Cytoplasmic lipid droplets are important organelles in nearly every eukaryotic and some prokaryotic cells. Storing and providing energy is their main function, but they do not work in isolation. They ...respond to stimuli initiated either on the cell surface or in the cytoplasm as conditions change. Cellular stresses such as starvation and invasion are internal insults that evoke changes in droplet metabolism and dynamics. This review will first outline lipid droplet assembly and then discuss how droplets respond to stress and in particular nutrient starvation. Finally, the role of droplets in viral and microbial invasion will be presented, where an unresolved issue is whether changes in droplet abundance promote the invader, defend the host, to try to do both. The challenges of stress and infection are often accompanied by changes in physical contacts between droplets and other organelles. How these changes may result in improving cellular physiology, an ongoing focus in the field, is discussed.
This review offers a concise summary of our current view of how cytoplasmic lipid droplets respond to cellular stresses and pathogen invasion, and cross‐talk with other organelles in order to maintain energy balance and cellular physiology.
The obligate intracellular parasite, Toxoplasma gondii, modulates host immunity in a variety of highly specific ways. Previous work revealed a polymorphic, injected parasite factor, ROP16, to be a ...key virulence determinant and regulator of host cell transcription. These properties were shown to be partially mediated by dysregulation of the host transcription factors STAT3 and STAT6, but the molecular mechanisms underlying this phenotype were unclear. Here, we use a Type I Toxoplasma strain deficient in ROP16 to show that ROP16 induces not only sustained activation but also an extremely rapid (within 1 min) initial activation of STAT6. Using recombinant wild-type and kinase-deficient ROP16, we demonstrate in vitro that ROP16 has intrinsic tyrosine kinase activity and is capable of directly phosphorylating the key tyrosine residue for STAT6 activation, Tyr641. Furthermore, ROP16 co-immunoprecipitates with STAT6 from infected cells. Taken together, these data strongly suggest that STAT6 is a direct substrate for ROP16 in vivo.
Reversible phosphorylation by protein kinases is one of the core mechanisms by which biological signals are propagated and processed. Mitogen-activated protein kinases, or MAPKs, are conserved ...throughout eukaryotes where they regulate cell cycle, development, and stress response. Here, we review advances in our understanding of the function and biochemistry of MAPK signaling in apicomplexan parasites. As expected for well-conserved signaling modules, MAPKs have been found to have multiple essential roles regulating both Toxoplasma tachyzoite replication and sexual differentiation in Plasmodium. However, apicomplexan MAPK signaling is notable for the lack of the canonical kinase cascade that normally regulates the networks, and therefore must be regulated by a distinct mechanism. We highlight what few regulatory relationships have been established to date, and discuss the challenges to the field in elucidating the complete MAPK signaling networks in these parasites.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Schistosomiasis is a neglected tropical disease that infects 240 million people. With no vaccines and only one drug available, new therapeutic targets are needed. The causative agents, schistosomes, ...are intravascular flatworm parasites that feed on blood and lay eggs, resulting in pathology. The function of the parasite's various tissues in successful parasitism are poorly understood, hindering identification of therapeutic targets. Using single-cell RNA sequencing (RNA-seq), we characterize 43,642 cells from the adult schistosome and identify 68 distinct cell populations, including specialized stem cells that maintain the parasite's blood-digesting gut. These stem cells express the gene
, which is required for gut maintenance, blood feeding, and pathology in vivo. Together, these data provide molecular insights into the organ systems of this important pathogen and identify potential therapeutic targets.
Primary cilia are important organizing centers that control diverse cellular processes. Apicomplexan parasites like
have a specialized cilium-like structure called the conoid that organizes the ...secretory and invasion machinery critical for the parasites' lifestyle. The proteins that initiate the biogenesis of this structure are largely unknown. We identified the
orthologue of the conserved kinase ERK7 as essential to conoid assembly. Parasites in which ERK7 has been depleted lose their conoids late during maturation and are immotile and thus unable to invade new host cells. This is the most severe phenotype to conoid biogenesis yet reported, and is made more striking by the fact that ERK7 is not a conoid protein, as it localizes just basal to the structure. ERK7 has been recently implicated in ciliogenesis in metazoan cells, and our data suggest that this kinase has an ancient and central role in regulating ciliogenesis throughout Eukaryota.
The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the ...interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The apical complex is a specialized collection of cytoskeletal and secretory machinery in apicomplexan parasites, which include the pathogens that cause malaria and toxoplasmosis. Its structure and ...mechanism of motion are poorly understood. We used cryo-FIB-milling and cryo-electron tomography to visualize the 3D-structure of the apical complex in its protruded and retracted states. Averages of conoid-fibers revealed their polarity and unusual nine-protofilament arrangement with associated proteins connecting and likely stabilizing the fibers. Neither the structure of the conoid-fibers nor the architecture of the spiral-shaped conoid complex change during protrusion or retraction. Thus, the conoid moves as a rigid body, and is not spring-like and compressible, as previously suggested. Instead, the apical-polar-rings (APR), previously considered rigid, dilate during conoid protrusion. We identified actin-like filaments connecting the conoid and APR during protrusion, suggesting a role during conoid movements. Furthermore, our data capture the parasites in the act of secretion during conoid protrusion.
Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. ...Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.
The Red Queen hypothesis proposes that there is an evolutionary arms race between host and pathogen. One possible example of such a phenomenon could be the recently discovered interaction between ...host defense proteins known as immunity-related GTPases (IRGs) and a family of rhoptry pseudokinases (ROP5) expressed by the protozoan parasite, Toxoplasma gondii. Mouse IRGs are encoded by an extensive and rapidly evolving family of over 20 genes. Similarly, the ROP5 family is highly polymorphic and consists of 4–10 genes, depending on the strain of Toxoplasma. IRGs are known to be avidly bound and functionally inactivated by ROP5 proteins, but the molecular basis of this interaction/inactivation has not previously been known. Here we show that ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG and that this produces a profound allosteric change in the IRG structure. This has two dramatic effects: 1) it prevents oligomerization of the IRG, and 2) it alters the orientation of two threonine residues that are targeted by the Toxoplasma Ser/Thr kinases, ROP17 and ROP18. ROP5s are highly specific in the IRGs that they will bind, and the fact that it is the most highly polymorphic surface of ROP5 that binds the IRG strongly supports the notion that these two protein families are co-evolving in a way predicted by the Red Queen hypothesis.
Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, ...infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.
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