Studies on the pro-oxidative properties and neurotoxic potential of Angeli's salt-derived nitroxyl (HNO/NO-)

The biological function of nitric oxide and its oxidized forms has received a great deal of attention over the past two decades. However much less attention has been focused on the reduced nitric oxide, nitroxyl (HNO). Unlike NO, HNO is highly reactive species and thus it needs to be generated by using donor compounds under experimental conditions. Currently there is only one donor available, Angeli s salt, which releases HNO in a controlled fashion under pysiological conditions. Prior studies have shown the pro-oxidative and cytotoxic potential of Angeli s salt compared to NO donors. The high reactivity of HNO with cysteine thiols is considered to form the biochemical basis for its unique properties compared to other nitrogen oxides. Such thiol modification cold result in disturbances of vital cellular functions and subsequently to death of disturbance sensitive cells, such as neurons. Therefore modification of proteins and lipids was studied in vitro and the potential neurotoxicity was studied in vivo by local infusion of Angeli s salt into the rat central nervous system. The results show that under aerobic in vitro conditions, HNO can, subsequent to autoxidation, cause irreversible oxidative modification of proteins and lipids. These effects are not however seen in cell culture or following infusion of Angeli s salt directly into the rat central nervous tissue likely due to presence of lower oxygen and higher thiol concentration. However, due to high reactivity with thiols, HNO can cause irreversible inactivation of cysteine modification sensitive enzymes such as cysteine proteases papain in vitro and cathepsin B in cell culture. Furthermore it was shown that infusion of HNO releasing Angeli s salt into the rat central nervous system causes necrotic cell death and motor dysfunction following infusion into the lumbal intrathecal space. In conclusion, the acute neurotoxic potential of Angeli s salt was shown to be relatively low, but still higher compared to NO donors. HNO was shown to affect numerous cellular processes which could result in neurotoxicity if HNO was produced in vivo.

Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.