Mono-ADP-ribosylation by PARP10 inhibits Chikungunya virus nsP2 proteolytic activity and viral replication.

Replication of viruses requires interaction with host cell factors and repression of innate immunity. Recent findings suggest that a subset of intracellular mono-ADP-ribosylating PARPs, which are induced by type I interferons, possess antiviral activity. Moreover, certain RNA viruses, including Chikungunya virus (CHIKV), encode mono-ADP-ribosylhydrolases. Together, this suggests a role for mono-ADP-ribosylation (MARylation) in host-virus conflicts, but the relevant substrates have not been identified. We addressed which PARP restricts CHIKV replication and identified PARP10 and PARP12. For PARP10, this restriction was dependent on catalytic activity. Replication requires processing of the non-structural polyprotein nsP1-4 by the protease located in nsP2 and the assembly of the four individual nsP1-nsP4 into a functional replication complex. PARP10 and PARP12 inhibited the production of nsP3, indicating a defect in polyprotein processing. The nsP3 protein encodes a macrodomain with de-MARylation activity, which is essential for replication. In support for MARylation affecting polyprotein processing, de-MARylation defective CHIKV replicons revealed reduced production of nsP2 and nsP3. We hypothesized that MARylation regulates the proteolytic function of nsP2. Indeed, we found that nsP2 is MARylated by PARP10 and, as a consequence, its proteolytic activity was inhibited. NsP3-dependent de-MARylation reactivated the protease. Hence, we propose that PARP10-mediated MARylation prevents polyprotein processing and consequently virus replication. Together, our findings provide a mechanistic explanation for the role of the viral MAR hydrolase in CHIKV replication.

Transcript and protein expression analysis of proteases in the blood stages of Plasmodium falciparum.

Proteases are crucial enzymes with varying roles in living organisms. In the malaria parasite Plasmodium falciparum, the role of proteases has been deciphered mainly in the asexual blood stages and shown to represent promising drug targets. However, little is known about their functions in the sexual blood stages, which are important for transmission of the disease from the human to the mosquito vector. Determination of their stage-specific expression during the malaria life-cycle is crucial for the effective design of multi-stage anti-malaria drugs aimed at eradicating the disease. In this study, we screened the P. falciparum genome database for putative proteases and determined the transcript and protein expression profiles of selected proteases in the plasmodial blood stages using semi-quantitative RT-PCR and indirect immunofluorescence assay. Database mining identified a total of 148 putative proteases, out of which 18 were demonstrated to be expressed in the blood stages on the transcript level; for 12 of these proteins synthesis was confirmed. While three of these proteases exhibit gametocyte-specific expression, two are restricted to the asexual blood stages and seven are found in both stages, making them interesting multi-stage drug targets.

Bistacrine derivatives as new potent antimalarials.

Linking two tacrine molecules results in a tremendous increase of activity against Plasmodia in comparison to the monomer. This finding prompted the synthesis of a library of monomeric and dimeric tacrine derivatives in order to derive structure-activity relationships. The most active compounds towards chloroquine sensitive Plasmodium strain 3D7 and chloroquine resistant strain Dd2 show IC50 values in the nanomolar range of concentration, low cytotoxicity and target the cysteine protease falcipain-2, which is essential for parasite growth.

Protein and RNA ADP-ribosylation detection is influenced by sample preparation and reagents used.

The modification of substrates with ADP-ribose (ADPr) is important in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid-ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts containing ADPr-proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the reactivity of available reagents with diverse ADP-ribosylated protein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross-reactivity with adenylylated RNA, AMPylated proteins, and metabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample preparation methods and have defined the reagents suitable for specific methods and substrates.

The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development.

The enzymes glyoxalase 1 and 2 (Glo1 and Glo2) are found in most eukaryotes and catalyze the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Four glyoxalases are encoded in the genome of the malaria parasite Plasmodium falciparum, the cytosolic enzymes PfGlo1 and PfcGlo2, the apicoplast enzyme PftGlo2, and an inactive Glo1-like protein that also carries an apicoplast-targeting sequence. Inhibition or knockout of the Plasmodium glyoxalases was hypothesized to lead to an accumulation of 2-oxoaldehydes and advanced glycation end-products (AGE) in the host-parasite unit and to result in parasite death. Here, we generated clonal P. falciparum strain 3D7 knockout lines for PFGLO1 and PFcGLO2 using the CRISPR-Cas9 system. Although 3D7Δglo1 knockout clones had an increased susceptibility to external glyoxal, all 3D7Δglo1 and 3D7Δcglo2 knockout lines were viable and showed no significant growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, increased gametocyte commitment in the knockout lines. In summary, PfGlo1 and PfcGlo2 are dispensable during asexual blood-stage development while the loss of PfcGlo2 may induce the formation of transmissible gametocytes. These combined data show that PfGlo1 and PfcGlo2 are most likely not suited as targets for selective drug development.