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The EMBO Journal Apr 2024Ten-eleven translocation (TET) proteins are dioxygenases that convert 5-methylcytosine (5mC) into 5-hydroxylmethylcytosine (5hmC) in DNA and RNA. However, their...
Ten-eleven translocation (TET) proteins are dioxygenases that convert 5-methylcytosine (5mC) into 5-hydroxylmethylcytosine (5hmC) in DNA and RNA. However, their involvement in adult stem cell regulation remains unclear. Here, we identify a novel enzymatic activity-independent function of Tet in the Drosophila germline stem cell (GSC) niche. Tet activates the expression of Dpp, the fly homologue of BMP, in the ovary stem cell niche, thereby controlling GSC self-renewal. Depletion of Tet disrupts Dpp production, leading to premature GSC loss. Strikingly, both wild-type and enzyme-dead mutant Tet proteins rescue defective BMP signaling and GSC loss when expressed in the niche. Mechanistically, Tet interacts directly with Bap55 and Stat92E, facilitating recruitment of the Polybromo Brahma associated protein (PBAP) complex to the dpp enhancer and activating Dpp expression. Furthermore, human TET3 can effectively substitute for Drosophila Tet in the niche to support BMP signaling and GSC self-renewal. Our findings highlight a conserved novel catalytic activity-independent role of Tet as a scaffold protein in supporting niche signaling for adult stem cell self-renewal.
Topics: Animals; Female; Humans; Cell Differentiation; Drosophila; Drosophila melanogaster; Drosophila Proteins; Germ Cells; Stem Cell Niche; Stem Cells; Dioxygenases
PubMed: 38499787
DOI: 10.1038/s44318-024-00074-9 -
Molecular Biology of the Cell Aug 2023Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway...
Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in-silico approach, we identified Striatin interacting protein (Strip), MOB kinase activator 4, and fibroblast growth factor receptor 1 oncogene partner 2 as conserved mediators of muscle tissue maintenance. We performed affinity purification-mass spectrometry (AP-MS) experiments with Strip as a bait protein and copurified additional Striatin-interacting phosphatase and kinase (STRIPAK) complex members from larval muscle tissue. NUAK family kinase 1 (NUAK) and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using proximity ligation assays. To understand the functional significance of the STRIPAK-NUAK-Stv complex, we employed a sensitized genetic assay combined with RNA interference (RNAi) to demonstrate that both and function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Autophagy-related 8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in muscles, while lysosome biogenesis and activity were unaffected. Our results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue.
Topics: Animals; Drosophila melanogaster; Carrier Proteins; Autophagy; Muscles; Cytoplasm; Drosophila Proteins
PubMed: 37379167
DOI: 10.1091/mbc.E23-01-0006 -
Science Advances Jul 2023PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the...
PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.
Topics: Animals; Male; Drosophila; RNA, Small Interfering; Drosophila melanogaster; MicroRNAs; Piwi-Interacting RNA; Drosophila Proteins; Spermatogenesis
PubMed: 37467338
DOI: 10.1126/sciadv.adh0397 -
ENeuro Aug 2023As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2...
As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2 (MFN2)/mitochondrial assembly regulatory factor (Marf), plays a role in mitochondrial fusion, mutations of which are implicated in age-related human diseases, including several neurodegenerative disorders. However, the regulation of MFN2/Marf-mediated mitochondrial fusion, as well as the pathologic mechanism of neurodegeneration, is not clearly understood. Here, we identified a novel interaction between MFN2/Marf and microtubule affinity-regulating kinase 4 (MARK4)/PAR-1. In the larval neuromuscular junction, muscle-specific overexpression of MFN2/Marf decreased the number of synaptic boutons, and the loss of MARK4/PAR-1 alleviated the synaptic defects of MFN2/Marf overexpression. Downregulation of MARK4/PAR-1 rescued the mitochondrial hyperfusion phenotype caused by MFN2/Marf overexpression in the muscles as well as in the cultured cells. In addition, knockdown of MARK4/PAR-1 rescued the respiratory dysfunction of mitochondria induced by MFN2/Marf overexpression in mammalian cells. Together, our results indicate that the interaction between MFN2/Marf and MARK4/PAR-1 is fine-tuned to maintain synaptic integrity and mitochondrial homeostasis, and its dysregulation may be implicated in neurologic pathogenesis.
Topics: Animals; Humans; Drosophila; Drosophila Proteins; GTP Phosphohydrolases; Mammals; Microtubules; Mitochondria; Mitochondrial Proteins; Protein Serine-Threonine Kinases; Synapses
PubMed: 37550059
DOI: 10.1523/ENEURO.0409-22.2023 -
Current Opinion in Neurobiology Feb 2024The descending neurons connecting the fly's brain to its ventral nerve cord respond to sensory stimuli and evoke motor programs of varying complexity. Anatomical... (Review)
Review
The descending neurons connecting the fly's brain to its ventral nerve cord respond to sensory stimuli and evoke motor programs of varying complexity. Anatomical characterization of the descending neurons and their synaptic connections suggests how these circuits organize movements, while optogenetic manipulation of their activity reveals what behaviors they can induce. Monitoring their responses to sensory stimuli or during behavior performance indicates what information they may encode. Recent advances in all three approaches make the descending neurons an excellent place to better understand the sensorimotor integration and transformation required for nervous systems to govern the motor sequences that constitute animal behavior.
Topics: Animals; Drosophila; Neurons; Brain; Drosophila Proteins; Behavior, Animal
PubMed: 38096757
DOI: 10.1016/j.conb.2023.102822 -
The EMBO Journal Dec 2023The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system....
The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system. Although this interplay is fundamental for the function of the immune system, the underlying mechanisms remain elusive. Our data show that the pro-inflammatory polarization of Drosophila macrophages is coupled to the production of the insulin antagonist ImpL2 through the activity of the transcription factor HIF1α. ImpL2 production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual's resistance to infection. We demonstrated that analogically to Drosophila, mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of ImpL2/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection.
Topics: Animals; Insulin Antagonists; Drosophila; Insulin; Macrophages; Bacterial Infections; Insulin Resistance; Mammals; Insulin-Like Growth Factor Binding Proteins; Drosophila Proteins
PubMed: 37807855
DOI: 10.15252/embj.2023114086 -
ELife May 2024Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in...
Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch-Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in , making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in -deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or -methyladenosine (mA) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to deficiency. Overall, our results suggest that could be used in different ways to better understand LND and seek a cure for this dramatic disease.
Topics: Animals; Drosophila melanogaster; Lesch-Nyhan Syndrome; Purines; Disease Models, Animal; Behavior, Animal; Hypoxanthine Phosphoribosyltransferase; Drosophila Proteins; Locomotion
PubMed: 38700995
DOI: 10.7554/eLife.88510 -
Human Molecular Genetics May 2024Neuromuscular disorders caused by dysfunction of the mitochondrial respiratory chain are common, severe and untreatable. We recovered a number of mitochondrial genes,...
Neuromuscular disorders caused by dysfunction of the mitochondrial respiratory chain are common, severe and untreatable. We recovered a number of mitochondrial genes, including electron transport chain components, in a large forward genetic screen for mutations causing age-related neurodegeneration in the context of proteostasis dysfunction. We created a model of complex I deficiency in the Drosophila retina to probe the role of protein degradation abnormalities in mitochondrial encephalomyopathies. Using our genetic model, we found that complex I deficiency regulates both the ubiquitin/proteasome and autophagy/lysosome arms of the proteostasis machinery. We further performed an in vivo kinome screen to uncover new and potentially druggable mechanisms contributing to complex I related neurodegeneration and proteostasis failure. Reduction of RIOK kinases and the innate immune signaling kinase pelle prevented neurodegeneration in complex I deficiency animals. Genetically targeting oxidative stress, but not RIOK1 or pelle knockdown, normalized proteostasis markers. Our findings outline distinct pathways controlling neurodegeneration and protein degradation in complex I deficiency and introduce an experimentally facile model in which to study these debilitating and currently treatment-refractory disorders.
Topics: Animals; Proteostasis; Electron Transport Complex I; Mitochondria; Drosophila Proteins; Mitochondrial Diseases; Disease Models, Animal; Humans; Proteasome Endopeptidase Complex; Autophagy; Oxidative Stress; Drosophila melanogaster; Mutation; Lysosomes; Drosophila; Signal Transduction
PubMed: 38324746
DOI: 10.1093/hmg/ddae018 -
Genes & Development Jun 2024Gene transcription is intimately linked to chromatin state and histone modifications. However, the enzymes mediating these post-translational modifications have many... (Review)
Review
Gene transcription is intimately linked to chromatin state and histone modifications. However, the enzymes mediating these post-translational modifications have many additional, nonhistone substrates, making it difficult to ascribe the most relevant modification. In this issue of , Crain and colleagues (doi:10.1101/gad.351698.124) have combined a powerful histone replacement system with mutational analysis of a chromatin regulator and a chromatin reader in Importantly, they discovered that genes controlled by the histone 4 lysine 20 (H4K20) methyltransferase Set8 and the protein recognizing H4K20 monomethylation, L(3)mbt, differ substantially from those affected by mutation of H4K20 itself. This demonstrates that H4K20 is not the key substrate for Set8 but that methylation of other, unidentified proteins mediates its effects on transcription.
Topics: Animals; Drosophila melanogaster; Chromatin; Histones; Drosophila Proteins; Histone-Lysine N-Methyltransferase; Methylation; Protein Processing, Post-Translational
PubMed: 38866554
DOI: 10.1101/gad.351969.124 -
Nature Communications Apr 2024Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that...
Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. Here we show that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. We find that endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. We show that Cdk8 promotes the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson's disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, we propose that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission.
Topics: Animals; Humans; Phosphorylation; Drosophila; Drosophila melanogaster; Drosophila Proteins; Mitochondrial Dynamics; Cyclin-Dependent Kinases; Cyclin-Dependent Kinase 8
PubMed: 38637532
DOI: 10.1038/s41467-024-47623-8