Protocol for using TRIBE to study RNA-protein interactions and nuclear organization in mammalian cells

β-Catenin functions as an adherens junction protein for cell–cell adhesion and as a signaling protein. β-catenin function is dependent on its stability, which is regulated by protein–protein interactions that stabilize β-catenin or target it for proteasome-mediated degradation. In this study, we show that β-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster. β-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase β-TrCP. While β-catenin phosphorylation was pH independent, higher pHi induced increased β-TrCP binding and decreased β-catenin stability. An evolutionarily conserved histidine in β-catenin (found in the β-TrCP DSGIHS destruction motif) is required for pH-dependent binding to β-TrCP. Expressing a cancer-associated H36R–β-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic β-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of β-catenin stability, functioning in coincidence with phosphorylation.

Download Full-text

TULP1 and TUB Are Required for Specific Localization of PRCD to Photoreceptor Outer Segments

International Journal of Molecular Sciences ◽

10.3390/ijms21228677 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8677

Author(s):

Lital Remez ◽

Ben Cohen ◽

Mariela J. Nevet ◽

Leah Rizel ◽

Tamar Ben-Yosef

Keyword(s):

Protein Interactions ◽

Mammalian Cells ◽

Outer Segment ◽

Protein Protein Interactions ◽

Photoreceptor Outer Segment ◽

Photoreceptor Outer Segments ◽

Outer Segments ◽

Yeast Two Hybrid System ◽

Specific Localization ◽

Recruitment System

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

Download Full-text

Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0701987104 ◽

2007 ◽

Vol 104 (24) ◽

pp. 10264-10269 ◽

Cited By ~ 87

Author(s):

X. Xu ◽

M. Soutto ◽

Q. Xie ◽

S. Servick ◽

C. Subramanian ◽

...

Keyword(s):

Energy Transfer ◽

Protein Interactions ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bioluminescence Resonance Energy Transfer

Download Full-text

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.114.10.1893 ◽

2001 ◽

Vol 114 (10) ◽

pp. 1893-1900 ◽

Cited By ~ 3

Author(s):

S. Lusa ◽

T.S. Blom ◽

E.L. Eskelinen ◽

E. Kuismanen ◽

J.E. Mansson ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Interactions ◽

Mammalian Cells ◽

Intracellular Transport ◽

Ldl Cholesterol ◽

Normal Cells ◽

Recycling Endosomes ◽

Regulate Protein ◽

Niemann Pick ◽

Accumulation Characteristic

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

Download Full-text

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

Molecular and Cellular Biology ◽

10.1128/mcb.00876-15 ◽

2015 ◽

Vol 36 (5) ◽

pp. 794-808 ◽

Cited By ~ 17

Author(s):

Sarah M. Pedersen ◽

Waipan Chan ◽

Rakhi P. Jattani ◽

deMauri S. Mackie ◽

Joel L. Pomerantz

Keyword(s):

Ubiquitin Ligase ◽

Protein Interactions ◽

Mammalian Cells ◽

E3 Ubiquitin Ligase ◽

Antigen Receptor ◽

Resonance Energy ◽

B Cell Lymphoma ◽

Receptor Signaling ◽

Antigen Receptor Signaling

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activityin vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.

Download Full-text

Methionine adenosyltransferases in cancers: Mechanisms of dysregulation and implications for therapy

Experimental Biology and Medicine ◽

10.1177/1535370217740860 ◽

2017 ◽

Vol 243 (2) ◽

pp. 107-117 ◽

Cited By ~ 15

Author(s):

Lauren Y Maldonado ◽

Diana Arsene ◽

José M Mato ◽

Shelly C Lu

Keyword(s):

Protein Interactions ◽

Mammalian Cells ◽

Human Cancer ◽

Methionine Adenosyltransferase ◽

Therapeutic Interventions ◽

Cancer Development ◽

Growth And Survival ◽

Liver Cancers ◽

Mat Genes

Methionine adenosyltransferase genes encode enzymes responsible for the biosynthesis of S-adenosylmethionine, the principal biological methyl donor and precursor of polyamines and glutathione. Mammalian cells express three genes – MAT1A, MAT2A, and MAT2B – with distinct expression and functions. MAT1A is mainly expressed in the liver and maintains the differentiated states of both hepatocytes and bile duct epithelial cells. Conversely, MAT2A and MAT2B are widely distributed in non-parenchymal cells of the liver and extrahepatic tissues. Increasing evidence suggests that methionine adenosyltransferases play significant roles in the development of cancers. Liver cancers, namely hepatocellular carcinoma and cholangiocarcinoma, involve dysregulation of all three methionine adenosyltransferase genes. MAT1A reduction is associated with increased oxidative stress, progenitor cell expansion, genomic instability, and other mechanisms implicated in tumorigenesis. MAT2A/MAT2B induction confers growth and survival advantage to cancerous cells, enhancing tumor migration. Highlighted examples from colon, gastric, breast, pancreas and prostate cancer studies further underscore methionine adenosyltransferase genes’ role beyond the liver in cancer development. In this subset of extra-hepatic cancers, MAT2A and MAT2B are induced via different regulatory mechanisms. Understanding the role of methionine adenosyltransferase genes in tumorigenesis helps identify attributes of these genes that may serve as valuable targets for therapy. While S-adenosylmethionine, and its metabolite, methylthioadenosine, have been largely explored as therapeutic interventions, targets aimed at regulation of MAT gene expression and methionine adenosyltransferase protein–protein interactions are now surfacing as potential effective strategies for treatment and chemoprevention of cancers. Impact statement This review examines the role of methionine adenosyltransferases (MATs) in human cancer development, with a particular focus on liver cancers in which all three MAT genes are implicated in tumorigenesis. An overview of MAT genes, isoenzymes and their regulation provide context for understanding consequences of dysregulation. Highlighting examples from liver, colon, gastric, breast, pancreas and prostate cancers underscore the importance of understanding MAT’s tumorigenic role in identifying future targets for cancer therapy.

Download Full-text

Mapping Transient Protein Interactions at the Nanoscale in Living Mammalian Cells

ACS Nano ◽

10.1021/acsnano.8b01227 ◽

2018 ◽

Vol 12 (10) ◽

pp. 9842-9854 ◽

Cited By ~ 8

Author(s):

Herlinde De Keersmaecker ◽

Rafael Camacho ◽

David Manuel Rantasa ◽

Eduard Fron ◽

Hiroshi Uji-i ◽

...

Keyword(s):

Protein Interactions ◽

Mammalian Cells

Download Full-text

Shaping the regulation of the p53 mRNA tumour suppressor: the co-evolution of genetic signatures

BMC Cancer ◽

10.1186/s12885-019-6118-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Konstantinos Karakostis ◽

Robin Fåhraeus

Keyword(s):

Protein Interactions ◽

Rna Structure ◽

Mammalian Cells ◽

Rna World ◽

Genetic Diseases ◽

Protein Protein Interactions ◽

Activation Domain ◽

Interacting Protein ◽

Protein Motifs ◽

Synonymous Mutations

Abstract Structured RNA regulatory motifs exist from the prebiotic stages of the RNA world to the more complex eukaryotic systems. In cases where a functional RNA structure is within the coding sequence a selective pressure drives a parallel co-evolution of the RNA structure and the encoded peptide domain. The p53-MDM2 axis, describing the interactions between the p53 tumor suppressor and the MDM2 E3 ubiquitin ligase, serves as particularly useful model revealing how secondary RNA structures have co-evolved along with corresponding interacting protein motifs, thus having an impact on protein – RNA and protein – protein interactions; and how such structures developed signal-dependent regulation in mammalian systems. The p53(BOX-I) RNA sequence binds the C-terminus of MDM2 and controls p53 synthesis while the encoded peptide domain binds MDM2 and controls p53 degradation. The BOX-I peptide domain is also located within p53 transcription activation domain. The folding of the p53 mRNA structure has evolved from temperature-regulated in pre-vertebrates to an ATM kinase signal-dependent pathway in mammalian cells. The protein – protein interaction evolved in vertebrates and became regulated by the same signaling pathway. At the same time the protein - RNA and protein - protein interactions evolved, the p53 trans-activation domain progressed to become integrated into a range of cellular pathways. We discuss how a single synonymous mutation in the BOX-1, the p53(L22 L), observed in a chronic lymphocyte leukaemia patient, prevents the activation of p53 following DNA damage. The concepts analysed and discussed in this review may serve as a conceptual mechanistic paradigm of the co-evolution and function of molecules having roles in cellular regulation, or the aetiology of genetic diseases and how synonymous mutations can affect the encoded protein.

Download Full-text