scholarly journals The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release

2020 ◽  
Author(s):  
Juanma Ramírez ◽  
Miguel Morales ◽  
Nerea Osinalde ◽  
Imanol Martínez-Padrón ◽  
Ugo Mayor ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Neurotransmitter Release ◽  
Quantitative Proteomics ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Sensitive Factor ◽  
Active Zones ◽  
Protein Ensembles ◽  
Mutant Phenotypes

ABSTRACTAriadne-1 (Ari-1) is an essential E3 ubiquitin-ligase whose neuronal substrates are yet to be identified. We have used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics to identify putative Ari-1 substrates in Drosophila heads. Sixteen candidates met the established criteria. Amongst those, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF). Using an in vivo GFP pulldown approach, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons. The interaction results in the monoubiquitination of Comt/NSF. We also report that Ari-1 loss of function mutants display a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants is enhanced in a Ca2+ dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. The distinct Ari-1 mutant phenotypes in spontaneous versus evoked release indicate that NSF activity discriminates the two corresponding protein ensembles that mediate each mode of release. Our results, thus, provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.

scholarly journals A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Sonal Nagarkar-Jaiswal ◽  
Pei-Tseng Lee ◽  
Megan E Campbell ◽  
Kuchuan Chen ◽  
Stephanie Anguiano-Zarate ◽  
...  
Keyword(s):  
Null Mutant ◽  
Loss Of Function ◽  
Severe Loss ◽  
New Strategy ◽  
Mutant Phenotypes

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

scholarly journals UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology

2019 ◽  
Vol 116 (25) ◽  
pp. 12500-12505 ◽  
Author(s):  
Jie Wang ◽  
Sen-Sen Lou ◽  
Tingting Wang ◽  
Rong-Jie Wu ◽  
Guangying Li ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Dendritic Spine ◽  
Isotope Labeling ◽  
Neurodevelopmental Disorder ◽  
Critical Role ◽  
Motor Impairment ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Pp2a Activity

Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am−/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11–13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am−/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am−/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.

GFI1 Is a Tumor Suppressor in Myeloid Progenitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2942-2942
Author(s):  
Aditya Chaubey ◽  
Shane Hormon ◽  
Chinavenmeni S. Velu ◽  
Tristan Bourdeau ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Myeloid Leukemia ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Increased Risk ◽  
Myeloid Leukemias ◽  
Dose Dependent ◽  
Anterior Posterior

Abstract In severe congenital neutropenia (SCN) patients and mice with Growth factor independent-1 (Gfi1) loss of function, arrested progenitors are suspended in a hyperproliferative state while terminal granulpoiesis is blocked. SCN patients are at increased risk for the development of acute myeloid leukemia. We demonstrate that Gfi1 directly targets HoxA9, Pbx1 and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and increased persistence in vivo and in vitro. Limiting HoxA9 alleles corrects, in a dose dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1. Moreover, in a manner conserved in Drosophila anterior/posterior patterning, we demonstrate that these factors can compete for occupancy of DNA sequences encoding composite Gfi1-HoxA9-Pbx1-Meis1 binding sites. Finally, the expression of Gfi1 and HoxA9 are inverse and stratify human myeloid leukemias, suggesting a role for HoxA9- Gfi1 antagonism in human AML. In agreement with this, a myeloproliferative disorder progresses into a rapid, lethal and transplantable myeloid leukemia in a Gfi1−/− setting. We conclude that the lifespan and oncogenic transformation of hematopoietic progenitor cells is regulated through a conserved competition between Gfi1 and HoxA9-Pbx1-Meis1.

scholarly journals β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction

2021 ◽  
Author(s):  
Zachary T Hilt ◽  
Preeti Maurya ◽  
Laura Tesoro ◽  
Daphne N Pariser ◽  
Sara K Ture ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Dependent Manner ◽  
Elevated Plasma ◽  
Imaging Flow Cytometry ◽  
Tgfβ Receptor

Rationale: Circulating monocytes can have pro-inflammatory or pro-reparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet derived beta-2 microglobulin (β2M) and transforming growth factor beta (TGFβ) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGFβ regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type (WT) and platelet specific β2M knockout (Plt-β2M -/- ) mice were treated intravenously with either β2M or TGFβ to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased pro-inflammatory monocytes, while increased plasma TGFβ increased pro-reparative monocytes. TGFβ receptor (TGFβR) inhibition blunted monocyte responses to both β2M and TGFβ in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGFβ promoted SMAD nuclear translocation, but decreased non-canonical/inflammatory (JNK and NFκB nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGFβ, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked non-canonical SMAD-independent monocyte signaling and skewed monocytes towards a pro-reparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGFβ dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor, but induce SMAD-dependent canonical signaling (TGFβ) versus non-canonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGFβ is increased in fibrotic diseases.

scholarly journals PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection

2021 ◽  
Author(s):  
Salik Miskat Borbora ◽  
R.S. Rajmani ◽  
Kithiganahalli N Balaji
Keyword(s):  
Ubiquitin Ligase ◽  
Lipid Accumulation ◽  
Ex Vivo ◽  
E3 Ubiquitin Ligase ◽  
Mycobacterial Infection ◽  
Drug Regimen ◽  
Loss Of Function ◽  
Methyl Transferase ◽  
Accumulation Of Lipids

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), triggers enhanced accumulation of lipids to generate foamy macrophages (FMs). This process has been often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in those involved in lipid efflux genes. Here, we define an Mtb-orchestrated modulation of the ubiquitination mechanism of lipid accumulation markers to enhance lipid accretion during infection. We find that Mtb infection represses the expression of the E3 ubiquitin ligase, ITCH, resulting in the sustenance of key lipid accrual molecules viz. ADRP and CD36, that are otherwise targeted by ITCH for proteasomal degradation. In line, overexpressing ITCH in Mtb-infected cells was found to suppress Mtb-induced lipid accumulation. Molecular analyses including loss-of-function and ChIP assays demonstrated a role for the concerted action of the transcription factor YY1 and the arginine methyl transferase PRMT5 in restricting the expression of Itch gene by conferring repressive symmetrical H4R3me2 marks on its promoter. Consequently, siRNA-mediated depletion of YY1 or PRMT5 rescued ITCH expression, thereby compromising the levels of Mtb-induced ADRP and CD36 and limiting FM formation during infection. Accumulation of lipids within the host has been implicated as a pro-mycobacterial process that aids in pathogen persistence and dormancy. In our study, perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in primary murine macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights on the role of PRMT5 and YY1 in augmenting mycobacterial pathogenesis. Thus, we posit that our observations could help design novel adjunct therapies and combinatorial drug regimen for effective anti-TB strategies.

scholarly journals Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

2021 ◽  
Vol 17 (2) ◽  
pp. e1009291
Author(s):  
Yuli Talyansky ◽  
Travis B. Nielsen ◽  
Jun Yan ◽  
Ulrike Carlino-Macdonald ◽  
Gisela Di Venanzio ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Bacterial Pathogen ◽  
Specific Gene ◽  
Dependent Manner ◽  
Bacterial Burden ◽  
Carbohydrate Structure ◽  
Loss Of Function ◽  
Capsule Assembly

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

scholarly journals The PDZ Binding Motif of Human Papillomavirus Type 16 E6 Induces PTPN13 Loss, Which Allows Anchorage-Independent Growth and Synergizes with Ras for Invasive Growth

2007 ◽  
Vol 82 (5) ◽  
pp. 2493-2500 ◽  
Author(s):  
William C. Spanos ◽  
Andrew Hoover ◽  
George F. Harris ◽  
Shu Wu ◽  
Guinevere L. Strand ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Tyrosine Phosphatase ◽  
Binding Motif ◽  
Ras Signaling ◽  
Invasive Growth ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Hpv16 E6 ◽  
Anchorage Independent Growth

ABSTRACT The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Rasv12, resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

scholarly journals Isthmin1, a secreted signaling protein, acts downstream of diverse embryonic patterning centers in development

2020 ◽  
Author(s):  
Gokul Kesavan ◽  
Florian Raible ◽  
Mansi Gupta ◽  
Anja Machate ◽  
Dilara Yilmaz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gene Expression Pattern ◽  
Correlation Spectroscopy ◽  
Dependent Manner ◽  
Embryonic Patterning ◽  
Loss Of Function ◽  
Concentration Dependent Manner ◽  
Developmental Defects ◽  
Extracellular Signals

AbstractExtracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.

Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1925-1933 ◽  
Author(s):  
Baohe Shen ◽  
James L. Manley
Keyword(s):  
Dependent Manner ◽  
Loss Of Function ◽  
Concentration Dependent Manner ◽  
Downstream Target ◽  
Toll Signaling ◽  
Early Embryos ◽  
High Concentrations ◽  
L2 Cells

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll10b), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll10b, and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.

scholarly journals Ovarian insufficiency and CTNNB1 mutations drive malignant transformation of endometrial hyperplasia with altered PTEN/PI3K activities

2019 ◽  
Vol 116 (10) ◽  
pp. 4528-4537 ◽  
Author(s):  
Jumpei Terakawa ◽  
Vanida Ann Serna ◽  
Makoto Mark Taketo ◽  
Takiko Daikoku ◽  
Adrian A. Suarez ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Biological Significance ◽  
Myometrial Invasion ◽  
Premenopausal Women ◽  
Driver Mutations ◽  
Low Grade ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Normal Endometrium

Endometrioid endometrial carcinomas (EECs) carry multiple driver mutations even when they are low grade. However, the biological significance of these concurrent mutations is unknown. We explored the interactions among three signature EEC mutations: loss-of-function (LOF) mutations inPTEN, gain-of-function (GOF) mutations of phosphoinositide 3-kinase (PI3K), andCTNNB1exon 3 mutations, utilizing in vivo mutagenesis of the mouse uterine epithelium. While epithelial cells with a monoallelic mutation in any one of three genes failed to propagate in the endometrium, any combination of two or more mutant alleles promoted the growth of epithelium, causing simple hyperplasia, in a dose-dependent manner. Notably,Ctnnb1exon 3 deletion significantly increased the size of hyperplastic lesions by promoting the growth of PTEN LOF and/or PI3K GOF mutant cells through the activation of neoadenogenesis pathways. Although these three mutations were insufficient to cause EEC in intact female mice, castration triggered malignant transformation, leading to myometrial invasion and serosal metastasis. Treatment of castrated mice with progesterone or estradiol attenuated the neoplastic transformation. This study demonstrates that multiple driver mutations are required for premalignant cells to break the growth-repressing field effect of normal endometrium maintained by ovarian steroids and thatCTNNB1exon 3 mutations play critical roles in the growth of preneoplastic cells within the endometrium of premenopausal women and in the myometrial invasion of EECs in menopausal women.

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