57 results on '"Prag G"'
Search Results
2. STRUCTURE OF UBIQUITYLATED-RPN10 FROM YEAST;
- Author
-
Keren-Kaplan, T., primary, Attali, I., additional, Levin-Kravets, O., additional, and Prag, G., additional
- Published
- 2016
- Full Text
- View/download PDF
3. CRYSTAL STRUCTURE OF THE ZEBRA FISH ENTH DOMAIN FROM EPSIN1 IN 1.41 ANGSTROM RESOLUTION
- Author
-
Levin-Kravets, O., primary and Prag, G., additional
- Published
- 2016
- Full Text
- View/download PDF
4. STRUCTURE OF YEAST ENT1 ENTH DOMAIN
- Author
-
Tanner, N., primary and Prag, G., additional
- Published
- 2016
- Full Text
- View/download PDF
5. A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation
- Author
-
Langowski, J, Zhao, H, Ghirlando, R, Alfonso, C, Arisaka, F, Attali, I, Bain, DL, Bakhtina, MM, Becker, DF, Bedwell, GJ, Bekdemir, A, Besong, TMD, Birck, C, Brautigam, CA, Brennerman, W, Byron, O, Bzowska, A, Chaires, JB, Chaton, CT, Coelfen, H, Connaghan, KD, Crowley, KA, Curth, U, Daviter, T, Dean, WL, Diez, AI, Ebel, C, Eckert, DM, Eisele, LE, Eisenstein, E, England, P, Escalante, C, Fagan, JA, Fairman, R, Finn, RM, Fischle, W, Garcia de la Torre, J, Gor, J, Gustafsson, H, Hall, D, Harding, SE, Hernandez Cifre, JG, Herr, AB, Howell, EE, Isaac, RS, Jao, S-C, Jose, D, Kim, S-J, Kokona, B, Kornblatt, JA, Kosek, D, Krayukhina, E, Krzizike, D, Kusznir, EA, Kwon, H, Larson, A, Laue, TM, Le Roy, A, Leech, AP, Lilie, H, Luger, K, Luque-Ortega, JR, Ma, J, May, CA, Maynard, EL, Modrak-Wojcik, A, Mok, Y-F, Muecke, N, Nagel-Steger, L, Narlikar, GJ, Noda, M, Nourse, A, Obsil, T, Park, CK, Park, J-K, Pawelek, PD, Perdue, EE, Perkins, SJ, Perugini, MA, Peterson, CL, Peverelli, MG, Piszczek, G, Prag, G, Prevelige, PE, Raynal, BDE, Rezabkova, L, Richter, K, Ringel, AE, Rosenberg, R, Rowe, AJ, Rufer, AC, Scott, DJ, Seravalli, JG, Solovyova, AS, Song, R, Staunton, D, Stoddard, C, Stott, K, Strauss, HM, Streicher, WW, Sumida, JP, Swygert, SG, Szczepanowski, RH, Tessmer, I, Toth, RT, Tripathy, A, Uchiyama, S, Uebel, SFW, Unzai, S, Gruber, AV, von Hippel, PH, Wandrey, C, Wang, S-H, Weitzel, SE, Wielgus-Kutrowska, B, Wolberger, C, Wolff, M, Wright, E, Wu, Y-S, Wubben, JM, Schuck, P, Langowski, J, Zhao, H, Ghirlando, R, Alfonso, C, Arisaka, F, Attali, I, Bain, DL, Bakhtina, MM, Becker, DF, Bedwell, GJ, Bekdemir, A, Besong, TMD, Birck, C, Brautigam, CA, Brennerman, W, Byron, O, Bzowska, A, Chaires, JB, Chaton, CT, Coelfen, H, Connaghan, KD, Crowley, KA, Curth, U, Daviter, T, Dean, WL, Diez, AI, Ebel, C, Eckert, DM, Eisele, LE, Eisenstein, E, England, P, Escalante, C, Fagan, JA, Fairman, R, Finn, RM, Fischle, W, Garcia de la Torre, J, Gor, J, Gustafsson, H, Hall, D, Harding, SE, Hernandez Cifre, JG, Herr, AB, Howell, EE, Isaac, RS, Jao, S-C, Jose, D, Kim, S-J, Kokona, B, Kornblatt, JA, Kosek, D, Krayukhina, E, Krzizike, D, Kusznir, EA, Kwon, H, Larson, A, Laue, TM, Le Roy, A, Leech, AP, Lilie, H, Luger, K, Luque-Ortega, JR, Ma, J, May, CA, Maynard, EL, Modrak-Wojcik, A, Mok, Y-F, Muecke, N, Nagel-Steger, L, Narlikar, GJ, Noda, M, Nourse, A, Obsil, T, Park, CK, Park, J-K, Pawelek, PD, Perdue, EE, Perkins, SJ, Perugini, MA, Peterson, CL, Peverelli, MG, Piszczek, G, Prag, G, Prevelige, PE, Raynal, BDE, Rezabkova, L, Richter, K, Ringel, AE, Rosenberg, R, Rowe, AJ, Rufer, AC, Scott, DJ, Seravalli, JG, Solovyova, AS, Song, R, Staunton, D, Stoddard, C, Stott, K, Strauss, HM, Streicher, WW, Sumida, JP, Swygert, SG, Szczepanowski, RH, Tessmer, I, Toth, RT, Tripathy, A, Uchiyama, S, Uebel, SFW, Unzai, S, Gruber, AV, von Hippel, PH, Wandrey, C, Wang, S-H, Weitzel, SE, Wielgus-Kutrowska, B, Wolberger, C, Wolff, M, Wright, E, Wu, Y-S, Wubben, JM, and Schuck, P
- Abstract
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
- Published
- 2015
6. Non-covalent assembly of monoubiquitin that mimics K11 poly-ubiquitin
- Author
-
Levin-Kravets, O., primary and Prag, G., additional
- Published
- 2015
- Full Text
- View/download PDF
7. S5A HOMOLOG
- Author
-
Keren-Kaplan, T., primary and Prag, G., additional
- Published
- 2015
- Full Text
- View/download PDF
8. High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis
- Author
-
Papanikolau, Y Prag, G Tavlas, G Vorgias, CE Oppenheim, AB Petratos, K
- Abstract
Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic enzyme, which cleaves beta -1,4-glycosidic bonds of the natural biopolymer chitin to generate di-N-acetyl-chitobiose. The refined structure of ChiA at 1.55 Angstrom shows that residue Asp313, which is located near the catalytic proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa- N-acetyl-glucosamine have been refined at high resolution and the interactions with the substrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical “chair” to “boat” conformational change on the sugar residue bound to the - 1 subsite. According to our results, we suggest that residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosidic bond, Asp313 that interacts with Asp311 flips to its alternative position where it interacts with Glu315 thus forcing the substrate acetamido group of - 1 sugar to rotate around the C2-N2 bond. As a result of these structural changes, the water molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido group is displaced to a position that allows the completion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed “substrate assisted” catalysis.
- Published
- 2001
9. Structures of chitobiase mutants complexed with the substrate di-N-acetyl-D-glucosamine: The catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540
- Author
-
Prag, G Papanikolau, Y Tavlas, G Vorgias, CE Petratos, K and Oppenheim, AB
- Abstract
The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from Serratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to family 20 of glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton donor and by the acetamido group of the substrate as a nucleophile. We investigated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemical characterization and by structural analyses of chitobiase substrate co-crystals. We found that both residues are essential for chitoblase activity. The mutations, however, led to subtle changes in the catalytic site. Our results support the model that Glu540 acts as the proton donor and that Asp539 acts in several different ways. Asp539 restrains the acetamido group of the substrate in a specific orientation by forming a hydrogen bond with N2 of the non-reduced (-1) sugar. In addition, this residue participates in substrate binding. It is also required for the correct positioning of Glu540 and may provide additional negative charge at the active site. Thus, these biochemical and structural studies provide a molecular explanation for the functional importance and conservation of these residues. (C) 2000 Academic Press.
- Published
- 2000
10. Structural studies of chitinase A from S. marcescens complexed with substrates and the inhibitor
- Author
-
Papanikolau, Y. Prag, G. Tavlas, G. Papadovasilaki, M. and Dimitriou, S. Vorgias, C. E. Oppenheim, A. B. Petratos, K.
- Published
- 2000
11. The Vps27/Hse1 complex is a GAT domain-based scaffold for ubiquitin-dependent sorting
- Author
-
Prag, G., primary and Hurley, J.H., additional
- Published
- 2007
- Full Text
- View/download PDF
12. Mechanism of Ubiquitin Recognition by the CUE Domain of VPS9
- Author
-
Prag, G., primary, Misra, S., additional, Jones, E.A., additional, Ghirlando, R., additional, Davies, B.A., additional, Horazdovsky, B.F., additional, and Hurley, J.H., additional
- Published
- 2003
- Full Text
- View/download PDF
13. CRYSTAL STRUCTURE OF CHITINASE A MUTANT Y390F COMPLEXED WITH HEXA-N-ACETYLCHITOHEXAOSE (NAG)6
- Author
-
Papanikolau, Y., primary, Prag, G., additional, Tavlas, G., additional, Vorgias, C.E., additional, Oppenheim, A.B., additional, and Petratos, K., additional
- Published
- 2001
- Full Text
- View/download PDF
14. Structural studies of chitinase A fromS. marcescenscomplexed with substrates and the inhibitor allosamidin
- Author
-
Papanikolau, Y., primary, Prag, G., additional, Tavlas, G., additional, Papadovasilaki, M., additional, Dimitriou, S., additional, Vorgias, C. E., additional, Oppenheim, A. B., additional, and Petratos, K., additional
- Published
- 2000
- Full Text
- View/download PDF
15. Cryoextraction: A novel approach to remove aspirated chewing gum
- Author
-
Edmundo Rubio, Prag Gupta, Susanti Ie, and Michael Boyd
- Subjects
Bronchoscopy ,cryotherapy ,cryoextraction ,foreign body ,gum ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 ,Diseases of the respiratory system ,RC705-779 - Abstract
The extraction of aspirated foreign bodies can prove challenging at times, requiring even rigid bronchoscopy. Cryotherapy probes have been reported to help with extraction of foreign bodies. We present a case where successful "cryoextraction" was performed on an aspirated chewing gum. The case highlights the fact that this technique is useful to extract all materials that have water content. This technique can be performed through flexible bronchoscopy and can save patients from more aggressive approaches.
- Published
- 2013
- Full Text
- View/download PDF
16. TMEM16F regulates pathologic α-synuclein secretion and spread in cellular and mouse models of Parkinson's disease.
- Author
-
Cohen-Adiv S, Amer-Sarsour F, Berdichevsky Y, Boxer E, Goldstein O, Gana-Weisz M, Tripathi U, Rike WA, Prag G, Gurevich T, Giladi N, Stern S, Orr-Urtreger A, Friedmann-Morvinski D, and Ashkenazi A
- Abstract
One of the main hallmarks of Parkinson's disease (PD) pathology is the spread of the aggregate-prone protein α-synuclein (α-syn), which can be detected in the plasma and cerebrospinal fluid of patients as well as in the extracellular environment of neuronal cells. The secreted α-syn can exhibit "prion-like" behavior and transmission to naïve cells can promote conformational changes and pathology. The precise role of plasma membrane proteins in the pathologic process of α-syn is yet to be fully resolved. The TMEM16 family of lipid scramblases and ion channels has been recently associated with cancer and infectious diseases but is less known for its role in aging-related diseases. To elucidate the role of TMEM16F in α-syn spread, we transduced neurons derived from TMEM16F knockout mice with a reporter system that enables the distinction between donor and recipient neurons of pathologic α-synA53T. We found that the spread of α-synA53T was reduced in neurons derived from TMEM16F-knockout mice. These findings were recapitulated in vivo in a mouse model of PD, where attenuated α-synA53T spread was observed when TMEM16F was ablated. Moreover, we identified a single nucleotide polymorphism in TMEM16F of Ashkenazi Jewish PD patients resulting in a missense Ala703Ser mutation with enhanced lipid scramblase activity. This mutation is associated with altered regulation of α-synA53T extracellular secretion in cellular models of PD. Our study highlights TMEM16F as a novel regulator of α-syn spread and as a potential therapeutic target in synucleinopathies., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)
- Published
- 2024
- Full Text
- View/download PDF
17. Dithiothreitol-based protein equalisation in the context of multiple myeloma: Enhancing proteomic analysis and therapeutic insights.
- Author
-
Domingos IF, Carvalho LB, Lodeiro C, Gerivaz R, Prag G, Micaglio E, Muchtar E, Santos HM, and Capelo JL
- Subjects
- Humans, Proteome analysis, Proteome metabolism, Multiple Myeloma drug therapy, Multiple Myeloma metabolism, Dithiothreitol chemistry, Proteomics methods
- Abstract
In this study, we employed the dithiothreitol-based protein equalisation technique and analytical proteomics to better understand myeloma diseases by comparing the proteomes of pellets and supernatants formed upon application of DTT on serum samples. The number of unique proteins found in pellets was 252 for healthy individuals and 223 for multiple myeloma patients. The comparison of these proteomes showed 97 dysregulated proteins. The unique proteins found for supernatants were 264 for healthy individuals and 235 for multiple myeloma patients. The comparison of these proteomes showed 87 dysregulated proteins. The analytical proteomic comparison of both groups of dysregulated proteins is translated into parallel dysregulated pathways, including chaperone-mediated autophagy and protein folding, addressing potential therapeutic interventions. Future research endeavours in personalised medicine should prioritize refining analytical proteomic methodologies using serum dithiothreitol-based protein equalisation to explore innovative therapeutic strategies. We highlight the advanced insights gained from this analytical strategy in studying multiple myeloma, emphasising its complex nature and the critical role of personalised, targeted analytical techniques in enhancing therapeutic efficacy in personalised medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
18. Proximal Co-Translation Facilitates Detection of Weak Protein-Protein Interactions.
- Author
-
Kordonsky A, Gabay M, Rosinoff A, Avishid R, Flornetin A, Deouell N, Abd Alkhaleq T, Efron N, Milshtein S, Shifman JM, Gal M, and Prag G
- Subjects
- Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae genetics, Protein Interaction Mapping methods, Protein Interaction Domains and Motifs, Protein Domains, Binding Sites, Protein Binding, Saccharomyces cerevisiae Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Protein Biosynthesis, Ubiquitin metabolism
- Abstract
Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein-protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions.
- Published
- 2024
- Full Text
- View/download PDF
19. RING/U-box E3 protein BIR1 interacts with and ubiquitinates barley growth repressor BROAD LEAF1.
- Author
-
Soltani O, Jöst M, Hoffie I, Hensel G, Kappel C, Prag G, McKim S, Kumlehn J, and Lenhard M
- Subjects
- Gene Expression Regulation, Plant, Protein Binding, Two-Hybrid System Techniques, Plants, Genetically Modified, Proteolysis, Cell Nucleus metabolism, Hordeum metabolism, Hordeum genetics, Hordeum growth & development, Plant Proteins metabolism, Plant Proteins genetics, Ubiquitination, Proteasome Endopeptidase Complex metabolism, Ubiquitin-Protein Ligases metabolism, Ubiquitin-Protein Ligases genetics, Plant Leaves metabolism, Plant Leaves growth & development, Plant Leaves genetics
- Abstract
Establishment of final leaf size in plants relies on the precise regulation of 2 interconnected processes, cell division and cell expansion. The barley (Hordeum vulgare) protein BROAD LEAF1 (BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here, we used a yeast 2-hybrid screen to identify the BLF1-INTERACTING RING/U-BOX 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the 2 proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP-expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knockout mutants suggested the involvement of BIR1 in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)
- Published
- 2024
- Full Text
- View/download PDF
20. A deleterious variant of INTS1 leads to disrupted sleep-wake cycles.
- Author
-
Confino S, Wexler Y, Medvetzky A, Elazary Y, Ben-Moshe Z, Reiter J, Dor T, Edvardson S, Prag G, Harel T, and Gothilf Y
- Subjects
- Animals, Child, Female, Humans, Male, Amino Acid Sequence, Larva genetics, Pedigree, Wakefulness physiology, Wakefulness genetics, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Circadian Rhythm genetics, Sleep genetics, Sleep physiology, Zebrafish genetics
- Abstract
Sleep disturbances are common among children with neurodevelopmental disorders. Here, we report a syndrome characterized by prenatal microcephaly, intellectual disability and severe disruption of sleep-wake cycles in a consanguineous family. Exome sequencing revealed homozygous variants (c.5224G>A and c.6506G>T) leading to the missense mutations E1742K and G2169V in integrator complex subunit 1 (INTS1), the core subunit of the Integrator complex. Conservation and structural analyses suggest that G2169V has a minor impact on the structure and function of the complex, while E1742K significantly alters a negatively charged conserved patch on the surface of the protein. The severe sleep-wake cycles disruption in human carriers highlights a new aspect of Integrator complex impairment. To further study INTS1 pathogenicity, we generated Ints1-deficient zebrafish lines. Mutant zebrafish larvae displayed abnormal circadian rhythms of locomotor activity and sleep, as is the case with the affected humans. Furthermore, Ints1-deficent larvae exhibited elevated levels of dopamine β-hydroxylase (dbh) mRNA in the locus coeruleus, a wakefulness-inducing brainstem center. Altogether, these findings suggest a significant, likely indirect, effect of INTS1 and the Integrator complex on maintaining circadian rhythms of locomotor activity and sleep homeostasis across vertebrates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)
- Published
- 2024
- Full Text
- View/download PDF
21. K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins.
- Author
-
Magits W, Steklov M, Jang H, Sewduth RN, Florentin A, Lechat B, Sheryazdanova A, Zhang M, Simicek M, Prag G, Nussinov R, and Sablina A
- Subjects
- Humans, Signal Transduction, Protein Serine-Threonine Kinases metabolism, Protein Serine-Threonine Kinases genetics, Animals, p120 GTPase Activating Protein metabolism, p120 GTPase Activating Protein genetics, Mice, Cell Line, Tumor, GTP Phosphohydrolases metabolism, GTP Phosphohydrolases genetics, Lysine metabolism, Membrane Proteins metabolism, Membrane Proteins genetics, ras Proteins metabolism, ras Proteins genetics, Neurofibromin 1, Ubiquitination, Proto-Oncogene Proteins p21(ras) metabolism, Proto-Oncogene Proteins p21(ras) genetics, Protein Binding
- Abstract
The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
22. CRL4 DCAF1 ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication.
- Author
-
Grossmann J, Kratz AS, Kordonsky A, Prag G, and Hoffmann I
- Subjects
- Humans, beta-Transducin Repeat-Containing Proteins genetics, beta-Transducin Repeat-Containing Proteins metabolism, Cell Cycle, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Centrioles metabolism, Ubiquitin metabolism
- Abstract
Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4
DCAF1 , E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFβ-TrCP , the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of β-TrCP. Thus, the SCFSlimb/β-TrCP pathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1 , which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication., (© 2024 Grossmann et al.)- Published
- 2024
- Full Text
- View/download PDF
23. Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.
- Author
-
Amer-Sarsour F, Falik D, Berdichevsky Y, Kordonsky A, Eid S, Rabinski T, Ishtayeh H, Cohen-Adiv S, Braverman I, Blumen SC, Laviv T, Prag G, Vatine GD, and Ashkenazi A
- Subjects
- Humans, Animals, Mice, Ubiquitination, Mutation, Ubiquitin genetics, Ubiquitin metabolism, Ubiquitin-Activating Enzymes genetics, Ubiquitin-Activating Enzymes metabolism, Peptides
- Abstract
Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
24. Physiological Functions of the Ubiquitin Ligases Nedd4-1 and Nedd4-2.
- Author
-
Rotin D and Prag G
- Subjects
- Humans, Nedd4 Ubiquitin Protein Ligases genetics, Nedd4 Ubiquitin Protein Ligases metabolism, Ubiquitin-Protein Ligases metabolism, Ubiquitination, Endosomal Sorting Complexes Required for Transport genetics, Endosomal Sorting Complexes Required for Transport metabolism, Ubiquitin metabolism
- Abstract
The Nedd4 family of E3 ubiquitin ligases, consisting of a C2-WW(n)-HECT domain architecture, includes the closely related Nedd4/Nedd4-1 and Nedd4L/Nedd4-2, which play critical roles in human physiology and pathophysiology.This review focuses on the regulation of enzymatic activity of these Nedd4 proteins, as well as on their roles in regulating stability and function of membrane and other signaling proteins, such as ion channels, ion transporters, and growth factor receptors. The diseases caused by impairment of such regulation are discussed, as well as opportunities and challenges for targeting these enzymes for therapy.
- Published
- 2024
- Full Text
- View/download PDF
25. Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli .
- Author
-
Florentin A, Kordonsky A, Yariv E, Avishid R, Efron N, Akogwu E, and Prag G
- Abstract
Protein-protein interactions and protein modifications play central roles in all living organisms. Of the more than 200 types of post-translational modifications, ubiquitylation is the most abundant, and it profoundly regulates the functionality of the eukaryotic proteome. Various in vitro and in vivo methodologies to study protein interactions and modifications have been developed, each presenting distinctive benefits and caveats. Here, we present a comprehensive protocol for applying a split-Chloramphenicol Acetyl-Transferase (split-CAT) based system, to study protein-protein interactions and ubiquitylation in E. coli . Functional assembly of bait and prey proteins tethered to the split-CAT fragments result in antibiotic resistance and growth on selective media. We demonstrate assays for protein interactions, protein ubiquitylation, and the system response to small compound modulators. To facilitate data collection, we provide an updated Scanner Acquisition Manager Program for Laboratory Experiments (SAMPLE; https://github.com/PragLab/SAMPLE ) that can be employed to monitor the growth of various microorganisms, including E. coli and S. cerevisiae . The advantage posed by this system lies in its sensitivity to a wide range of chloramphenicol concentrations, which allows the detection of a large spectrum of protein-protein interactions, without the need for their purification. The tight linkage between binding or ubiquitylation and growth enables the estimation of apparent relative affinity, and represents the system's quantitative characteristics. Graphical abstract., Competing Interests: Competing interests GP has equity in Coltac therapeutics LTD, other authors declare no competing interests. Company and patents: Patents: US10982252, US20200385706, and US20210356467., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)
- Published
- 2022
- Full Text
- View/download PDF
26. Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.
- Author
-
Levin-Kravets O, Kordonsky A, Shusterman A, Biswas S, Persaud A, Elias S, Langut Y, Florentin A, Simpson-Lavy KJ, Yariv E, Avishid R, Sror M, Almog O, Marshanski T, Kadosh S, Ben David N, Manori B, Fischer Z, Lilly J, Borisova E, Ambrozkiewicz MC, Tarabykin V, Kupiec M, Thaker M, Rotin D, and Prag G
- Subjects
- Enzyme Activation, Escherichia coli genetics, Escherichia coli metabolism, Protein Processing, Post-Translational, Proteolysis, Biological Assay methods, Chloramphenicol O-Acetyltransferase genetics, Chloramphenicol O-Acetyltransferase metabolism, Gene Expression, Genes, Reporter, Ubiquitin-Protein Ligases metabolism, Ubiquitination
- Abstract
Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
27. Deubiquitylating enzymes in neuronal health and disease.
- Author
-
Amer-Sarsour F, Kordonsky A, Berdichevsky Y, Prag G, and Ashkenazi A
- Subjects
- Humans, Neurodegenerative Diseases genetics, Ubiquitination genetics
- Abstract
Ubiquitylation and deubiquitylation play a pivotal role in protein homeostasis (proteostasis). Proteostasis shapes the proteome landscape in the human brain and its impairment is linked to neurodevelopmental and neurodegenerative disorders. Here we discuss the emerging roles of deubiquitylating enzymes in neuronal function and survival. We provide an updated perspective on the genetics, physiology, structure, and function of deubiquitylases in neuronal health and disease.
- Published
- 2021
- Full Text
- View/download PDF
28. HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy.
- Author
-
Elias A, Kassis H, Elkader SA, Gritsenko N, Nahmad A, Shir H, Younis L, Shannan A, Aihara H, Prag G, Yagil E, and Kolot M
- Subjects
- Bacteriophage HK022 enzymology, DNA Nucleotidyltransferases genetics, Genome, Human genetics, Humans, Bacteriophage HK022 genetics, Genetic Therapy, Integrases genetics, Recombination, Genetic genetics
- Abstract
HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2020
- Full Text
- View/download PDF
29. An Integrative Synthetic Biology Approach to Interrogating Cellular Ubiquitin and Ufm Signaling.
- Author
-
Li C, Han T, Guo R, Chen P, Peng C, Prag G, and Hu R
- Subjects
- Cell Line, Cloning, Molecular, HEK293 Cells, HeLa Cells, Humans, Open Reading Frames, Protein Processing, Post-Translational, Signal Transduction, Two-Hybrid System Techniques, Ubiquitin-Protein Ligases genetics, Synthetic Biology methods, Ubiquitin metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Global identification of substrates for PTMs (post-translational modifications) represents a critical but yet dauntingly challenging task in understanding biology and disease pathology. Here we presented a synthetic biology approach, namely 'YESS', which coupled Y2H (yeast two hybrid) interactome screening with PTMs reactions reconstituted in bacteria for substrates identification and validation, followed by the functional validation in mammalian cells. Specifically, the sequence-independent Gateway
® cloning technique was adopted to afford simultaneous transfer of multiple hit ORFs (open reading frames) between the YESS sub-systems. In proof-of-evidence applications of YESS, novel substrates were identified for UBE3A and UFL1, the E3 ligases for ubiquitination and ufmylation, respectively. Therefore, the YESS approach could serve as a potentially powerful tool to study cellular signaling mediated by different PTMs.- Published
- 2020
- Full Text
- View/download PDF
30. Ubiquitin Signaling and Degradation of Aggregate-Prone Proteins.
- Author
-
Galves M, Rathi R, Prag G, and Ashkenazi A
- Subjects
- Animals, Humans, Models, Molecular, Protein Aggregates, Proteins chemistry, Proteins genetics, Proteolysis, Signal Transduction, Ubiquitin chemistry, Proteins metabolism, Ubiquitin metabolism
- Abstract
Mutant protein aggregation and misfolding is often correlated with toxicity in neurodegenerative diseases. Aggregate-prone proteins are tagged by ubiquitin that signals them for destruction by the proteasome or autophagy, two key pathways for protein degradation and proteostasis. Here, we review recent studies showing that the regulation of aggregate-prone proteins by ubiquitin signaling is more complex than initially postulated. We discuss how the ubiquitin code of aggregate-prone proteins is written by specific E3 ubiquitin ligases and edited by deubiquitylating enzymes (DUBs) in cells and in brain tissues, as well as how this affects protein degradation. These studies have advanced our understanding of the specificity of the ubiquitin system and provide new information about its relevance to neurodegenerative diseases and therapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
31. Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.
- Author
-
Volpe M, Levinton N, Rosenstein N, Prag G, and Ben-Aroya S
- Subjects
- Anaphase-Promoting Complex-Cyclosome genetics, Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome genetics, Cell Cycle, Gene Expression Regulation, Fungal, Humans, Protein Binding, Saccharomyces cerevisiae Proteins genetics, Substrate Specificity, Ubiquitin metabolism, Ubiquitination, Anaphase-Promoting Complex-Cyclosome metabolism, Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
One of the challenges encountered by the protein quality control machinery is the need to ensure that members of multiprotein complexes are available in the correct proportions. In this study, we demonstrate that the ubiquitin proteasome system (UPS) mediates the degradation of Apc11, the catalytic core subunit of the anaphase promoting complex/cyclosome (APC/C). In vitro studies have shown that Apc11, together with its E2 enzyme, is sufficient to ubiquitinate substrates independently of the APC/C. Here, we establish that this can occur in living yeast cells. We show that the tight controls regulating the function of the fully assembled APC/C can be circumvented when its substrates are ubiquitinated by the excess levels of Apc11 independently of the assembled complex. We thus suggest that the UPS-mediated degradation of Apc11 is an overlooked mechanism ensuring that proper function of the APC/C is limited to suitably delimited holoenzymes and that an imbalance in protein expression may result in detrimental gain-of-function activity, rather than merely the disruption of protein complex stoichiometry.-Volpe, M., Levinton, N., Rosenstein, N., Prag, G., Ben-Aroya, S. Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.
- Published
- 2019
- Full Text
- View/download PDF
32. Remodeling Membrane Binding by Mono-Ubiquitylation.
- Author
-
Tanner N, Kleifeld O, Nachman I, and Prag G
- Subjects
- Models, Molecular, Protein Binding, Protein Domains, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Vesicular Transport Proteins chemistry, Vesicular Transport Proteins metabolism, Cell Membrane metabolism, Ubiquitination
- Abstract
Ubiquitin (Ub) receptors respond to ubiquitylation signals. They bind ubiquitylated substrates and exert their activity in situ. Intriguingly, Ub receptors themselves undergo rapid ubiquitylation and deubiquitylation. Here we asked what is the function of ubiquitylation of Ub receptors? We focused on yeast epsin, a Ub receptor that decodes the ubiquitylation signal of plasma membrane proteins into an endocytosis response. Using mass spectrometry, we identified lysine-3 as the major ubiquitylation site in the epsin plasma membrane binding domain. By projecting this ubiquitylation site onto our crystal structure, we hypothesized that this modification would compete with phosphatidylinositol-4,5-bisphosphate (PIP
2 ) binding and dissociate epsin from the membrane. Using an E. coli -based expression of an authentic ubiquitylation apparatus, we purified ubiquitylated epsin. We demonstrated in vitro that in contrast to apo epsin, the ubiquitylated epsin does not bind to either immobilized PIPs or PIP2 -enriched liposomes. To test this hypothesis in vivo, we mimicked ubiquitylation by the fusion of Ub at the ubiquitylation site. Live cell imaging demonstrated that the mimicked ubiquitylated epsin dissociates from the membrane. Our findings suggest that ubiquitylation of the Ub receptors dissociates them from their products to allow binding to a new ubiquitylated substrates, consequently promoting cyclic activity of the Ub receptors.- Published
- 2019
- Full Text
- View/download PDF
33. A mutagenesis analysis of Tim50, the major receptor of the TIM23 complex, identifies regions that affect its interaction with Tim23.
- Author
-
Dayan D, Bandel M, Günsel U, Nussbaum I, Prag G, Mokranjac D, Neupert W, and Azem A
- Subjects
- Mitochondrial Membrane Transport Proteins chemistry, Mitochondrial Precursor Protein Import Complex Proteins, Models, Molecular, Mutation, Protein Binding, Protein Conformation, Saccharomyces cerevisiae Proteins chemistry, Temperature, Membrane Transport Proteins metabolism, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Membrane Transport Proteins metabolism, Mutagenesis, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Maintenance of the mitochondrial proteome depends on import of newly made proteins from the cytosol. More than half of mitochondrial proteins are made as precursor proteins with N-terminal extensions called presequences and use the TIM23 complex for translocation into the matrix, the inner mitochondrial membrane and the intermembrane space (IMS). Tim50 is the central receptor of the complex that recognizes precursor proteins in the IMS. Additionally, Tim50 interacts with the IMS domain of the channel forming subunit, Tim23, an interaction that is essential for protein import across the mitochondrial inner membrane. In order to gain deeper insight into the molecular function of Tim50, we used random mutagenesis to determine residues that are important for its function. The temperature-sensitive mutants isolated were defective in import of TIM23-dependent precursor proteins. The residues mutated map to two distinct patches on the surface of Tim50. Notably, mutations in both patches impaired the interaction of Tim50 with Tim23. We propose that two regions of Tim50 play a role in its interaction with Tim23 and thereby affect the import function of the complex.
- Published
- 2019
- Full Text
- View/download PDF
34. Anti-cancer binary system activated by bacteriophage HK022 integrase.
- Author
-
Elias A, Gritsenko N, Gorovits R, Spector I, Prag G, Yagil E, and Kolot M
- Abstract
The binary system presented in this work is based on the bacteriophage HK022 integrase recombinase that activates the expression of a silenced Diphtheria toxin gene, both controlled by the cancer specific hTERT promoter. Using a lung cancer mice model, assays of different apoptotic and anti-apoptotic factors have demonstrated that the Integrase based binary system is highly specific towards cancer cells and more efficient compared to the conventional mono system whose toxin is directly expressed under hTERT . In a mice survival test, this binary system demonstrated longer persistence compared to the untreated and the mono treated ones. The reason underlying the advantage of this binary system over the mono system seems to be an overexpression of various hTERT suppressing factors induced by the mono system., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.
- Published
- 2018
- Full Text
- View/download PDF
35. E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins.
- Author
-
Levin-Kravets O, Keren-Kaplan T, Attali I, Sharon I, Tanner N, Shapira D, Rathi R, Persaud A, Shohat N, Shusterman A, and Prag G
- Subjects
- Gene Order, Genetic Vectors genetics, Models, Molecular, Protein Conformation, Proteins chemistry, Ubiquitin metabolism, Ubiquitin-Activating Enzymes metabolism, Ubiquitin-Conjugating Enzymes metabolism, Ubiquitin-Protein Ligases metabolism, Ubiquitination, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Proteins genetics, Proteins isolation & purification, Proteins metabolism
- Abstract
Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.
- Published
- 2018
- Full Text
- View/download PDF
36. Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases.
- Author
-
Attali I, Tobelaim WS, Persaud A, Motamedchaboki K, Simpson-Lavy KJ, Mashahreh B, Levin-Kravets O, Keren-Kaplan T, Pilzer I, Kupiec M, Wiener R, Wolf DA, Rotin D, and Prag G
- Subjects
- Humans, Microfilament Proteins chemistry, Nedd4 Ubiquitin Protein Ligases, Potassium Channels, Voltage-Gated chemistry, Proteasome Endopeptidase Complex chemistry, Receptor, Fibroblast Growth Factor, Type 1 chemistry, Saccharomyces cerevisiae Proteins chemistry, Endosomal Sorting Complexes Required for Transport metabolism, Microfilament Proteins metabolism, Potassium Channels, Voltage-Gated metabolism, Proteasome Endopeptidase Complex metabolism, Protein Multimerization, Receptor, Fibroblast Growth Factor, Type 1 metabolism, Saccharomyces cerevisiae Proteins metabolism, Ubiquitin-Protein Ligases metabolism, Ubiquitination
- Abstract
Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac I
KS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition., (© 2017 The Authors.)- Published
- 2017
- Full Text
- View/download PDF
37. A bacterial genetic selection system for ubiquitylation cascade discovery.
- Author
-
Levin-Kravets O, Tanner N, Shohat N, Attali I, Keren-Kaplan T, Shusterman A, Artzi S, Varvak A, Reshef Y, Shi X, Zucker O, Baram T, Katina C, Pilzer I, Ben-Aroya S, and Prag G
- Subjects
- Drug Resistance, Bacterial genetics, Escherichia coli enzymology, Escherichia coli metabolism, Models, Molecular, Plasmids, Signal Transduction genetics, Ubiquitin metabolism, Deubiquitinating Enzymes genetics, Escherichia coli genetics, Escherichia coli Proteins genetics, Selection, Genetic, Thiolester Hydrolases genetics, Ubiquitin-Protein Ligases genetics, Ubiquitination genetics
- Abstract
About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.
- Published
- 2016
- Full Text
- View/download PDF
38. Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism.
- Author
-
Keren-Kaplan T, Zeev Peters L, Levin-Kravets O, Attali I, Kleifeld O, Shohat N, Artzi S, Zucker O, Pilzer I, Reis N, Glickman MH, Ben-Aroya S, and Prag G
- Subjects
- DNA analysis, Microscopy, Electron, Molecular Conformation, Open Reading Frames, Proteasome Endopeptidase Complex genetics, Protein Binding, Protein Conformation, Protein Domains, Protein Interaction Mapping, Saccharomyces cerevisiae Proteins genetics, Surface Plasmon Resonance, Ubiquitination, Gene Expression Regulation, Fungal, Proteasome Endopeptidase Complex chemistry, Proteasome Endopeptidase Complex metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Ubiquitin chemistry
- Abstract
Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors.
- Published
- 2016
- Full Text
- View/download PDF
39. Tetrameric Assembly of Monoubiquitin Accurately Mimics the Lys11 Polyubiquitin Chain Structure.
- Author
-
Levin-Kravets O, Shohat N, and Prag G
- Subjects
- Amino Acid Substitution, Humans, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Mutation, Missense, Signal Transduction, Structure-Activity Relationship, Ubiquitin genetics, Ubiquitin metabolism, Multiprotein Complexes chemistry, Protein Multimerization, Ubiquitin chemistry
- Abstract
Specific lysine residues on the ubiquitin surface were selected during the course of evolution to form different polyubiquitin chain structures that signal diverse cellular processes. A vast number of ubiquitin receptors specifically recognize and decode the signals conferred by these polyubiquitin chains. The mechanisms of formation and the structure of Lys11-linked ubiquitin, which signals for cell-cycle and innate immune control, have been elucidated. Here, we present a new crystal structure of monomeric ubiquitin that accurately mimics one of the structures of Lys11-linked ubiquitin. Analysis of the ubiquitin:ubiquitin interface demonstrates structural fitness and specificity. The interaction is exclusively hydrophilic, leaving the Ile44 hydrophobic patch, a major recognition site for ubiquitin receptors, exposed. These noncovalent ubiquitin:ubiquitin interactions are nearly identical to those reported for Lys11-linked ubiquitin and seem to play a significant role in stabilizing the crystal structure without the isopeptide bond. In vitro cross-linking analysis with wild-type ubiquitin or its mutants partially mimics the interactions in the crystal. We suggest that these interactions may play a biological role in transmitting Lys11-linked ubiquitin chain-type cellular signals.
- Published
- 2015
- Full Text
- View/download PDF
40. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.
- Author
-
Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL, Bakhtina MM, Becker DF, Bedwell GJ, Bekdemir A, Besong TM, Birck C, Brautigam CA, Brennerman W, Byron O, Bzowska A, Chaires JB, Chaton CT, Cölfen H, Connaghan KD, Crowley KA, Curth U, Daviter T, Dean WL, Díez AI, Ebel C, Eckert DM, Eisele LE, Eisenstein E, England P, Escalante C, Fagan JA, Fairman R, Finn RM, Fischle W, de la Torre JG, Gor J, Gustafsson H, Hall D, Harding SE, Cifre JG, Herr AB, Howell EE, Isaac RS, Jao SC, Jose D, Kim SJ, Kokona B, Kornblatt JA, Kosek D, Krayukhina E, Krzizike D, Kusznir EA, Kwon H, Larson A, Laue TM, Le Roy A, Leech AP, Lilie H, Luger K, Luque-Ortega JR, Ma J, May CA, Maynard EL, Modrak-Wojcik A, Mok YF, Mücke N, Nagel-Steger L, Narlikar GJ, Noda M, Nourse A, Obsil T, Park CK, Park JK, Pawelek PD, Perdue EE, Perkins SJ, Perugini MA, Peterson CL, Peverelli MG, Piszczek G, Prag G, Prevelige PE, Raynal BD, Rezabkova L, Richter K, Ringel AE, Rosenberg R, Rowe AJ, Rufer AC, Scott DJ, Seravalli JG, Solovyova AS, Song R, Staunton D, Stoddard C, Stott K, Strauss HM, Streicher WW, Sumida JP, Swygert SG, Szczepanowski RH, Tessmer I, Toth RT 4th, Tripathy A, Uchiyama S, Uebel SF, Unzai S, Gruber AV, von Hippel PH, Wandrey C, Wang SH, Weitzel SE, Wielgus-Kutrowska B, Wolberger C, Wolff M, Wright E, Wu YS, Wubben JM, and Schuck P
- Subjects
- Calibration, Reproducibility of Results, Ultracentrifugation methods, Ultracentrifugation standards
- Abstract
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
- Published
- 2015
- Full Text
- View/download PDF
41. Decreased susceptibility to chlorhexidine and prevalence of disinfectant resistance genes among clinical isolates of Staphylococcus epidermidis.
- Author
-
Prag G, Falk-Brynhildsen K, Jacobsson S, Hellmark B, Unemo M, and Söderquist B
- Subjects
- Anti-Infective Agents, Local therapeutic use, Humans, Microbial Sensitivity Tests, Prevalence, Quaternary Ammonium Compounds therapeutic use, Retrospective Studies, Skin microbiology, Staphylococcal Infections genetics, Chlorhexidine therapeutic use, Disinfectants therapeutic use, Drug Resistance, Bacterial genetics, Staphylococcal Infections drug therapy, Staphylococcus epidermidis drug effects
- Abstract
Staphylococcus epidermidis is a versatile agent, being both a commensal and a nosocomial pathogen usually with an opportunistic role in association with implanted foreign body materials. Pre-operative antiseptic preparation is an important strategy for reducing the risk of complications such as surgical site infection (SSI). Currently, the most widely used antiseptics are alcohols, quaternary ammonium compounds (QACs), and the bisbiguanide chlorhexidine. Occurrence of resistance to the latter agent has drawn increasing attention. The aim of this study was to investigate if decreased susceptibility to chlorhexidine among S. epidermidis was present in our setting, a Swedish university hospital. Staphylococcus epidermidis (n = 143), retrospectively collected, were obtained from prosthetic joint infections (PJI) (n = 61), post-operative infections after cardiac surgery (n = 31), and the skin of the chest after routine disinfection prior to cardiac surgery (n = 27). In addition, 24 commensal isolates were included. Minimum inhibitory concentration (MIC) of chlorhexidine was determined on Mueller Hinton agar plates supplemented with serial dilutions of chlorhexidine. Five QAC resistance genes, qacA/B, smr, qacH, qacJ, and qacG, were detected using PCR. Decreased susceptibility to chlorhexidine was found in 54% of PJI isolates, 68% of cardiac isolates, 21% of commensal isolates, and 7% of skin isolates from cardiac patients, respectively. The qacA/B gene was present in 62/143 isolates (43%), smr in 8/143 (6%), and qacH in one isolate (0.7%). The qacA/B gene was found in 52% of PJI isolates, 61% of cardiac isolates, 25% of commensal isolates, and 19% of the skin isolates. In conclusion, decreased susceptibility to chlorhexidine, as well as QAC resistance genes, were prevalent among S. epidermidis isolates associated with deep SSIs., (© 2014 APMIS. Published by John Wiley & Sons Ltd.)
- Published
- 2014
- Full Text
- View/download PDF
42. Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding.
- Author
-
Keren-Kaplan T, Attali I, Estrin M, Kuo LS, Farkash E, Jerabek-Willemsen M, Blutraich N, Artzi S, Peri A, Freed EO, Wolfson HJ, and Prag G
- Subjects
- Animals, Humans, Infectious Anemia Virus, Equine genetics, Mice, Models, Biological, Protein Structure, Tertiary, Infectious Anemia Virus, Equine metabolism, Multienzyme Complexes chemistry, Multienzyme Complexes genetics, Multienzyme Complexes metabolism, Mutation, Ubiquitin-Protein Ligases chemistry, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Virus Release physiology
- Abstract
The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a K(d) of 119 μM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC(50) of 27.6 μM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding.
- Published
- 2013
- Full Text
- View/download PDF
43. Purification and crystallization of mono-ubiquitylated ubiquitin receptor Rpn10.
- Author
-
Keren-Kaplan T and Prag G
- Subjects
- Crystallization, Proteasome Endopeptidase Complex isolation & purification, Saccharomyces cerevisiae Proteins isolation & purification, Proteasome Endopeptidase Complex chemistry, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins chemistry
- Abstract
Protein ubiquitylation controls nearly all cellular pathways in eukaryotes. A repertoire of proteins named ubiquitin (Ub) receptors harbouring ubiquitin-binding domains (UBDs) recognize ubiquitylated proteins. These Ub receptors decode the Ub signal by tethering a UBD or UBDs to a functional domain or domains, thus linking the ubiquitylated target to a specific function. The rapid dynamics of ubiquitylation/deubiquitylation has impeded the characterization of ubiquitylated proteins. To bypass this obstacle, a recently developed synthetic system that reconstructs the entire eukaryotic ubiquitylation cascade in Escherichia coli was used to purify the mono-ubiquitylated form of the regulatory proteasomal non-ATPase subunit (Ub-Rpn10) from Saccharomyces cerevisiae. Here, the first crystallization and data collection of Ub-Rpn10 is reported. Purified Ub-Rpn10 was crystallized in 12%(w/v) PEG 20,000, 0.1 M MES pH 6.5 and yielded thin rhombus-shaped crystals. X-ray analysis revealed that these crystals belonged to the monoclinic system C2, with unit-cell parameters a = 107.3, b = 49.7, c = 81.3 Å, α = γ = 90.0, β = 130.5°. A full synchrotron data set has been collected, merged and scaled with a diffraction limit of 3.14 Å.
- Published
- 2012
- Full Text
- View/download PDF
44. Purification and crystallization of yeast Ent1 ENTH domain.
- Author
-
Tanner N and Prag G
- Subjects
- Cloning, Molecular, Crystallization, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins isolation & purification, Vesicular Transport Proteins genetics, Vesicular Transport Proteins isolation & purification, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins chemistry, Vesicular Transport Proteins chemistry
- Abstract
Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6 Å and a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS-PAGE analysis of dissolved crystals.
- Published
- 2012
- Full Text
- View/download PDF
45. Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria.
- Author
-
Keren-Kaplan T, Attali I, Motamedchaboki K, Davis BA, Tanner N, Reshef Y, Laudon E, Kolot M, Levin-Kravets O, Kleifeld O, Glickman M, Horazdovsky BF, Wolf DA, and Prag G
- Subjects
- Adaptor Proteins, Vesicular Transport metabolism, Affinity Labels, Cloning, Molecular methods, Fluorescence Resonance Energy Transfer, Genetic Vectors genetics, Guanine Nucleotide Exchange Factors, Guanosine Diphosphate metabolism, Plant Proteins genetics, Plant Proteins metabolism, Proteasome Endopeptidase Complex metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Substrate Specificity, Ubiquitin-Activating Enzymes genetics, Ubiquitin-Activating Enzymes metabolism, Ubiquitin-Conjugating Enzymes genetics, Ubiquitin-Conjugating Enzymes metabolism, Ubiquitin-Protein Ligase Complexes genetics, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Vesicular Transport Proteins, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, Escherichia coli metabolism, Protein Processing, Post-Translational physiology, Synthetic Biology methods, Ubiquitin metabolism, Ubiquitin-Protein Ligase Complexes metabolism, Ubiquitination physiology
- Abstract
Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.
- Published
- 2012
- Full Text
- View/download PDF
46. The hetero-hexameric nature of a chloroplast AAA+ FtsH protease contributes to its thermodynamic stability.
- Author
-
Moldavski O, Levin-Kravets O, Ziv T, Adam Z, and Prag G
- Subjects
- ATP-Dependent Proteases isolation & purification, ATP-Dependent Proteases metabolism, Amino Acid Sequence, Arabidopsis genetics, Arabidopsis Proteins isolation & purification, Arabidopsis Proteins metabolism, Enzyme Stability, Epitopes metabolism, Mass Spectrometry, Membrane Proteins isolation & purification, Membrane Proteins metabolism, Metalloendopeptidases metabolism, Metalloproteases metabolism, Models, Molecular, Molecular Sequence Data, Multienzyme Complexes metabolism, Plants, Genetically Modified, Protein Subunits chemistry, Protein Subunits isolation & purification, Protein Subunits metabolism, Protein Transport, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Thermodynamics, Thylakoids enzymology, ATP-Dependent Proteases chemistry, Arabidopsis enzymology, Arabidopsis Proteins chemistry, Chloroplasts enzymology, Membrane Proteins chemistry, Metalloendopeptidases chemistry, Metalloproteases chemistry, Protein Multimerization
- Abstract
FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4:2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that 'type B' subunits (FtsH2 and FtsH8) were 2-3 fold more abundant than 'type A' (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two 'type A' and four 'type B' subunits.
- Published
- 2012
- Full Text
- View/download PDF
47. A phage display system designed to detect and study protein-protein interactions.
- Author
-
Bair CL, Oppenheim A, Trostel A, Prag G, and Adhya S
- Subjects
- Genetic Techniques, Genetic Vectors, Oligopeptides metabolism, Plasmids, Protein Binding, Proteomics, Bacteriophage lambda metabolism, Peptide Library, Proteins metabolism, Two-Hybrid System Techniques, Ubiquitin metabolism
- Abstract
Analysing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described.
- Published
- 2008
- Full Text
- View/download PDF
48. The Vps27/Hse1 complex is a GAT domain-based scaffold for ubiquitin-dependent sorting.
- Author
-
Prag G, Watson H, Kim YC, Beach BM, Ghirlando R, Hummer G, Bonifacino JS, and Hurley JH
- Subjects
- Amino Acid Sequence, Chromatography, Gel, Crystallography, X-Ray, Endosomal Sorting Complexes Required for Transport, Immunoprecipitation, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation genetics, Protein Binding, Protein Structure, Tertiary, Protein Transport, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Cytoplasmic and Nuclear metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Sequence Homology, Amino Acid, Ubiquitin metabolism, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Receptors, Cytoplasmic and Nuclear chemistry, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins chemistry, Vesicular Transport Proteins chemistry
- Abstract
The yeast Vps27/Hse1 complex and the homologous mammalian Hrs/STAM complex deliver ubiquitinated transmembrane proteins to the ESCRT endosomal-sorting pathway. The Vps27/Hse1 complex directly binds to ubiquitinated transmembrane proteins and recruits both ubiquitin ligases and deubiquitinating enzymes. We have solved the crystal structure of the core responsible for the assembly of the Vps27/Hse1 complex at 3.0 A resolution. The structure consists of two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The two GAT domains are connected by an antiparallel coiled coil, forming a 90 A-long barbell-like structure. This structure places the domains of Vps27 and Hse1 that recruit ubiquitinated cargo and deubiquitinating enzymes close to each other. Coarse-grained Monte Carlo simulations of the Vps27/Hse1 complex on a membrane show how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.
- Published
- 2007
- Full Text
- View/download PDF
49. Ubiquitin-binding domains.
- Author
-
Hurley JH, Lee S, and Prag G
- Subjects
- Animals, Humans, Kinetics, Models, Molecular, Polyubiquitin chemistry, Polyubiquitin metabolism, Proteasome Endopeptidase Complex metabolism, Protein Binding, Protein Conformation, Protein Processing, Post-Translational, Protein Transport, Structure-Activity Relationship, Ubiquitin chemistry, Ubiquitin-Protein Ligase Complexes metabolism, Protein Structure, Tertiary, Ubiquitin metabolism
- Abstract
The covalent modification of proteins by ubiquitination is a major regulatory mechanism of protein degradation and quality control, endocytosis, vesicular trafficking, cell-cycle control, stress response, DNA repair, growth-factor signalling, transcription, gene silencing and other areas of biology. A class of specific ubiquitin-binding domains mediates most of the effects of protein ubiquitination. The known membership of this group has expanded rapidly and now includes at least sixteen domains: UBA, UIM, MIU, DUIM, CUE, GAT, NZF, A20 ZnF, UBP ZnF, UBZ, Ubc, UEV, UBM, GLUE, Jab1/MPN and PFU. The structures of many of the complexes with mono-ubiquitin have been determined, revealing interactions with multiple surfaces on ubiquitin. Inroads into understanding polyubiquitin specificity have been made for two UBA domains, whose structures have been characterized in complex with Lys48-linked di-ubiquitin. Several ubiquitin-binding domains, including the UIM, CUE and A20 ZnF (zinc finger) domains, promote auto-ubiquitination, which regulates the activity of proteins that contain them. At least one of these domains, the A20 ZnF, acts as a ubiquitin ligase by recruiting a ubiquitin-ubiquitin-conjugating enzyme thiolester adduct in a process that depends on the ubiquitin-binding activity of the A20 ZnF. The affinities of the mono-ubiquitin-binding interactions of these domains span a wide range, but are most commonly weak, with Kd>100 microM. The weak interactions between individual domains and mono-ubiquitin are leveraged into physiologically relevant high-affinity interactions via several mechanisms: ubiquitin polymerization, modification multiplicity, oligomerization of ubiquitinated proteins and binding domain proteins, tandem-binding domains, binding domains with multiple ubiquitin-binding sites and co-operativity between ubiquitin binding and binding through other domains to phospholipids and small G-proteins.
- Published
- 2006
- Full Text
- View/download PDF
50. Structural mechanism for ubiquitinated-cargo recognition by the Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding proteins.
- Author
-
Prag G, Lee S, Mattera R, Arighi CN, Beach BM, Bonifacino JS, and Hurley JH
- Subjects
- ADP-Ribosylation Factors genetics, Adaptor Proteins, Vesicular Transport genetics, Amino Acid Sequence, Binding Sites, Crystallography, X-Ray, Golgi Apparatus metabolism, Humans, In Vitro Techniques, Models, Molecular, Molecular Sequence Data, Molecular Structure, Multiprotein Complexes, Mutagenesis, Site-Directed, Protein Conformation, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Static Electricity, Ubiquitin chemistry, Ubiquitin metabolism, Vesicular Transport Proteins chemistry, Vesicular Transport Proteins metabolism, ADP-Ribosylation Factors chemistry, ADP-Ribosylation Factors metabolism, Adaptor Proteins, Vesicular Transport chemistry, Adaptor Proteins, Vesicular Transport metabolism
- Abstract
The Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding (GGA) proteins are clathrin adaptors that mediate the sorting of transmembrane-cargo molecules at the trans-Golgi network and endosomes. Cargo proteins can be directed into the GGA pathway by at least two different types of sorting signals: acidic cluster-dileucine motifs and covalent modification by ubiquitin. The latter modification is recognized by the GGAs through binding to their GAT [GGA and TOM (target of Myb)] domain. Here we report the crystal structure of the GAT domain of human GGA3 in a 1:1 complex with ubiquitin at 2.8-A resolution. Ubiquitin binds to a hydrophobic and acidic patch on helices alpha1 and alpha2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Glu-230, Met-231, Asp-244, Glu-246, Leu-247, Glu-250, and Leu-251. The GAT-binding surface on ubiquitin is a hydrophobic patch centered on Ile-44 that is also responsible for binding most other ubiquitin effectors. The ubiquitin-binding site observed in the crystal is distinct from the Rabaptin-5-binding site on helices alpha2 and alpha3 of the GAT domain. Mutational analysis and modeling of the ubiquitin-Rabaptin-5-GAT ternary complex indicates that ubiquitin and Rabaptin-5 can bind to the GAT domain at two different sites without any steric conflict. This ability highlights the GAT domain as a hub for interactions with multiple partners in trafficking.
- Published
- 2005
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.