Expression and processing of Plasmodium berghei SERA3 during liver stages.

Cysteine proteases mediate liberation of Plasmodium berghei merozoites from infected hepatocytes. In an attempt to identify the responsible parasite proteases, we screened the genome of P. berghei for cysteine protease-encoding genes. RT-PCR analyses revealed that transcription of four out of five P. berghei serine repeat antigen (PbSERA) genes was strongly upregulated in late liver stages briefly before the parasitophorous vacuole membrane ruptured to release merozoites into the host cell cytoplasm, suggesting a role of PbSERA proteases in these processes. In order to characterize PbSERA3 processing, we raised an antiserum against a non-conserved region of the protein and generated a transgenic P. berghei strain expressing a TAP-tagged PbSERA3 under the control of the endogenous promoter. Immunofluorescence assays revealed that PbSERA3 leaks into the host cell cytoplasm during merozoite development, where it might contribute to host cell death or activate host cell proteases that execute cell death. Importantly, processed PbSERA3 has been detected by Western blot analysis in cell extracts of schizont-infected cells and merozoite-infected detached hepatic cells.

Characterization of Endoproteolytic Processing of Dynorphins by Proprotein Convertases using Mouse Spinal Cord S9 Fractions and Mass Spectrometry

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous Big Dynorphin (BDyn) and Dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild type (WT), PC1-/+ and PC2-/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments Dynorphin 1-19, Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7 and C-terminal processing of Dyn A generates Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even partial inhibition of PC1 or PC2 may impair the endogenous opioid system.