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1.
Redox Biol ; 37: 101686, 2020 10.
Article in English | MEDLINE | ID: mdl-32911434

ABSTRACT

NRF2 (NFE2L2) is a cytoprotective transcription factor associated with >60 human diseases, adverse drug reactions and therapeutic resistance. To provide insight into the complex regulation of NRF2 responses, 1962 predicted NRF2-partner interactions were systematically tested to generate an experimentally defined high-density human NRF2 interactome. Verification and conditional stratification of 46 new NRF2 partners was achieved by co-immunoprecipitation and the novel integration of quantitative data from dual luminescence-based co-immunoprecipitation (DULIP) assays and live-cell fluorescence cross-correlation spectroscopy (FCCS). The functional impact of new partners was then assessed in genetically edited loss-of-function (NRF2-/-) and disease-related gain-of-function (NRF2T80K and KEAP1-/-) cell-lines. Of the new partners investigated >77% (17/22) modified NRF2 responses, including partners that only exhibited effects under disease-related conditions. This experimentally defined binary NRF2 interactome provides a new vision of the complex molecular networks that govern the modulation and consequence of NRF2 activity in health and disease.


Subject(s)
Gene Expression Regulation , NF-E2-Related Factor 2 , Cell Line , Humans , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Transcriptional Activation
2.
J Cell Sci ; 133(11)2020 06 11.
Article in English | MEDLINE | ID: mdl-32381682

ABSTRACT

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.This article has an associated First Person interview with the first author of the paper.


Subject(s)
Glucocorticoids , Receptors, Glucocorticoid , Cell Movement , Cytosol , Gene Expression , Glucocorticoids/pharmacology , Histone Deacetylase 6 , Receptors, Glucocorticoid/genetics
3.
Nat Commun ; 10(1): 2835, 2019 06 27.
Article in English | MEDLINE | ID: mdl-31249377

ABSTRACT

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Neural Stem Cells/metabolism , Neurogenesis , Repressor Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Female , Gene Expression Regulation, Developmental , Male , Mice/embryology , Mice/metabolism , Mice, Inbred ICR , Mice, Knockout , Neural Stem Cells/chemistry , Neural Stem Cells/cytology , Repressor Proteins/chemistry , Repressor Proteins/genetics , Single-Cell Analysis
4.
Cancers (Basel) ; 11(2)2019 Feb 23.
Article in English | MEDLINE | ID: mdl-30813438

ABSTRACT

Although hypoxia is known to contribute to several aspects of tumour progression, relatively little is known about the effects of hypoxia on cancer-associated myofibroblasts (CAMs), or the consequences that conditional changes in CAM function may have on tumour development and metastasis. To investigate this issue in the context of gastric cancer, a comparative multiomic analysis was performed on populations of patient-derived myofibroblasts, cultured under normoxic or hypoxic conditions. Data from this study reveal a novel set of CAM-specific hypoxia-induced changes in gene expression and secreted proteins. Significantly, these signatures are not observed in either patient matched adjacent tissue myofibroblasts (ATMs) or non-cancer associated normal tissue myofibroblasts (NTMs). Functional characterisation of different myofibroblast populations shows that hypoxia-induced changes in gene expression not only enhance the ability of CAMs to induce cancer cell migration, but also confer pro-tumorigenic (CAM-like) properties in NTMs. This study provides the first global mechanistic insight into the molecular changes that contribute to hypoxia-induced pro-tumorigenic changes in gastric stromal myofibroblasts.

5.
Carcinogenesis ; 40(4): 500-512, 2019 06 10.
Article in English | MEDLINE | ID: mdl-30624614

ABSTRACT

There is increasing evidence that stromal myofibroblasts play a key role in the tumour development however, the mechanisms by which they become reprogrammed to assist in cancer progression remain unclear. As cultured cancer-associated myofibroblasts (CAMs) retain an ability to enhance the proliferation and migration of cancer cells in vitro, it is possible that epigenetic reprogramming of CAMs within the tumour microenvironment may confer long-term pro-tumourigenic changes in gene expression. This study reports the first comparative multi-omics analysis of cancer-related changes in gene expression and DNA methylation in primary myofibroblasts derived from gastric and oesophageal tumours. In addition, we identify novel CAM-specific DNA methylation signatures, which are not observed in patient-matched adjacent tissue-derived myofibroblasts, or corresponding normal tissue-derived myofibroblasts. Analysis of correlated changes in DNA methylation and gene expression shows that different patterns of gene-specific DNA methylation have the potential to confer pro-tumourigenic changes in metabolism, cell signalling and differential responses to hypoxia. These molecular signatures provide new insights into potential mechanisms of stromal reprogramming in gastric and oesophageal cancer, while also providing a new resource to facilitate biomarker identification and future hypothesis-driven studies into mechanisms of stromal reprogramming and tumour progression in solid tumours.


Subject(s)
Biomarkers, Tumor/genetics , Epigenesis, Genetic , Esophageal Neoplasms/pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Myofibroblasts/pathology , Stomach Neoplasms/pathology , Cell Movement , Cell Proliferation , DNA Methylation , Epigenomics , Esophageal Neoplasms/genetics , Humans , Myofibroblasts/metabolism , Stomach Neoplasms/genetics , Tumor Cells, Cultured , Tumor Microenvironment
6.
Nucleic Acids Res ; 45(8): 4756-4767, 2017 05 05.
Article in English | MEDLINE | ID: mdl-28132029

ABSTRACT

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated.


Subject(s)
Herpesvirus 8, Human/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , SOXB1 Transcription Factors/chemistry , Crystallography, X-Ray , Gene Expression , Herpesvirus 8, Human/genetics , Host-Pathogen Interactions/genetics , Humans , Life Cycle Stages/genetics , Protein Conformation , RNA, Messenger/genetics , SOXB1 Transcription Factors/genetics
7.
PLoS Pathog ; 12(11): e1005977, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27870901

ABSTRACT

Trichinella spiralis is a muscle-specific parasitic worm that is uniquely intracellular. T. spiralis reprograms terminally differentiated skeletal muscle cells causing them to de-differentiate and re-enter the cell cycle, a process that cannot occur naturally in mammalian skeletal muscle cells, but one that holds great therapeutic potential. Although the host ubiquitin pathway is a common target for viruses and bacteria during infection, its role in parasite pathogenesis has been largely overlooked. Here we demonstrate that the secreted proteins of T. spiralis contain E2 Ub-conjugating and E3 Ub-ligase activity. The E2 activity is attributed to TsUBE2L3, a novel and conserved T. spiralis enzyme located in the secretory organ of the parasite during the muscle stages of infection. TsUBE2L3 cannot function with any T.spiralis secreted E3, but specifically binds to a panel of human RING E3 ligases, including the RBR E3 ARIH2 with which it interacts with a higher affinity than the mammalian ortholog UbcH7/UBE2L3. Expression of TsUBE2L3 in skeletal muscle cells causes a global downregulation in protein ubiquitination, most predominantly affecting motor, sarcomeric and extracellular matrix proteins, thus mediating their stabilization with regards to proteasomal degradation. This effect is not observed in the presence of the mammalian ortholog, suggesting functional divergence in the evolution of the parasite protein. These findings demonstrate the first example of host-parasite interactions via a parasite-derived Ub conjugating enzyme; an E2 that demonstrates a novel muscle protein stabilization function.


Subject(s)
Helminth Proteins/metabolism , Host-Parasite Interactions/physiology , Muscle, Skeletal/pathology , Muscle, Skeletal/parasitology , Trichinellosis/enzymology , Ubiquitin-Conjugating Enzymes/metabolism , Animals , Chromatography, Liquid , HEK293 Cells , Humans , Immunoprecipitation , Rats , Rats, Sprague-Dawley , Tandem Mass Spectrometry , Trichinella spiralis , Ubiquitin , Ubiquitination/physiology
8.
Biochem Soc Trans ; 43(4): 621-6, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26551702

ABSTRACT

In most tissues, cells are exposed to frequent changes in levels of oxidative stress and inflammation. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor-κB (NF-κB) are the two key transcription factors that regulate cellular responses to oxidative stress and inflammation respectively. Pharmacological and genetic studies suggest that there is functional cross-talk between these two important pathways. The absence of Nrf2 can exacerbate NF-κB activity leading to increased cytokine production, whereas NF-κB can modulate Nrf2 transcription and activity, having both positive and negative effects on the target gene expression. This review focuses on the potentially complex molecular mechanisms that link the Nrf2 and NF-κB pathways and the importance of designing more effective therapeutic strategies to prevent or treat a broad range of neurological disorders.


Subject(s)
NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Nervous System Diseases/metabolism , Animals , Cytokines/metabolism , Gene Expression Regulation , Humans , Nervous System Diseases/drug therapy , Nervous System Diseases/genetics , Oxidative Stress , Signal Transduction
9.
Cell Cycle ; 14(6): 920-30, 2015.
Article in English | MEDLINE | ID: mdl-25590999

ABSTRACT

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5-6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.


Subject(s)
Carrier Proteins/metabolism , Multiprotein Complexes/metabolism , Neoplasm Proteins/metabolism , Peptide Fragments/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Chromatography, Liquid , HEK293 Cells , Humans , Immunoprecipitation , Models, Biological , Molecular Sequence Data , Neoplasm Proteins/chemistry , Peptide Fragments/chemistry , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Multimerization , RING Finger Domains , Tandem Mass Spectrometry , Tripartite Motif Proteins , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/chemistry
10.
Free Radic Biol Med ; 78: 202-12, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25445704

ABSTRACT

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.


Subject(s)
Apoptosis/drug effects , Carcinoma, Hepatocellular/drug therapy , Gene Expression Regulation/drug effects , Liver Neoplasms/drug therapy , NF-E2-Related Factor 2/antagonists & inhibitors , NF-E2-Related Factor 2/metabolism , Quassins/pharmacology , Animals , Autophagy , Blotting, Western , Brucea/chemistry , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Proliferation/drug effects , Cells, Cultured , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice , NF-E2-Related Factor 2/genetics , Oxidation-Reduction , Oxidative Stress , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects
11.
PeerJ ; 2: e618, 2014.
Article in English | MEDLINE | ID: mdl-25374775

ABSTRACT

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and Results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

12.
J Biol Chem ; 287(51): 43007-18, 2012 Dec 14.
Article in English | MEDLINE | ID: mdl-23105109

ABSTRACT

The opposing regulators of ubiquitylation status, E3 ligases and deubiquitylases, are often found to be associated in complexes. Here we report on a novel interaction between the E3 ligase BRAP (also referred to as IMP), a negative regulator of the MAPK scaffold protein KSR, and two closely related deubiquitylases, USP15 and USP4. We map the interaction to the N-terminal DUSP-UBL domain of USP15 and the coiled coil region of BRAP. USP15 as well as USP4 oppose the autoubiquitylation of BRAP, whereas BRAP promotes the ubiquitylation of USP15. Importantly, USP15 but not USP4 depletion destabilizes BRAP by promoting its proteasomal degradation, and BRAP-protein levels can be rescued by reintroducing catalytically active but not inactive mutant USP15. Unexpectedly, USP15 depletion results in a decrease in amplitude of MAPK signaling in response to EGF and PDGF. We provide evidence for a model in which the dominant effect of prolonged USP15 depletion upon signal amplitude is due to a decrease in CRAF levels while allowing for the possibility that USP15 may also function to dampen MAPK signaling through direct stabilization of a negative regulator, the E3 ligase BRAP.


Subject(s)
Endopeptidases/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Ubiquitin-Protein Ligases/metabolism , Biocatalysis/drug effects , Cell Line , Enzyme Activation/drug effects , Enzyme Stability/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Platelet-Derived Growth Factor/pharmacology , Proteasome Endopeptidase Complex/metabolism , Protein Binding/drug effects , Proteolysis/drug effects , Proto-Oncogene Proteins c-raf/genetics , RNA, Small Interfering/metabolism , Transcription, Genetic/drug effects , Two-Hybrid System Techniques , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Proteases , Ubiquitination/drug effects
13.
Dev Cell ; 22(5): 979-88, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22521722

ABSTRACT

VAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show that the linker of the δ-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to δ-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of δ-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type δ-adaptin but not by δ-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.


Subject(s)
Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex delta Subunits/metabolism , Protein Transport/physiology , R-SNARE Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Membrane/metabolism , Crystallography, X-Ray , Endocytosis , Endosomes/metabolism , Fibroblasts , Flow Cytometry , Humans , Mice , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary
14.
Biochem J ; 423(1): 31-9, 2009 Sep 14.
Article in English | MEDLINE | ID: mdl-19580544

ABSTRACT

The HSPs (hereditary spastic paraplegias) are genetic conditions in which there is distal degeneration of the longest axons of the corticospinal tract, resulting in spastic paralysis of the legs. The gene encoding spartin is mutated in Troyer syndrome, an HSP in which paralysis is accompanied by additional clinical features. There has been controversy over the subcellular distribution of spartin. We show here that, at steady state, endogenous spartin exists in a cytosolic pool that can be recruited to endosomes and to lipid droplets. Cytosolic endogenous spartin is mono-ubiquitinated and we demonstrate that it interacts via a PPXY motif with the ubiquitin E3 ligases AIP4 [atrophin-interacting protein 4; ITCH (itchy E3 ubiquitin protein ligase homologue] [corrected] and AIP5 (WWP1). Surprisingly, the PPXY motif, AIP4 and AIP5 are not required for spartin's ubiquitination, and so we propose that spartin acts as an adaptor for these proteins. Our results suggest that spartin is involved in diverse cellular functions, which may be of relevance to the complex phenotype seen in Troyer syndrome.


Subject(s)
Endosomes/metabolism , Lipid Metabolism , Proteins/metabolism , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Cycle Proteins , HeLa Cells , Humans , Lipid Metabolism/physiology , Liposomes/metabolism , Mice , PC12 Cells , Protein Binding , Rats , Tumor Cells, Cultured , Ubiquitination
15.
Genome Res ; 19(10): 1905-11, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19549727

ABSTRACT

In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3-CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a approximately 93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations.


Subject(s)
Metabolic Networks and Pathways , Ubiquitin-Conjugating Enzymes/metabolism , Amino Acid Sequence , False Positive Reactions , Humans , K562 Cells , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Interaction Domains and Motifs/genetics , Sequence Analysis, Protein/methods , Sequence Homology, Amino Acid , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/physiology , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
16.
Brief Funct Genomic Proteomic ; 8(1): 1-11, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19282470

ABSTRACT

One of the greatest challenges of the post-genomic era is the construction of a more comprehensive human protein interaction map. While this process may take many years to complete, the development of stringent high throughput techniques and the emergence of complementary assays mean that the aim of building a detailed binary map of the human interactome is now a very realistic goal. In particular, methods which facilitate the analysis of large numbers of membrane-protein interactions mean that it will be possible to construct more extensive networks, which in turn provide new insights into the functional connectivity between intra- and extra-cellular processes. This is important as many therapeutic strategies are designed to elicit effects via 'tractable' cell-surface proteins. Therefore, the construction of maps depicting the complexity of trans-cellular communication networks will not only improve our understanding of physiological processes, it will also aid the design of rational therapeutic strategies, with fewer potential side effects. This review aims to provide a basic insight into the approaches currently being used to construct binary human protein interaction networks, with particular reference to newer techniques, which have the potential to extend network coverage and aid the conditional annotation of interactome-scale protein interaction maps.


Subject(s)
Computational Biology/methods , Protein Interaction Mapping/instrumentation , Protein Interaction Mapping/methods , Proteins/metabolism , Proteomics , Gene Expression Regulation , Gene Regulatory Networks , Genome , Humans , Protein Binding/genetics , Proteins/genetics , Proteome/genetics , Proteome/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Software , Two-Hybrid System Techniques
17.
Cell ; 134(5): 817-27, 2008 Sep 05.
Article in English | MEDLINE | ID: mdl-18775314

ABSTRACT

SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Clathrin-Coated Vesicles/metabolism , R-SNARE Proteins/chemistry , R-SNARE Proteins/metabolism , Adaptor Proteins, Vesicular Transport/chemistry , Amino Acid Sequence , Animals , Cell Membrane/metabolism , Endocytosis , Humans , Mice , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Protein Transport , Two-Hybrid System Techniques
18.
Genome Res ; 18(4): 517-20, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18381898

ABSTRACT

Eukaryotic genomes encode large numbers of proteins that are either secreted or have exposed extracellular domains. It is highly likely that these proteins facilitate many important biological processes: however, as yet, most remain uncharacterized. Progress in this area of research has been impaired by the lack of a robust screening system that can be used to investigate interactions between large numbers of different extracellular proteins. In this issue, Bushell et al. introduce AVEXIS (avidity-based extracellular interaction screen), a high-throughput screening procedure, which can be used to identify even weak extracellular protein interactions with extremely high confidence. This assay represents an important development in the field of network biology. By combining data from the AVEXIS system with data produced by classical or variant yeast two-hybrid methods, it will be possible to assemble binary protein interaction networks that connect extracellular and intracellular processes. This information will dramatically increase our ability to understand a wide range of physiological processes and facilitate the development of better therapeutic strategies.


Subject(s)
Membrane Proteins/metabolism , Protein Interaction Mapping/methods , Humans
19.
J Biol Chem ; 282(42): 30929-37, 2007 Oct 19.
Article in English | MEDLINE | ID: mdl-17711858

ABSTRACT

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.


Subject(s)
Endopeptidases/metabolism , Endosomes/metabolism , ErbB Receptors/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Vesicular Transport Proteins/metabolism , Amino Acid Sequence/genetics , Endopeptidases/genetics , Endosomal Sorting Complexes Required for Transport , ErbB Receptors/genetics , HeLa Cells , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Protein Transport/physiology , RNA, Small Interfering/genetics , Sequence Deletion , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitination/physiology , Vesicular Transport Proteins/genetics
20.
Curr Biol ; 17(8): 711-6, 2007 Apr 17.
Article in English | MEDLINE | ID: mdl-17398095

ABSTRACT

The small GTPase Arf6 regulates endocytosis, actin dynamics, and cell adhesion, and one of its major activators is the exchange factor Arf nucleotide-binding site opener (ARNO), also called cytohesin-2 [1, 2]. ARNO must be recruited from the cytosol to the plasma membrane in order to activate Arf6, and in addition to a Sec7 nucleotide-exchange domain it contains a C-terminal pleckstrin homology (PH) domain that binds phosphoinositides [3, 4]. ARNO and its three relatives, cytohesin-1, Grp1/cytohesin-3, and cytohesin-4, are expressed as two splice variants, with either two or three glycines in a loop in the phosphoinositide-binding pocket of the PH domain [5, 6]. The diglycine form binds PtdIns(3,4,5)P(3) with high affinity and mediates recruitment of cytohesins to the plasma membrane in response to insulin and growth factors [7, 8]. However, the triglycine form has only micromolar affinity for both PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2), affinities that are insufficient to confer membrane recruitment, raising the question of how the triglycine forms of cytohesins are regulated [5, 9]. Here we show that three related Arf-like GTPases of unknown function, Arl4a, Arl4c, and Arl4d, are able to recruit ARNO and other cytohesins to the plasma membrane by binding to their PH domains irrespective of whether they are in the diglycine or triglycine form. The Arl4 family thus defines a signal-transduction pathway that can mediate the plasma-membrane recruitment of cytohesins independently of a requirement for the generation of PtdIns(3,4,5)P(3).


Subject(s)
ADP-Ribosylation Factors/metabolism , Cell Membrane/metabolism , GTPase-Activating Proteins/metabolism , Amino Acid Sequence , DNA, Complementary , HeLa Cells , Humans , Molecular Sequence Data , Protein Isoforms/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Two-Hybrid System Techniques
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