. These findings establish that LGI1–ADAM22 instructs MAGUKs to organize transsynaptic nanocolumns and guarantee the stable brain activity.     

Interferon α2–Thymosin α1 Fusion Protein (IFNα2–Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2–thymosin α1 fusion protein (IFNα2–Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2–Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2–Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2–Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2–Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.     

Human recombinant interleukin-1 beta stimulates glycosaminoglycan production in human synovial fibroblast cultures

Human recombinant interleukin-1 beta (rIL-1 beta) stimulated glycosaminoglycan (GAG) production in human synovial fibroblast cultures. A dose-dependent increase in GAG production was found, to a maximum of 500%. Increase was detected at doses as low as 1 pg/ml of rIL-1 beta, reached a maximum at 10-100 pg/ml, and was apparent 10 hours after addition of rIL-1 beta. Stimulation of GAG was always accompanied by increased accumulation of prostaglandin E (PGE) in culture media and by increased collagenase production in approximately one-half the experiments. Indomethacin (5 micrograms/ml) completely inhibited PGE stimulation by rIL-1 beta, but only partially inhibited that of GAG overproduction and had no effect on collagenase production. Hydrocortisone (2 micrograms/ml) inhibited stimulation of all 3 parameters. Stimulation of hyaluronate in synovial cultures prevailed over that of sulfated GAG, which occurred to a lesser extent. Our results support earlier suggestions that interleukin-1 is a major active mononuclear cell factor that is capable of inducing profound changes in connective tissue cell function.     

Antiferromagnet–Ferromagnet Transition in Fe1−xCuxNbO4

Iron niobates, pure and substituted with copper (Fe_1−xCu_xNbO₄ with x = 0–0.15), were prepared by the solid-state method and characterized by X-ray diffraction, Raman spectroscopy, and magnetic measurements. The results of the structural characterizations revealed the high solubility of Cu ions in the structure and better structural stability compared to the pure sample. The analysis of the magnetic properties showed that the antiferromagnetic–ferromagnetic transition was caused by the insertion of Cu²⁺ ions into the FeNbO₄ structure. The pure FeNbO₄ structure presented an antiferromagnetic ordering state, with a Néel temperature of approximately 36.81K. The increase in substitution promoted a change in the magnetic ordering, with the state passing to a weak ferromagnetic order with a transition temperature (T_c) higher than the ambient temperature. The origin of the ferromagnetic ordering could be attributed to the increase in super-exchange interactions between Fe/Cu ions in the Cu²⁺-O-Fe³⁺ chains and the formation of bound magnetic polarons in the oxygen vacancies.     

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.     

Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Synucleinopathies, including Parkinson’s disease (PD), are associated with the accumulation of intracellular neuronal aggregates termed as Lewy bodies and Lewy neuritis, which contain high concentration of the protein α-synuclein (α-Syn) in an aggregated state (1, 2). The disease-relevant role of α-Syn is further highlighted by mutations in the α-Syn gene (SNCA) causing familial PD [i.e., A30P (3), E46K (4), H50Q (5), G51D (6), A53E (7), and A53T (8)] and the duplication or triplication of the SNCA leading to early-onset PD in affected families (9, 10). α-Syn is a 140-residue intrinsically disordered protein (IDP) in solution (11) but adopts a helical structure in the presence of acidic lipid surfaces (12, 13). The positively charged N terminus (residues 1 to 60) is rich in lysine residues and contains KTKEGV binding repeats associated with vesicle binding (14). Moreover, the N-terminal domain includes all known SNCA familial PD mutations. The central region (residues 61 to 95) defines the non-amyloid-β component (NAC) (15), which is essential for α-Syn aggregation (16), while the C terminus (residues 96 to 140) is highly negatively charged.In vitro, α-Syn forms polymorphic amyloid fibrils (17–19) with unique arrangements of cross-β-sheet motifs (20–22). When injected into model animals, these fibrils induce a PD-like pathology (23) where the aggregation pathway of α-Syn plays a key role in the development of the disease (24). A detailed analysis of the aggregation kinetics of α-Syn into amyloids is therefore important toward understanding the toxic mechanisms relevant for synucleinopathies.Amyloid formation of α-Syn is very sensitive to solution conditions, including pH (25), temperature (26), and salt concentration (27). It further requires the presence of an air–water interface (28) or negatively charged lipid membranes (29) for which α-Syn has a high affinity. Previous studies suggest that amyloid fibril growth of α-Syn occurs via a nucleation-dependent polymerization reaction (30). Following a fairly slow primary nucleus formation, α-Syn fibrils are elongated by addition of single monomers. In a next step, the amyloid fibrils multiply by fragmentation or can catalyze the formation of new amyloids from monomers on their surface—a process known as secondary nucleation that was first described for sickle cell anemia 40 y ago (31). Fragmentation and secondary nucleation critically depend on the fibril mass and accelerate the aggregation kinetics (30). In the case of α-Syn aggregation under quiescent condition fragmentation does not exist and only the described secondary nucleation process occurs. While detailed kinetic experiments showed no significant secondary nucleation at pH 7, it strongly contributes at pH values lower than 6 (25, 30). However, mechanistic or structural information of the secondary nucleation process in α-Syn aggregation has been lacking so far.In this study we investigated the structural properties of α-Syn monomer–fibril interactions by NMR and electron paramagnetic resonance (EPR) spectroscopy. Our results provide insights into how monomeric α-Syn transiently interacts in vitro via its positively charged N terminus with the negatively charged C-terminal residues of the α-Syn fibrils, giving detailed insights into the mechanism of the secondary nucleation process.     

Dpb11 activates the Mec1–Ddc2 complex

The Saccharomyces cerevisiae Mec1–Ddc2 checkpoint kinase complex (the ortholog to human ATR-ATRIP) is an essential regulator of genomic integrity. The S. cerevisiae BRCT repeat protein Dpb11 functions in the initiation of both DNA replication and cell cycle checkpoints. Here, we report a genetic and physical interaction between Dpb11 and Mec1–Ddc2. A C-terminal domain of Dpb11 is sufficient to associate with Mec1–Ddc2 and strongly stimulates the kinase activity of Mec1 in a Ddc2-dependent manner. Furthermore, Mec1 phosphorylates Dpb11 and thereby amplifies the stimulating effect of Dpb11 on Mec1–Ddc2 kinase activity. Thus, Dpb11 is a functional ortholog of human TopBP1, and the Mec1/ATR activation mechanism is conserved from yeast to humans.     

α-Tocopherol transfer protein stimulates the secretion of α-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway

Vitamin E (α-tocopherol) is a fat-soluble antioxidant that is transported by plasma lipoproteins in the body. α-Tocopherol taken up by the liver with lipoprotein is thought to be resecreted into the plasma in very low density lipoprotein (VLDL). α-Tocopherol transfer protein (αTTP), which was recently identified as a product of the causative gene for familial isolated vitamin E deficiency, is a cytosolic liver protein and plays an important role in the efficient recycling of plasma vitamin E. To throw light on the mechanism of αTTP-mediated α-tocopherol transfer in the liver cell, we devised an assay system using the hepatoma cell line McARH7777. Using this system, we found that the secretion of α-tocopherol was more efficient in cells expressing αTTP than in matched cells lacking αTTP. Brefeldin A, which effectively inhibits VLDL secretion by disrupting the Golgi apparatus, had no effect on α-tocopherol secretion, indicating that αTTP-mediated α-tocopherol secretion is not coupled to VLDL secretion. Among other agents tested, only 25-hydroxycholesterol, a modulator of cholesterol metabolism, inhibited α-tocopherol secretion. This inhibition is most likely mediated by oxysterol-binding protein. These results suggest that αTTP present in the liver cytosol functions to stimulate secretion of cellular α-tocopherol into the extracellular medium and that the reaction utilizes a novel non-Golgi-mediated pathway that may be linked to cellular cholesterol metabolism and/or transport.