The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to ...topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms.
the etiologic agent of the most common non-viral sexually transmitted infection worldwide. With an estimated annual prevalence of 276 million new cases, mixed infections with different parasite ...strains are expected. Although it is known that parasites interact with their host to enhance their own survival and transmission, evidence of mixed infections call into question the extent to which unicellular parasites communicate with each other. Here, we demonstrated that different
strains can communicate through the formation of cytoneme-like membranous cell connections. We showed that cytonemes formation of an adherent parasite strain (CDC1132) is affected in the presence of a different strain (G3 or B7RC2). Our findings provide evidence that this effect is contact-independent and that extracellular vesicles (EVs) are responsible, at least in part, of the communication among strains. We found that EVs isolated from G3, B7RC2, and CDC1132 strains contain a highly distinct repertoire of proteins, some of them involved in signaling and communication, among other functions. Finally, we showed that parasite adherence to host cells is affected by communication between strains as binding of adherent
CDC1132 strain to prostate cells is significantly higher in the presence of G3 or B7RC2 strains. We also observed that a poorly adherent parasite strain (G3) adheres more strongly to prostate cells in the presence of an adherent strain. The study of signaling, sensing, and cell communication in parasitic organisms will enhance our understanding of the basic biological characteristics of parasites, which may have important consequences in pathogenesis.
Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, ...components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T . gondii . In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0’s localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701–877 of BCC0 are essential for both its localization and function and that residues 1–899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0’s essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The inner membrane complex (IMC) of Toxoplasma gondii is essential for all phases of the parasite's life cycle. One of its most critical roles is to act as a scaffold for the assembly of daughter ...buds during replication by endodyogeny. While many daughter IMC proteins have been identified, most are recruited after bud initiation and are not essential for parasite fitness. Here, we report the identification of IMC43, a novel daughter IMC protein that is recruited at the earliest stages of daughter bud initiation. Using an auxin-inducible degron system we show that depletion of IMC43 results in aberrant morphology, dysregulation of endodyogeny, and an extreme defect in replication. Deletion analyses reveal a region of IMC43 that plays a role in localization and a C-terminal domain that is essential for the protein's function. TurboID proximity labelling and a yeast two-hybrid screen using IMC43 as bait identify 30 candidate IMC43 binding partners. We investigate two of these: the essential daughter protein IMC32 and a novel daughter IMC protein we named IMC44. We show that IMC43 is responsible for regulating the localization of both IMC32 and IMC44 at specific stages of endodyogeny and that this regulation is dependent on the essential C-terminal domain of IMC43. Using pairwise yeast two-hybrid assays, we determine that this region is also sufficient for binding to both IMC32 and IMC44. As IMC43 and IMC32 are both essential proteins, this work reveals the existence of a bud assembly complex that forms the foundation of the daughter IMC during endodyogeny.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab ...GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Trichomonas vaginalis
is a common sexually transmitted extracellular parasite that adheres to epithelial cells in the human urogenital tract. Extracellular vesicles (EVs) have been described as ...important players in the pathogenesis of this parasite as they deliver proteins and RNA into host cells and modulate parasite adherence. EVs are heterogeneous membrane vesicles released from virtually all cell types that collectively represent a new dimension of intercellular communication. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery contributes to several key mechanisms in which it reshapes membranes. Based on this, some components of the ESCRT have been implicated in EVs biogenesis in other cells. Here, we demonstrated that VPS32, a member of ESCRTIII complex, contribute to the biogenesis and cargo sorting of extracellular vesicles in the parasite
T. vaginalis.
Moreover, we observe that parasites overexpressing VPS32 have a striking increase in adherence to host cells compared to control parasites; demonstrating a key role for this protein in mediating host: parasite interactions. These results provide valuable information on the molecular mechanisms involved in extracellular vesicles biogenesis, cargo-sorting, and parasite pathogenesis.
Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric
port
...ement (PEXEL)/host-targeting motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here, we report that the non-exported parasitophorous vacuole protein UIS2 contains a
PEXEL motif that is processed in the
blood stage. While the N termini of exported proteins containing the PEXEL and immediately downstream ~10 residues are sufficient to mediate translocation into the RBC, the equivalent UIS2 N terminus does not promote the export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position, which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RIL↓DE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N terminus mediates export, not PEXEL processing
. Prompted by this observation, we further show that PEXEL sequences in the N termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export.IMPORTANCEHost erythrocyte remodeling by malaria parasite-exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite
, most exported proteins undergo proteolytic maturation via recognition of the pentameric
port
ement (PEXEL)/host-targeting motif by the aspartic protease Plasmepsin V, which exposes a mature N terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate that PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing, reinforcing that features of the mature N terminus, and not PEXEL cleavage, identify cargo for export. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.
The design of popular disposable electronic cigarettes (ECs) was analyzed, and the concentrations of WS-23, a synthetic coolant, in EC fluids were determined for 22 devices from 4 different brands. ...All products contained WS-23 in concentrations that ranged from 1.0 to 40.1 mg/mL (mean = 21.4 ± 9.2 mg/mL). To determine the effects of WS-23 on human bronchial epithelium in isolation of other chemicals, we exposed EpiAirway 3-D microtissues to WS-23 at the air liquid interface (ALI) using a cloud chamber that generated aerosols without heating. Proteomics analysis of exposed tissues revealed that the cytoskeleton was a major target of WS-23. BEAS-2B cells were exposed to WS-23 in submerged culture to validate the main results from proteomics. F-actin, which was visualized with phalloidin, decreased concentration dependently in WS-23 treated BEAS-2B cells, and cells became immotile in concentrations above 1.5 mg/mL. Gap closure, which depends on both cell proliferation and migration, was inhibited by 0.45 mg/mL of WS-23. These data show that WS-23 is being added to popular EC fluids at concentrations that can impair processes dependent on the actin cytoskeleton and disturb homeostasis of the bronchial epithelium. The unregulated use of WS-23 in EC products may harm human health.
Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) ...within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify MIP candidates. Using RNAi knockdown together with fitting of AlphaFold models into cryoET maps, we demonstrate that one of these candidates, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention.
The enrichment of biotinylated proteins using immobilized streptavidin has become a staple methodology for affinity purification-based proteomics. Many of these workflows rely upon tryptic digestion ...to elute streptavidin-captured moieties from the beads. The concurrent release of high amounts of streptavidin-derived peptides into the digested sample, however, can significantly hamper the effectiveness of downstream proteomic analyses by increasing the complexity and dynamic range of the mixture. Here, we describe a strategy for the chemical derivatization of streptavidin that renders it largely resistant to proteolysis by trypsin and thereby dramatically reduces the amount of streptavidin contamination in the sample. This rapid and robust approach improves the effectiveness of mass spectrometry-based characterization of streptavidin-purified samples making it broadly useful for a wide variety of applications. In addition, we show that this chemical protection strategy can also be applied to other affinity matrices including immobilized antibodies against HA epitopes.
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