Biology subjects

Wohlschlegel, J. A.

Publications and source records attributed to Wohlschlegel, J. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Acyl Carrier Protein is Essential for Apicoplast Biogenesis in Malaria Parasites Independent of Fatty Acid Synthesis

Acyl carrier protein (ACP) and its 4-phosphopantetheine prosthetic group canonically function as the soluble scaffold for acyl chain assembly and elongation during type-II fatty acid synthesis (FASII). Plasmodium malaria parasites retain a FASII pathway in the apicoplast organelle that has been the subject of considerable scrutiny and confusion. Although apicoplast FASII is essential for P. falciparum growth within mosquitoes and the human liver, this pathway is dispensable and largely inactive in blood-stage parasites that can scavenge host fatty acids. In contrast to FASII enzymes that can be disrupted without fitness defect, we report that knockout or ligand-dependent knockdown of apicoplast ACP is lethal to blood-stage P. falciparum, indicating an essential FASII-independent function. Loss of ACP impairs the biosynthesis of essential isoprenoid precursors and blocks apicoplast biogenesis. Using proximity biotinylation and biochemical interaction studies, we identified a key role for ACP in binding and stabilizing apicoplast pyruvate kinase II (PKII). This critical enzyme is the only known source of nucleoside triphosphates (NTPs) in this organelle and is required for isoprenoid synthesis and apicoplast biogenesis. Our work reveals that ACP knockdown results in destabilization and loss of PKII, which is sufficient to explain ACP essentiality in this stage. This work unveils essential ACP function at a key biochemical hub controlling broad apicoplast metabolism in malaria parasites that is independent of the canonical ACP role in FASII.

biochemistry↗

bioRxiv2025

Mapping the Glycogen Synthase 1 Interactome in Brown Adipocytes

Brown adipose tissue (BAT) is distinguished by its ability to dissipate energy through thermogenesis, a process with considerable therapeutic potential for obesity, type 2 diabetes, and related metabolic disorders. Although glycogen represents a relatively minor energy reserve in adipocytes compared with liver and muscle, emerging evidence shows that glycogen turnover increases sharply during metabolic transitions and is required for full thermogenic activation in brown adipocytes. The rate-limiting enzyme glycogen synthase 1 (GYS1) catalyzes glycogen chain elongation, and adipose-specific Gys1 deletion impairs adaptive thermogenesis, highlighting the importance of GYS1 in coordinating glucose and lipid metabolism; however, the mechanisms regulating GYS1 in BAT remain poorly defined. To address this gap, we used an unbiased proximity-labeling strategy in brown adipocytes by fusing GYS1 to the biotin ligase TurboID and identifying biotinylated proteins through mass spectrometry, revealing 425 putative interactors. These included canonical glycogen-handling proteins such as glycogenin and glycogen phosphorylase, validating the approach, while gene ontology analysis uncovered broader enrichment in cytoskeletal remodeling, vesicle trafficking, and transcriptional regulation, suggesting previously unrecognized roles for GYS1 beyond glycogen synthesis. Notably, we identified several novel candidate interactors, including CAST, PDAP1, and CCDC102A. Together, these findings define the most comprehensive GYS1 interactome reported to date and provide new insights into the molecular mechanisms linking glycogen metabolism to thermogenic function in BAT.

biochemistry↗

bioRxiv2025

Arabidopsis YEATS domain proteins facilitate DNA double strand break repair via homologous recombination pathways

DNA double-strand break (DSB) repair is required to maintain genome integrity and organismal fitness. This process occurs within chromatin and although accurate repair requires extensive changes in chromatin structure and composition, chromatin effectors facilitating DSB repair remain largely unknown in plants. Using reverse genetics, we identified two chromatin readers, YAF9A and YAF9B, as new factors required for DSB repair in Arabidopsis. While only YAF9B is induced by DNA damage, both YAF9A and YAF9B are required for DSB repair, including via homologous recombination (HR)-like pathways. Mechanistically, we found YAF9A and YAF9B denote distinct remodeling complexes: While YAF9A associates with both NuA4 and SWR1, YAF9B only associates with NuA4, defining a DNA-damage-specific version of this remodeling complex and revealing the first link between NuA4 and DSB repair in plants. We also uncovered direct roles for YAF9A and YAF9B in DNA repair, rather than indirect roles in gene regulation, as yaf9 mutants showed normal transcriptional responses to DNA damage. Finally, we demonstrate that the YAF9B reader domain is required for DSB repair. These results link YAF9A, YAF9B, and their respective remodeling complexes, to DSB repair in a histone reader-dependent manner, expanding our understanding of how chromatin effectors regulate DSB repair in plants.

plant biology↗

bioRxiv2025

Cytoplasmic and nuclear protein interaction networks of the synapto-nuclear messenger CRTC1 in neurons reveal cooperative chromatin binding between CREB1 and CRTC1, MEF2C and RFX3

Glutamatergic stimulation of excitatory neurons triggers the synapto-nuclear translocation of the cAMP response element (CRE) binding protein (CREB) regulated transcription coactivator 1 (CRTC1), resulting in the transcription of CREB1 target genes. Whether and how CRTC1 and CREB1 interact with other transcription factors to regulate activity-dependent transcription, and what the role of CRTC1 is in neurons beyond the activation of CREB1 regulated transcription, remains unknown. To address these questions in an unbiased manner, we used proximity labeling to identify CRTC1-proximal proteins in cytoplasmic and nuclear compartments of rodent forebrain neurons. The cytoplasmic CRTC1 proxisome included a variety of signaling pathways and downstream cellular processes involved in synaptic plasticity. In contrast, the nuclear CRTC1 proxisome included transcription factors that mediate activity-dependent transcription, chromatin factors, and splicing factors. Our data revealed that CRTC1 and CREB1 interact with MEF2C and RFX3 transcription factors in an activity-dependent manner. Thus, in chromatin immunoprecipitation-sequencing experiments, CREB1 was prebound to chromatin regions containing bZIP motifs in a manner that was unchanged by neuronal activity, while glutamatergic stimulation triggered the recruitment of CRTC1 and CREB1 to activity-dependent enhancers enriched in motifs for MEF2C and RFX3. Collectively, these results not only enhance our understanding of the role of cytoplasmic and nuclear CRTC1 in neurons, but also reveal a role for CRTC1 in promoting cooperativity of CREB1 with other transcription factors in response to synaptic activity.

neuroscience↗

bioRxiv2025

The regulation of Protein Phosphatase 4 by FBXO42 is required for cancer cell survival.

FBXO42 is a poorly characterized F-box protein that is essential in 15% of cancer cell lines from diverse tissue types. FBXO42 has been implicated in the regulation of mitosis and p53 signaling. High-throughput approaches indicate that FBXO42 function correlates with that of CCDC6, and that the two proteins interact physically, but the relationship between these proteins is not understood. Through a genome-wide CRISPR knockout screen, we found that mutation of FBXO42 is synthetically lethal with mutations in the {gamma}- tubulin ring complex proteins MZT1 and MZT2B, suggesting that cells with centrosome and/or mitotic spindle assembly dysfunction are more sensitive to FBXO42 loss. Furthermore, we found that FBXO42 and CCDC6 contribute to p53 activation in response to centrosome depletion. Using mass spectrometry-based proteomics, we found that FBXO42 binds, is required for the ubiquitination of, and negatively regulates the expression of PPP4C (protein phosphatase 4 catalytic subunit). FBXO42s interaction with PPP4C was independent of CCDC6. Similarly, we found that CCDC6 physically interacts with PPP4C independently of FBXO42 and does not affect PPP4C ubiquitination. Knockdown of PPP4C reduced FBXO42-CCDC6 interactions, suggesting that FBXO42 and CCDC6 may bind to and regulate PPP4C through separate mechanisms. Using gene knockdown rescue experiments, we confirmed that aberrant expression of PPP4C is a major driver of cell death in an FBXO42-essential Neuroblastoma cell line. These findings shed light on the function of two poorly understood proteins in regulating PP4 activity, p53 signaling, mitosis and cancer cell survival. A better understanding of FBXO42 and CCDC6 could inform the development of targeted cancer therapeutics.

cell biology↗

bioRxiv2025

The interactome of the Bakers' yeast peroxiredoxin Tsa1 implicates it in the redox regulation of intermediary metabolism, glycolysis and zinc homeostasis

Zinc (Zn) is an essential nutrient supporting a range of critical processes. In the yeast Saccharomyces cerevisiae, Zn deficiency induces a transcriptional response mediated by the Zap1 activator, which controls a regulon of [~]80 genes. A subset support zinc homeostasis by promoting zinc uptake and its distribution between compartments, while the remainder mediate an "adaptive response" to enhance fitness of zinc deficient cells. The peroxiredoxin Tsa1 is a Zap1-regulated adaptive factor essential for the growth of Zn deficient cells. Tsa1 can function as an antioxidant peroxidase, protein chaperone, or redox sensor: the latter activity oxidizes associated proteins via a redox relay mechanism. We previously reported that in Zn deficient cells, Tsa1 inhibits pyruvate kinase (Pyk1) to conserve phosphoenolpyruvate for aromatic amino acid synthesis. However, this regulation makes a relatively minor contribution to fitness in low zinc, suggesting that Tsa1 targets other pathways important to adaptation. Consistent with this model, the redox sensor function of Tsa1 was essential for growth of ZnD cells. Using an MBP-tagged version of Tsa1, we identified a redox-sensitive non-covalent interaction with Pyk1, and applied this system to identify multiple novel interacting partners. This interactome implicates Tsa1 in the regulation of critical processes including many Zn-dependent metabolic pathways. Interestingly, Zap1 was a preferred Tsa1 target, as Tsa1 strongly promoted the oxidation of Zap1 activation domain 2, and was essential for full Zap1 activity. Our findings reveal a novel posttranslational response to Zn deficiency, overlain on and interconnected with the Zap1-mediated transcriptional response. Graphical abstractIn ZnD cells, Tsa1 mediates metabolic adaptation by regulation of glycolytic enzymes Pyk1 and Fba1, and supports activity of the Zap1 transcriptional activator via oxidation of the AD2 domain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/638137v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1ef6858org.highwire.dtl.DTLVardef@ab9682org.highwire.dtl.DTLVardef@12644a3org.highwire.dtl.DTLVardef@1644fe0_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Mdm38/LETM1 couples ion homeostasis and proteostatic mechanisms in the inner mitochondrial membrane

The mitochondrial inner membrane is among the most protein-dense cellular membranes. Its functional integrity is maintained through a concerted action of several conserved mechanisms that are far from clear. Here, using the bakers yeast model, we functionally characterize Mdm38/LETM1, a disease-related protein implicated in mitochondrial translation and ion homeostasis, although the molecular basis of these connections remains elusive. Our findings reveal a novel role for Mdm38 in maintaining protein homeostasis within the inner membrane. Specifically, we demonstrate that Mdm38 is required for mitochondrial iron homeostasis and for signaling iron bioavailability from mitochondria to vacuoles. These processes are linked to the m- AAA quality control protease, whose unrestrained activity disrupts the assembly and stability of respiratory chain complexes in Mdm38-deficient cells. Our study highlights the central role of Mdm38 in mitochondrial biology and reveals how it couples proteostatic mechanisms to ion homeostasis across subcellular compartments.

biochemistry↗

bioRxiv2024

Secretome analysis of oligodendrocytes and precursors reveals their roles as contributors to the extracellular matrix and potential regulators of inflammation

Oligodendrocytes form myelin that ensheaths axons and accelerates the speed of action potential propagation. Oligodendrocyte progenitor cells (OPCs) proliferate and replenish oligodendrocytes. While the myelin-forming role of oligodendrocytes and OPCs is well-established, potential additional roles of these cells are yet to be fully explored. Here, we analyzed the secreted proteome of oligodendrocytes and OPCs in vitro to determine whether these cell types are major sources of secreted proteins in the central nervous system (CNS). Interestingly, we found that both oligodendrocytes and OPCs secret various extracellular matrix proteins. Considering the critical role of neuroinflammation in neurological disorders, we evaluated the responses and potential contributions of oligodendrocytes and OPCs to this process. By characterizing the secreted proteomes of these cells after pro-inflammatory cytokine treatment, we discovered the secretion of immunoregulators such as C2 and B2m. This finding sheds new light on the hitherto underappreciated role of oligodendrocytes and OPCs in actively modulating neuroinflammation. Our study provides a comprehensive and unbiased proteomic dataset of proteins secreted by oligodendrocyte and OPC under both physiological and inflammatory conditions. It revealed the potential of these cells to secrete matrix and signaling molecules, highlighting their multifaceted function beyond their conventional myelin-forming roles.

neuroscience↗

bioRxiv2024

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 [A]-resolution X-ray structure that revealed a highly -helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

biochemistry↗

bioRxiv2024

Characterization and functional analysis of Toxoplasma Golgi-associated proteins identified by proximity labelling

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles which play essential roles in the parasites lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here we identify a novel Golgi-localizing protein (ULP1) which contains structural homology to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness and replicative ability. Using ULP1 as bait for TurboID proximity labelling and immunoprecipitation, we identify eleven more novel Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii COG complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these eleven proteins on parasite fitness. Together, this work reveals a diverse set of novel T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites. ImportanceApicomplexan parasites such as Toxoplasma gondii infect a large percentage of the worlds population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite replication and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labelling to identify eleven additional Golgi-associated proteins which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention.

microbiology↗

bioRxiv2024

Microglia promote extracellular matrix deposition and restrict excitatory synapse numbers in the mesolimbic dopamine system during healthy aging

Synapse dysfunction is tightly linked to cognitive changes during aging. Emerging evidence suggests that microglia and the extracellular matrix (ECM) can potently regulate synapse integrity and plasticity. Yet the brain ECM, and its relationship with microglia, synapses, and cognition during aging remains virtually unexplored. Using ECM-optimized proteomic workflows and histological analyses, we discovered striking regional differences in ECM composition and aging-induced ECM remodeling across key basal ganglia nuclei. Moreover, we combine two distinct behavioral classification strategies with fixed-tissue confocal imaging and proteomic analysis to identify robust relationships between the hyaluronan- and proteoglycan-rich ECM and cognitive aging phenotypes. Finally, we provide evidence that aging midbrain microglia lose capacity to interact with and regulate the ECM, and that these aging-associated microglial changes are accompanied by local ECM accumulation and worse behavioral performance. Together, these foundational observations implicate changing microglia-ECM-synapse interactions as a key determinant of cognitive functioning during healthy aging.

neuroscience↗

bioRxiv2023

Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome

Neurons express different combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. Here we use epitope tagged endogenous NR subunits, expansion light-sheet microscopy, and EM connectomics to molecularly characterize synapses in Drosophila. We show that in directionally selective motion sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determines patterns of synaptic inputs. In support of this model, we identify a transmembrane protein associated selectively with a subset of spatially restricted synapses and demonstrate through genetic analysis its requirement for synapse formation. We propose that mechanisms which regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites.

neuroscience↗

bioRxiv2023

PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins

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 Plasmodium export element (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 bona fide PEXEL motif that is processed in the P. falciparum blood-stage. While the N-termini of exported proteins containing the PEXEL and immediately downstream [~]10 residues is sufficient to mediate translocation into the RBC, the equivalent UIS2 N-terminus does not promote 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{tau}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 per se. 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 Plasmodium falciparum, most exported proteins undergo proteolytic maturation via recognition of the pentameric Plasmodium export element (PEXEL)/Host Targeting motif by the aspartic protease Plasmepsin V (PMV) 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 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 by PMV, reinforcing that features of the mature N-terminus, and not PEXEL cleavage, identify cargo for export cargo. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

microbiology↗

bioRxiv2023

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection

Toxoplasma gondiis propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with {Delta}gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon gamma (IFN-{gamma}) in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-{kappa}B complex. While GRA15 similarly regulates NF-{kappa}B, infection with {Delta}gra83/{Delta}gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. ImportanceToxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasites ability to efficiently invade and regulate components of the hosts infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasmas ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasites diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogens tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cells response to limit infection.

microbiology↗

bioRxiv2023

Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels.

Despite their role as innate sentinels, macrophages are cellular reservoirs for chikungunya virus (CHIKV), a highly pathogenic arthropod-borne alphavirus that has caused unprecedented epidemics worldwide. Here, we took interdisciplinary approaches to elucidate the CHIKV determinants that subvert macrophages into virion dissemination vessels. Through comparative infection using chimeric alphaviruses and evolutionary selection analyses, we discovered for the first time that CHIKV glycoproteins E2 and E1 coordinate efficient virion production in macrophages with the domains involved under positive selection. We performed proteomics on CHIKV-infected macrophages to identify cellular proteins interacting with the precursor and/or mature forms of viral glycoproteins. We uncovered two E1-binding proteins, signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 (eIF3k), with novel inhibitory activities against CHIKV production. These results highlight how CHIKV E2 and E1 have been evolutionarily selected for viral dissemination likely through counteracting host restriction factors, making them attractive targets for therapeutic intervention.

microbiology↗

bioRxiv2023

Direct Tests of Cytochrome Function in the Electron Transport Chain of Malaria Parasites

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution. SIGNIFICANCE STATEMENTMitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.

microbiology↗

bioRxiv2023

Role of cytoneme-like structures and extracellular vesicles in Trichomonas vaginalis parasite: parasite communication

Trichomonas vaginalis, the etiologic agent of the most common non-viral sexually transmitted infection worldwide, colonizes the human urogenital tract where it remains extracellular and adheres to epithelial cells. With an estimated prevalence of 276 million new cases annually, mixed infections with different parasite strains are expected. Although it is considered as obvious that parasites interact with their host to enhance their own survival and transmission, evidence of mixed infection call into question the extent to which unicellular parasites communicate with each other. Here, we demonstrated that different T. vaginalis strains are able to communicate through the formation of cytoneme-like membranous cell connections. We showed that T. vaginalis adherent strains form abundant membrane protrusions and cytonemes formation of an adherent parasite strain (CDC1132) is affected in the presence of a different strain (G3 or B7RC2). Using a cell culture inserts assays, we demonstrated that the effect in cytoneme formation is contact independent and that extracellular vesicles (EVs) are responsible, at least in part, of the communication among strains. In this sense, 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 this communication between strains as binding of adherent T. vaginalis CDC1132 strain to prostate cells is significantly higher in the presence of G3 or B7RC2 strains. Demonstrating that interaction of isolates with distinct phenotypic characteristics may have significant clinical repercussions, 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 surely enhance our understanding of the basic biological characteristics of parasites that might have important consequences in pathogenesis.

microbiology↗

bioRxiv2022

FAP106 is an interaction hub required for stable assembly of conserved and lineage-specific proteins at the cilium inner junction

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 further use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify novel MIP candidates, and RNAi knockdown plus fitting of AlphaFold models into cryoET maps to demonstrate one of these, 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.

cell biology↗