Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway.

BACKGROUND: Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of Plasmodium falciparum CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2ß1 and PfCK2ß2. RESULTS: We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2ß1 and PfCK2ß2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2ß1 or HA-PfCK2ß2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2ß1- and PfCK2ß2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2ß1 and PfCK2ß2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α in vitro. CONCLUSIONS: Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.

The update of self-identity: Importance of assessing autobiographical memory in major depressive disorder.

Major depressive disorder is a leading global cause of disability. There is a growing interest for memory in mood disorders since it might constitute an original tool for prevention, diagnosis, and treatment. MDD is associated with impaired autobiographical memory characterized by a tendency to overgeneral memory, rather than vivid episodic self-defining memory, which is mandatory for problem-solving and projection in the future. This memory bias is maintained by three mechanisms: ruminations, avoidance, and impaired executive control. If we adopt a broader and comprehensive perspective, we can hypothesize that all those alterations have the potential to impair self-identity updating. We posit that this update requires a double referencing process: (1) to internalized self-representation and (2) to an externalized framework dealing with the representation of the consequence of actions. Diagnostic and therapeutic implications are discussed in the light of this model and the importance of assessing autobiographical memory in MDD is highlighted. This article is categorized under: Psychology > Memory Psychology > Brain Function and Dysfunction Neuroscience > Clinical.

Malaria: targeting parasite and host cell kinomes.

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.

Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase.

BACKGROUND: Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. METHODS: Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. RESULTS: This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo. CONCLUSIONS: While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

An essential role for the Plasmodium Nek-2 Nima-related protein kinase in the sexual development of malaria parasites.

The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission.

Functional analysis of protein kinase CK2 of the human malaria parasite Plasmodium falciparum.

Protein kinase CK2 (casein kinase 2) is a eukaryotic serine/threonine protein kinase with multiple substrates and roles in diverse cellular processes, including differentiation, proliferation, and translation. The mammalian holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We report the identification and characterization of a Plasmodium falciparum CK2alpha orthologue, PfCK2alpha, and two PfCK2beta orthologues, PfCK2beta1 and PfCK2beta2. Recombinant PfCK2alpha possesses protein kinase activity, exhibits similar substrate and cosubstrate preferences to those of CK2alpha subunits from other organisms, and interacts with both of the PfCK2beta subunits in vitro. Gene disruption experiments show that the presence of PfCK2alpha is crucial to asexual blood stage parasites and thereby validate the enzyme as a possible drug target. PfCK2alpha is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2alpha enzymes to a small molecule inhibitor. Taken together, our data identify PfCK2alpha as a potential target for antimalarial chemotherapeutic intervention.

Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.

The role of protein phosphorylation in the life cycle of malaria parasites is slowly emerging. Here we combine global phospho-proteomic analysis with kinome-wide reverse genetics to assess the importance of protein phosphorylation in Plasmodium falciparum asexual proliferation. We identify 1177 phosphorylation sites on 650 parasite proteins that are involved in a wide range of general cellular activities such as DNA synthesis, transcription and metabolism as well as key parasite processes such as invasion and cyto-adherence. Several parasite protein kinases are themselves phosphorylated on putative regulatory residues, including tyrosines in the activation loop of PfGSK3 and PfCLK3; we show that phosphorylation of PfCLK3 Y526 is essential for full kinase activity. A kinome-wide reverse genetics strategy identified 36 parasite kinases as likely essential for erythrocytic schizogony. These studies not only reveal processes that are regulated by protein phosphorylation, but also define potential anti-malarial drug targets within the parasite kinome.