Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

The human micro-vascular enclothelial cells in vitro interaction with atomic-nitrogen-doped diamond-like carbon thin films

This paper reports the initial response of atomic nitrogen doped diamond like carbon (DLC) to endothelial cells in vitro. The introduction of nitrogen atoms/molecules to the diamond like carbon structures leads to an atomic structural change favorable to the attachment of human micro-vascular enclothelial cells. Whilst the semi-conductivity induced by nitrogen in DLC is thought to play a part, the increase in the inion-bonded N atoms and N-2 molecules in the atomic doped species (with the exclusion of the charged species) seems to contribute to the improved attachment of human microvascular endothelial cells. The increased endothelial attachment is associated with a lower work function and slightly higher water contact angle in the atomic doped films, where the heavy charged particles are excluded. The films used in the study were synthesized by the RF PECVD technique followed by post deposition doping with nitrogen, and afterwards the films were characterized by XPS, Raman spectroscopy, SIMS and Kelvin probe. The water contact angles were measured, and the counts of the adherent endothelial cells on the samples were carried out. This study is relevant and contributory to improving biocompatibility of surgical implants and prostheses.

Two-electron QED contributions to the ground-state binding energy in He-like Kr34+ ions

The two-electron QED contributions to the ground-state binding energy of Kr34+ ions have been determined in two independent experiments performed with electron beam ion traps (EBIT) in Heidelberg (HD) and Tokyo (BT, Belfast-Tokyo collaboration). X rays arising from radiative recombination (RR) of free electrons to the ground state of initially bare Kr36+ and hydrogenlike Kr35+ ions were observed as a function of the interacting electron energy. The K edge absorption by thin Eu and W foils provided fixed photon energy references used to measure the difference in binding energy Delta E-2e between the H- and He-like Kr ions (Kr35+ and Kr34+, respectively). The two values agree well, yielding a final result of Delta E-2e=641.8 +/- 1.7 eV, confirming recent results of rigorous QED calculations. This accuracy is just of the order required to access screened radiative QED contributions.

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.