The plant mitochondrial TAT pathway is essential for complex III biogenesis
Ontology highlight
ABSTRACT: Investigation of the composition of Arabidopsis complex III in a knock-out line of mitochondrial TATB complemented with a truncated version of this protein
Project description:Hypoxia is an important driver of cancer progression, and rapidly growing tumors often result in intratumoral hypoxic regions. Hypoxia is associated with metabolic reprogramming and angiogenesis, resulting in enhanced tumor progression. Here, we aimed to study breast cancer hypoxia responses at the proteomic level, and we wanted to identify differences in protein secretion from luminal-like and basal-like cell lines before and after hypoxia.
Project description:Salivarian trypanosomes cause human sleeping sickness and economically important livestock diseases, transmitted by Tsetse flies and replicating extracellularly throughout the life cycle. The surface of "bloodstream forms", which live within mammals, is coated by a monolayer of a Variant Surface Glycoprotein (VSG). Switching of the expressed VSG gene enables the parasites to evade adaptive immunity. Up until now, the mechanism by which VSG mRNA stability is maintained was unknown. To identify proteins specifically interacting with VSG mRNA (and alpha tubulin as control), we performed RNA-antisense purifications (RAP) following instructions published by McHugh et al. 2018 with some modifications.
Project description:cell activation demands a significant boost in NAD+ levels, often outpacing the capacity of oxidative phosphorylation (OXPHOS). To explore how T cells manage this metabolic stress, we created T cell-specific ADP/ATP translocase-2 knockout (Ant2-/-) mice. Ant2, which is vital for ADP/ATP exchange between the mitochondria and cytoplasm, when deleted, disrupts OXPHOS by limiting ATP synthase function and blocking NAD+ replenishment. Surprisingly, Ant2-/- naïve T cells show increased activation, proliferation, and effector functions compared to wild-type T cells. Through metabolic profiling, we find these cells adopt a metabolic state similar to that of activated T cells, with heightened mitochondrial biogenesis and anabolic processes. Inhibiting ANT pharmacologically in wild-type T cells mirrors the Ant2-/- phenotype and enhances the effectiveness of adoptive T cell therapies in cancer treatment. These results suggest that Ant2-deficient T cells bypass the usual metabolic shifts necessary for activation, leading to greater T cell function and highlighting ANT inhibition as a potential therapeutic strategy for modulating immune responses.
Project description:Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases (DNMTs) and Polycomb Repressor Complexes (PRCs), bind to chromosomes throughout mitosis and their depletion results in increased chromosome size. Enzymes that catalyse H3K9 methylation, such as Suv39h1, Suv39h2, G9a, GLP are also retained by mitotic chromosomes. Surprisingly however, mutants lacking H3K9me3 have unusually small and compact mitotic chromosomes that are associated with increased H3S10ph and H3K27me3 levels. Chromosome size and centromere compaction in these mutants was rescued by providing exogenous Suv39h1, or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wildtype versus Suv39h1,2 double null ESCs revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis, and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.
Project description:The developmental program of seed formation and seedling development requires not only tight regulation of cell division and metabolism but also the adaption of organelles in structure and function. Therefore, changes in organellar protein composition is one crucial factor in development. Of particular interest in plants is the switch to photoautotrophic growth, for which biosynthesis and degradation of lipid droplets (LDs) play a critical role. We present here a bottom-up proteomics study analyzing eight different developmental phases during silique development, seed germination and seedling establishment. We investigated both total protein fractions and LD-enriched fractions for each time point. The overall changes in the seed and seedling proteome during germination and seedling establishment monitored in this study present a rich resource for researchers interested in different questions of early seedling biology. The analysis of the proteome of LDs using LD-enrichment factors allowed the identification of four LD-associated protein families, which were subsequently confirmed by a cell biological approach. In addition to protein discovery, our dataset allows for the study of the dynamics of LD proteins throughout the developmental phases analyzed. We found that the relative levels of oleosin stay stable, while many other proteins accumulate on LDs at later stages of seedling establishment. The methodology described here is shown to be well suited for describing a comprehensive and quantitative view of the Arabidopsis proteome across time, with a particular focus on proteins associated with LDs.
Project description:An animal’s ability to effectively capture prey and defend against predators is pivotal to its survival1. A key innovation in many predator-prey interactions is venom, which consists of many toxin proteins that shape its phenotype2. Its unusually direct relationship of gene-toxin-phenotype make it an appealing system for studies at the organismal level 3,4. In this work we use the sea anemone Nematostella vectensis as a model organism as it provides us with the opportunity to test for the first time how toxin-genotypes impact predator-prey interactions. Specifically, we compare a native-population5 and a transgenic line which both have significantly reduced levels of Nv1, the major toxin in adult Nematostella6, to animals with wildtype levels of Nv1. We demonstrate that the transgenic strain phenocopies native anemones lacking Nv1 as they both have impaired ability to defend themselves against grass shrimp, a native predator. Mummichog killifish, unexpectedly are attracted to Nematostella with wildtype-levels of Nv1, highlighting that Nv1 plays a complex role in shaping interspecific-interactions. Finally, we demonstrate an evolutionary tradeoff as the reduction of Nv1 levels causes faster growth and increased reproductive rates compared to Nematostella control lines. Overall, our results experimentally link organism’s venom to its physiology, reproduction and interspecific interactions.
Project description:An animal’s ability to effectively capture prey and defend against predators is pivotal to its survival1. A key innovation in many predator-prey interactions is venom, which consists of many toxin proteins that shape its phenotype2. Its unusually direct relationship of gene-toxin-phenotype make it an appealing system for studies at the organismal level 3,4. In this work we use the sea anemone Nematostella vectensis as a model organism as it provides us with the opportunity to test for the first time how toxin-genotypes impact predator-prey interactions. Specifically, we compare a native-population5 and a transgenic line which both have significantly reduced levels of Nv1, the major toxin in adult Nematostella6, to animals with wildtype levels of Nv1. We demonstrate that the transgenic strain phenocopies native anemones lacking Nv1 as they both have impaired ability to defend themselves against grass shrimp, a native predator. Mummichog killifish, unexpectedly are attracted to Nematostella with wildtype-levels of Nv1, highlighting that Nv1 plays a complex role in shaping interspecific-interactions. Finally, we demonstrate an evolutionary tradeoff as the reduction of Nv1 levels causes faster growth and increased reproductive rates compared to Nematostella control lines. Overall, our results experimentally link organism’s venom to its physiology, reproduction and interspecific interactions.
Project description:The unicellular eukaryote Trypanosoma brucei relies heavily on posttranscriptional regulatory mechanisms, as pol II transcription is polycistronic. The parasite has six isoforms of the cap-binding translation initiation factor eIF4E, and five eiF4Gs (PMID: 29077018), which potentially allow for differential mRNA target selection in order to fine-tune translation. EIF4E3 and EIF4E4 appear to be general initiation factors; we are investigating the EIF4E proteins that are presumed to have more specialized functions, i.e., EIF4E1, EIF4E2, EIF4E5, and EIF4E6. EIF4E1 interacts with 4E-interacting protein (4EIP) and not with any EIF4G; they functionally resemble mammalian 4E-HP and GIGYF2. 4EIP suppresses translation and provokes mRNA degradation. 4EIP and EIF4E1 are dispensable in slender “bloodstream forms” (BSFs), which multiply in mammals, but 4EIP is required for translation suppression in the growth-arrested “stumpy” BSF (PMID: 30124912). Meanwhile EIF4E1, but not 4EIP, is required for survival of “procyclic forms” (PCFs), which grow in Tsetse. Tethered EIF4E1 is suppressive only when 4EIP is present. We are investigating whether it can, without an EIF4G, activate translation in BSFs/PCFs, as well as how target mRNAs are selected in absence and presence of 4EIP, based on the proteins it associates with. Like EIF4E1, EIF4E2 does not interact with any EIF4G protein, but was found in association with a homolog of the histone mRNA stem-loop-binding protein, called SLBP2, in PCFs (PMID: 29288414). The protein binding partners in BSFs have not been addressed this far. EIF4E3, EIF4E4, EIF4E5, and EIF4E6 all stimulate expression when tethered. They interact with different EIF4G homologs, and each is essential in at least one life-cycle stage, indicating that each serves a distinct role. We have now evidence that EIF4E6 interacts specifically, not only with EIF4G5, but also with a previously characterized stimulatory complex containing MKT1, PBP1, and LSM12, which we aimed to confirm by this quantitative mass spectrometry approach. The complex is recruited to mRNAs via sequence-specific RNA-binding proteins (PMID: 24470144), offering a novel mechanism for specific translation activation by the EIF4E6-EIF4G5 complex. EIF4E5 on the other hand associates with either EIF4G1 or EIF4G2 to exert its functions. It is dispensable in the BSF, but essential in PCFs, where knock-down results in a motility-related phenotype. Furthermore, previous studies identified several 14-3-3 homologs to be associated with EIF4E5 in complex with either EIF4G1 or EIF4G2 in PCFs (PMID: 24962368). In the course of this study, PTP-tagged versions of EIF4E1+/- 4EIP (BSF, PCF), EIF4E2, EIF4E5, and EIF4E6 (BSF only) were pulled down and bound proteins were analyzed by quantitative mass spectrometry, with EIF4E3-PTP (BSF) and GFP-PTP (BSF, PCF) serving as controls.
Project description:Ras is a key switch controlling cell behavior. In the GTP-bound form, Ras interacts with numerous effectors in a mutually exclusive manner, where individual Ras-effectors are likely part of larger cellular (sub)complexes. The molecular details of these (sub)complexes and their alteration in specific contexts is not understood. Focusing on KRAS, we performed affinity purification (AP)-mass spectrometry (MS) experiments of exogenous expressed Flag-KRAS WT and three oncogenic mutants (‘genetic contexts’) in the human Caco-2 cell line, each exposed to 11 different culture media (‘culture contexts’) that mimic conditions relevant in the colon and colorectal cancer. We identified four effectors present in complex with KRAS in all genetic and growth contexts (‘context-general effectors’). Seven effectors are found in KRAS complexes in only some contexts (‘context-specific effectors’). Analyzing all interactors in complex with KRAS per condition, we find that the culture contexts had a larger impact on interaction rewiring than genetic contexts. We analyzed how changes in the interactome impact functional outcomes and created interactive shiny app for interactive visualization. We validated some of the functional differences in metabolism and proliferation. Finally, we used networks to evaluate how KRAS effectors are involved in the modulation of functions by random walk analyses of effector-mediated (sub)complexes. Altogether, our work shows the impact of environmental contexts on network rewiring, which provides insides into tissue-specific signaling mechanisms. This may also explain why KRAS oncogenic mutants may be causing cancer only in specific tissues despite KRAS being expressed in most cells and tissues.
Project description:Description: (Supplemental data for Project PXD035399) “Analysis of context-specific KRAS-effectors (sub)complexes in Caco-2 cells”. Proteome of Caco-2 cells, transfected with KRAS-G12D and stimulated with different conditions.