scholarly journals RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

2018 ◽  
Vol 4 (11) ◽  
pp. eaav0443 ◽  
Author(s):  
J.-M. Heo ◽  
A. Ordureau ◽  
S. Swarup ◽  
J. A. Paulo ◽  
K. Shen ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Quantitative Proteomics ◽  
Mitochondrial Outer Membrane ◽  
Ubiquitin Binding ◽  
Homologous Site ◽  
Dynamic Target ◽  
Interaction Proteomics ◽  
Gdp Dissociation Inhibitor

Removal of damaged mitochondria is orchestrated by a pathway involving the PINK1 kinase and the PARKIN ubiquitin ligase. Ubiquitin chains assembled by PARKIN on the mitochondrial outer membrane recruit autophagy cargo receptors in complexes with TBK1 protein kinase. While TBK1 is known to phosphorylate cargo receptors to promote ubiquitin binding, it is unknown whether TBK1 phosphorylates other proteins to promote mitophagy. Using global quantitative proteomics, we identified S72 in RAB7A, a RAB previously linked with mitophagy, as a dynamic target of TBK1 upon mitochondrial depolarization. TBK1 directly phosphorylates RAB7AS72, but not several other RABs known to be phosphorylated on the homologous residue by LRRK2, in vitro, and this modification requires PARKIN activity in vivo. Interaction proteomics using nonphosphorylatable and phosphomimetic RAB7A mutants revealed loss of association of RAB7AS72E with RAB GDP dissociation inhibitor and increased association with the DENN domain–containing heterodimer FLCN-FNIP1. FLCN-FNIP1 is recruited to damaged mitochondria, and this process is inhibited in cells expressing RAB7AS72A. Moreover, nonphosphorylatable RAB7A failed to support efficient mitophagy, as well as recruitment of ATG9A-positive vesicles to damaged mitochondria. These data reveal a novel function for TBK1 in mitophagy, which parallels that of LRRK2-mediated phosphorylation of the homologous site in distinct RABs to control membrane trafficking.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach ◽  
Telomeric Protein ◽  
Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

scholarly journals ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling

2019 ◽  
Author(s):  
Alfonso Bolado-Carrancio ◽  
Morwenna Muir ◽  
Ailith Ewing ◽  
Kenneth Macleod ◽  
William Gallagher ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Tumour Progression ◽  
Survival Rates ◽  
Breast Tumour ◽  
Breast Cancers ◽  
Breast Cancer Patients ◽  
Akt Activation ◽  
Gdp Dissociation Inhibitor

ABSTRACTISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. However, the mechanism by which ISG15 achieves this remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR). By regulating EGFR trafficking, ISGylation sustains Akt-signalling in vitro and in vivo. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in animal models and human breast cancers. We show that ISGylation can act as driver of tumour progression rather than merely being a marker of it.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2020 ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Binding Proteins ◽  
Protein Complexes ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach

AbstractTelomeres are bound by dedicated protein complexes, like shelterin in mammals, which protect telomeres from DNA damage. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 are expressed in the germline and during embryogenesis. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp-1; tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. Furthermore, we find that POT-1 bridges the double- stranded binders TEBP-1 and TEBP-2, with the single-stranded binders POT-2 and MRT-1. These results describe the first telomere-binding complex in C. elegans, with TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility and that mediate opposite telomere dynamics.

scholarly journals Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

2002 ◽  
Vol 159 (6) ◽  
pp. 1039-1049 ◽  
Author(s):  
Vicki A. Sciorra ◽  
Simon A. Rudge ◽  
Jiyao Wang ◽  
Stuart McLaughlin ◽  
JoAnne Engebrecht ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Membrane Trafficking ◽  
Dual Role ◽  
Biological Functions ◽  
Ph Domain ◽  
Catalytically Active ◽  
Acidic Phospholipids ◽  
Stimulation Of

Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling. PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair. Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2). We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions. Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes. As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo. Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion. Binding of PLD2 to model membranes is enhanced by acidic phospholipids. Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2. By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes. Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif.

scholarly journals Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria

2001 ◽  
Vol 12 (5) ◽  
pp. 1189-1198 ◽  
Author(s):  
Doron Rapaport ◽  
Rebecca D. Taylor ◽  
Michael Käser ◽  
Thomas Langer ◽  
Walter Neupert ◽  
...  
Keyword(s):  
Intermediate Stage ◽  
In Vivo Studies ◽  
Mitochondrial Outer Membrane ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Blue Native

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

2007 ◽  
Vol 292 (4) ◽  
pp. C1562-C1566 ◽  
Author(s):  
Christopher J. Guerriero ◽  
Ora A. Weisz
Keyword(s):  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Cell Function ◽  
Actin Dynamics ◽  
Cellular Functions ◽  
Intact Cells ◽  
Atp Levels ◽  
Dependent Inhibition

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo.

scholarly journals Global ubiquitylation analysis of mitochondria in primary neurons identifies physiological Parkin targets following activation of PINK1

2021 ◽  
Author(s):  
Odetta Antico ◽  
Alban Ordureau ◽  
Michael Stevens ◽  
Francois Singh ◽  
Marek Gierlinski ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Mitochondrial Outer Membrane ◽  
Culture Studies ◽  
Parkinsons Disease ◽  
Recessive Mutations ◽  
Ubiquitin E3 Ligase ◽  
Cytoplasmic Proteins ◽  
Online Resource

Autosomal recessive mutations in PINK1 and Parkin cause Parkinsons disease. How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell culture studies with few molecular studies in neurons. Herein we have undertaken a global proteomic- analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin activation. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1-dependent diGLY sites upregulated upon mitochondrial depolarisation in 22 proteins conserved across mouse and human systems. These proteins were exclusively localised at the mitochondrial outer membrane (MOM) including, CISD1, CPT1A, ACSL1, and FAM213A. We demonstrate that these proteins can be directly ubiquitylated by Parkin in vitro. We also provide evidence for a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation including SNX3, CAMK2A; and CAMK2B; indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarisation in neurons. Finally we have developed an online resource to visualise mitochondrial ubiquitin sites in neurons and search for ubiquitin components recruited to mitochondria upon mitochondrial depolarisation, MitoNUb. This analysis defines the ubiquitin architecture of damaged mitochondria and will aid in future studies to understand Parkin activation in neuronal subtypes. Our findings also suggest that monitoring ubiquitylation status of the 22 identified MOM proteins may represent robust biomarkers for PINK1 and Parkin activity in vivo.

scholarly journals The MiR-193a-5p/NCX2/AKT Axis Promotes Invasion and Metastasis of Osteosarcoma

2021 ◽  
Author(s):  
Ruiqi Chen ◽  
Yichong Ning ◽  
Guirong Zeng ◽  
Hao Zhou ◽  
Chao Tu ◽  
...  
Keyword(s):  
Colony Formation ◽  
Quantitative Proteomics ◽  
Epithelial Mesenchymal Transition ◽  
Ectopic Expression ◽  
Akt Inhibitor ◽  
Proteomics Analysis ◽  
Mesenchymal Transition ◽  
Intracellular Ca

Abstract Background: MiR-193a-5p has been observed to have oncogenic or tumor-suppressive function in different kinds of cancers. Na+/Ca2+ exchangers (NCX1, NCX2 and NCX3) normally extrude Ca2+ from the cell, and deregulation of the intracellular Ca2+ homeostasis is related to several kinds of diseases, including cancer. However, their roles and molecular mechanism in osteosarcoma are elusive.Methods: The expression of miR-193a-5p and NCX2 in osteosarcoma and corresponding adjacent noncancerous tissues was investigated by qRT-PCR. Colony formation assay, wound healing assay, transwell invasion assay and xenograft mouse model were used for in vitro and in vivo functional analyses. Tandem mass tag-based quantitative proteomics analysis was performed to identify the targets of miR-193a-5p.Results: This study showed that miR-193a-5p was upregulated in osteosarcoma tissues compared with the corresponding adjacent noncancerous tissues, and promoted colony formation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, as well as metastasis in vivo. Quantitative proteomics analysis identified NCX2 as a potential target of miR-193a-5p. Luciferase activity assay and Western blotting further confirmed that miR-193a-5p recognized the 3′-untranslated region of NCX2 mRNA, and negatively regulated NCX2 expression. NCX2 was downregulated in osteosarcoma tissues, and its expression was negatively correlated with miR-193a-5p levels. Ectopic expression of NCX2 in osteosarcoma cells could counteract the oncogenic effects of miR-193a-5p. These results indicate that miR-193a-5p exerts its effects by targeting NCX2. Further study demonstrated that NCX2 suppressed Ca2+-dependent Akt phosphorylation by decreasing intracellular Ca2+ efflux, then inhibited EMT process. Treatment with the antagomir against miR-193a-5p sensitized osteosarcoma to the Akt inhibitor afuresertib in a murine xenograft tumor model. Conclusion: This study revealed a miR-193a-5p/NCX2/AKT signaling axis in the progression of osteosarcoma, which may provide a new therapeutic target for osteosarcoma treatment.

Abstract 369: Engineering Rrm2 to Elevate 2-deoxy-ATP and Improve Cardiomyocyte Function

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Jason D Murray ◽  
Xuan Guan ◽  
Farid Moussavi-Harami ◽  
Sigurast S Olafsson ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Therapeutic Option ◽  
Induced Pluripotent Stem Cell ◽  
Left Ventricular ◽  
Ubiquitin Binding ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Induced Pluripotent ◽  
Cultured Cardiomyocytes

Overexpression of ribonucleotide reductase (RNR) in cardiomyocytes increases the amount of cytosolic 2-deoxy-ATP (dATP), which can be used by myosin and significantly increases contraction of cardiac muscle at all levels of calcium activation. Our group is working to develop enhanced dATP as a therapeutic option for heart failure. We have demonstrated that virally-mediated overexpression of RNR elevates dATP and increases the rate and magnitude of contraction and increases left ventricular contraction in normal hearts as well as rodent models of myocardial infarction and dilated cardiomyopathy. RNR is a heterotetramer containing two subunits, Rrm1 and Rrm2. While cardiomyocyte-specific overexpression of Rrm1 is stable, we have observed high variability in expression levels of the Rrm2 subunit in multiple disease models. We hypothesized that this variability was largely due to protein degradation via the ubiquitin-proteasome complex (UPC). We found that pharmacological inhibition of proteasome activity leads to increased expression of Rrm2 in virally-transduced cardiomyocytes in vitro. To confirm the hypothesis that the overexpressed Rrm2 is degraded via UPC-mediated degradation, we engineered mutations in specific ubiquitin-binding degrons of the Rrm2 gene. Transfecting human induced pluripotent stem cell-derived cardiomyocytes resulted in higher levels of Rrm2 than those overexpressing wild-type protein and resulted in higher levels of cytosolic dATP as measured by Liquid chromatography-mass spectrometry. Ongoing and planned experiments will compare the effects of this engineered mutation on Rrm2 overexpression, dATP production, and contractility in cultured cardiomyocytes. Our goal is to develop an improved RNR vector that will be resistant to degradation through the ubiquitin-proteasome pathway and therefore enable more stable and consistent RNR enzyme activity and deoxynucleotide levels of cardiomyocytes transduced in vivo.

scholarly journals ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes

1998 ◽  
Vol 142 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Chean Eng Ooi ◽  
Esteban C. Dell'Angelica ◽  
Juan S. Bonifacino
Keyword(s):  
Membrane Trafficking ◽  
Brefeldin A ◽  
Membrane Association ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

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