QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser(160), Glu(201), Asp(248), Asp(305) and His(319)), within which Glu(201) and Asp(248) were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu(201)...Asp(305) and Asp(248)...Asp(305), reduced the steady-state catalysis dramatically. The roles of these two COOH...COOH bonds on catalysis could be partly replaced by COOH...water bonds, but not by COOH...CONH(2) bonds, reminiscent of the low-barrier Asp...Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp(305), a residue located at the centre of the H-bond network, raised the K(m) value of the enzyme by 4.4-19-fold, but decreased the k(cat) value by 79-2842-fold, indicating that Asp(305) primarily plays a catalytic role. In addition, results from mutational studies on Ser(160) and His(319) suggest that these two residues might help to stabilize the conformations of Asp(248) and Asp(305) respectively. These data allow us to propose an essential proton transfer between Glu(201), Asp(305) and Asp(248) during the catalysis by animal QCs.
Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene ( HTT ), which causes the inherited neurodegenerative disorder, Huntington’s Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila “rough eye” phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.
Since the discovery of graphene, 2D materials are establishing a rapidly growing and promising field, with great potential for diverse applications given their excellent conductivity and high transparency. In particular, graphene can isolate external chemical reactions when applied to back-end-of-line (BEOL) interconnect metals, thereby preventing the oxidation of the interconnect metals. In addition, it can improve the conductivity and breakdown current density of interconnects. However, the thermal budget remains an important problem in BEOL interconnects. We demonstrate an advanced graphene deposition method using an in-house plasma plasma-enhanced chemical vapor deposition system, which offers low thermal budget and high stability. We also optimize carbon precursors to improve the quality of graphene on Ru and Co thin films. We show the improvement of electrical conductivity, electromigration lifetime, and maximum breakdown current density after capping Ru and Co interconnects with graphene. The electromigration lifetimes of the Ru and Co interconnects increase by 4 and 4.5 times, respectively, and their maximum breakdown current density increases by 17.6 and 10.6%, respectively. The results show that capping with graphene has a high potential in BEOL applications.
In recent years, many reports have demonstrated the high potential for multilayer graphene in semiconductor fabrication. As interconnects within semiconductors or electrodes for two-dimensional transistors, the preparation of large-area multilayer graphene is becoming increasingly important. Herein, we report a method for growing large-area multilayer graphene, which can achieve rapid heating and cooling. With the use of a high carbon concentration source, the preparation of multilayer graphene can be completed in a few seconds. This manufacturing method has the advantage of producing graphene with high quality, uniformity, and electrical conductivity. In commercial applications, this technology has great potential for the mass production and rapid fabrication of multilayer graphene. In addition, we found that the multilayer graphene produced by this method had cobalt atoms doped into the multilayer graphene during the process, resulting in its low resistivity. Combined with our intercalation technology, intercalated FeCl 3 in the graphene interlayer can reduce the resistivity of graphene to 3.55 μΩ cm, which is very close to the resistivity of copper bulk. This result makes multilayer graphene more promising for various applications.
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