scholarly journals Mandipropamid as a chemical inducer of proximity for in vivo applications

2021 ◽  
Author(s):  
Michael J. Ziegler ◽  
Klaus Yserentant ◽  
Valentin Dunsing ◽  
Volker Middel ◽  
Antoni J. Gralak ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Plant Hormone ◽  
Protein Translocation ◽  
Cell Permeability ◽  
Chemical Inducers ◽  
Chemically Induced ◽  
Endogenous Proteins ◽  
Living Organisms

AbstractDirect control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

scholarly journals A chemical strategy to control protein networks in vivo

2020 ◽  
Author(s):  
Michael J. Ziegler ◽  
Klaus Yserentant ◽  
Volker Middel ◽  
Valentin Dunsing ◽  
Antoni J. Gralak ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Cell Permeability ◽  
Chemical Inducers ◽  
Cell Culture Systems ◽  
Chemically Induced ◽  
Endogenous Proteins ◽  
Living Organisms ◽  
Chemical Strategy ◽  
Control Protein

ABSTRACTDirect control of protein interaction by chemically induced protein proximity (CIPP) holds great potential for cell- and synthetic biology as well as therapeutic applications. However, toxicity, low cell-permeability and lack of orthogonality currently limit the use of available chemical inducers of proximity (CIP). We present ‘Mandi’, a novel CIP and demonstrate its applicability in cell culture systems as well as living organisms for protein translocation, protein network shuttling and manipulation of endogenous proteins.

scholarly journals Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

1999 ◽  
Vol 10 (2) ◽  
pp. 329-344 ◽  
Author(s):  
Martin Dünnwald ◽  
Alexander Varshavsky ◽  
Nils Johnsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Polypeptide Chain ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Living Cells ◽  
C Terminus ◽  
Identical Test ◽  
Split Ubiquitin

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum ofSaccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

scholarly journals Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

2022 ◽  
Author(s):  
Lewis A Macdonald ◽  
Gillian C A Taylor ◽  
Jennifer M Brisbane ◽  
Ersi Christodoulou ◽  
Lucy Scott ◽  
...  
Keyword(s):  
Mouse Models ◽  
Protein Function ◽  
Mammalian Cells ◽  
Degradation Kinetics ◽  
Cell Types ◽  
Mouse Genetics ◽  
Chemical Genetic ◽  
Endogenous Proteins ◽  
Specific Degradation

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II, two essential regulators of mitotic chromosome structure, revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

scholarly journals In Vivo Dynamics of Swi6 in Yeast: Evidence for a Stochastic Model of Heterochromatin

2004 ◽  
Vol 24 (8) ◽  
pp. 3157-3167 ◽  
Author(s):  
Thierry Cheutin ◽  
Stanislaw A. Gorski ◽  
Karen M. May ◽  
Prim B. Singh ◽  
Tom Misteli
Keyword(s):  
Stochastic Model ◽  
Fission Yeast ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Large Scale ◽  
Quantitative Estimation ◽  
Pericentric Heterochromatin ◽  
Heterochromatin Protein ◽  
Structural Heterochromatin

ABSTRACT The mechanism for transcriptional silencing of pericentric heterochromatin is conserved from fission yeast to mammals. Silenced genome regions are marked by epigenetic methylation of histone H3, which serves as a binding site for structural heterochromatin proteins. In the fission yeast Schizosaccharomyces pombe, the major structural heterochromatin protein is Swi6. To gain insight into Swi6 function in vivo, we have studied its dynamics in the nucleus of living yeast. We demonstrate that, in contrast to mammalian cells, yeast heterochromatin domains undergo rapid, large-scale motions within the nucleus. Similar to the situation in mammalian cells, Swi6 does not permanently associate with these chromatin domains but binds only transiently to euchromatin and heterochromatin. Swi6 binding dynamics are dependent on growth status and on the silencing factors Clr4 and Rik1, but not Clr1, Clr2, or Clr3. By comparing the kinetics of mutant Swi6 proteins in swi6− and swi6+ strains, we demonstrate that homotypic protein-protein interactions via the chromoshadow domain stabilize Swi6 binding to chromatin in vivo. Kinetic modeling allowed quantitative estimation of residence times and indicated the existence of at least two kinetically distinct populations of Swi6 in heterochromatin. The observed dynamics of Swi6 binding are consistent with a stochastic model of heterochromatin and indicate evolutionary conservation of heterochromatin protein binding properties from mammals to yeast.

scholarly journals A new method for the selection of protein interactions in mammalian cells

2000 ◽  
Vol 348 (3) ◽  
pp. 585-590 ◽  
Author(s):  
Eileen ROJO-NIERSBACH ◽  
Debra MORLEY ◽  
Stephanie HECK ◽  
Norbert LEHMING
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Homo Sapiens ◽  
Fibroblast Cell ◽  
New Method ◽  
Suitable Alternative ◽  
Processing Enzymes ◽  
Selection Of

In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor ĸB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1α, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT- (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.

scholarly journals Monitoring protein–protein interactions in mammalian cells by trans-SUMOylation

2011 ◽  
Vol 438 (3) ◽  
pp. 495-503 ◽  
Author(s):  
Ratnesh K. Srivastav ◽  
Susan Schwede ◽  
Malte Klaus ◽  
Jessica Schwermann ◽  
Matthias Gaestel ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Glutathione Transferase ◽  
Mitogen Activated Protein Kinase ◽  
Protein Protein Interactions ◽  
Sumoylation Site ◽  
Endogenous Expression ◽  
Mitogen Activated Protein

Protein–protein interactions are essential for almost all cellular processes, hence understanding these processes mainly depends on the identification and characterization of the relevant protein–protein interactions. In the present paper, we introduce the concept of TRS (trans-SUMOylation), a new method developed to identify and verify protein–protein interactions in mammalian cells in vivo. TRS utilizes Ubc9-fusion proteins that trans-SUMOylate co-expressed interacting proteins. Using TRS, we analysed interactions of 65 protein pairs co-expressed in HEK (human embryonic kidney)-293 cells. We identified seven new and confirmed 16 known protein interactions, which were determined via endogenous SUMOylation sites of the binding partners or by using SUMOylation-site tags respectively. Four of the new protein interactions were confirmed by GST (glutathione transferase) pull-down and the p38α–Edr2 interaction was verified by co-localization analysis. Functionally, this p38α–Edr2 interaction could possibly be involved in the recruitment of p38α to the polycomb chromatin-remodelling complex to phosphorylate Bmi1. We also used TRS to characterize protein-interaction domains of the protein kinase pairs p38α–MK2 [MK is MAPK (mitogen-activated protein kinase)-activated protein kinase] and ERK3 (extracellular-signal-regulated kinase 3)–MK5 and of the p38α–p53 complex. The ability of TRS to monitor protein interactions in mammalian cells in vivo at levels similar to endogenous expression makes it an excellent new tool that can help in defining the protein interactome of mammalian cells.

scholarly journals In Vivo Biosynthesis of Inorganic Nanomaterials Using Eukaryotes—A Review

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3246 ◽  
Author(s):  
Ashiqur Rahman ◽  
Julia Lin ◽  
Francisco E. Jaramillo ◽  
Dennis A. Bazylinski ◽  
Clayton Jeffryes ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Eukaryotic Cells ◽  
Synthesis Conditions ◽  
The Past ◽  
Living Biomass ◽  
Inorganic Nanomaterials ◽  
Living Organisms ◽  
The Many ◽  
Yeast Fungus

Bionanotechnology, the use of biological resources to produce novel, valuable nanomaterials, has witnessed tremendous developments over the past two decades. This eco-friendly and sustainable approach enables the synthesis of numerous, diverse types of useful nanomaterials for many medical, commercial, and scientific applications. Countless reviews describing the biosynthesis of nanomaterials have been published. However, to the best of our knowledge, no review has been exclusively focused on the in vivo biosynthesis of inorganic nanomaterials. Therefore, the present review is dedicated to filling this gap by describing the many different facets of the in vivo biosynthesis of nanoparticles (NPs) using living eukaryotic cells and organisms—more specifically, live plants and living biomass of several species of microalgae, yeast, fungus, mammalian cells, and animals. It also highlights the strengths and weaknesses of the synthesis methodologies and the NP characteristics, bio-applications, and proposed synthesis mechanisms. This comprehensive review also brings attention to enabling a better understanding between the living organisms themselves and the synthesis conditions that allow their exploitation as nanobiotechnological production platforms as these might serve as a robust resource to boost and expand the bio-production and use of desirable, functional inorganic nanomaterials.

scholarly journals Jun Dimerization Protein 2 Functions as a Progesterone Receptor N-Terminal Domain Coactivator

2002 ◽  
Vol 22 (15) ◽  
pp. 5451-5466 ◽  
Author(s):  
Suzanne E. Wardell ◽  
Viroj Boonyaratanakornkit ◽  
James S. Adelman ◽  
Ami Aronheim ◽  
Dean P. Edwards
Keyword(s):  
Progesterone Receptor ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Physiological Role ◽  
Target Cells ◽  
Basic Leucine Zipper ◽  
Interacting Protein

ABSTRACT The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined. By interaction cloning, we identified an AP-1 family member, Jun dimerization protein 2 (JDP-2), as a novel PR-interacting protein. JDP-2 was first defined as a c-Jun interacting protein that functions as an AP-1 repressor. PR and JDP-2 interact directly in vitro through the DNA binding domain (DBD) of PR and the basic leucine zipper (bZIP) region of JDP-2. The two proteins also physically associate in mammalian cells, as detected by coimmunoprecipitation, and are recruited in vivo to a progesterone-inducible target gene promoter, as detected by a chromatin immunoprecipitation (ChIP) assay. In cell transfection assays, JDP-2 substantially increased hormone-dependent PR-mediated transactivation and worked primarily by stimulating AF-1 activity. JDP-2 is a substantially stronger coactivator of AF-1 than SRC-1 and stimulates AF-1 independent of SRC-1 pathways. The PR DBD is necessary but not sufficient for JDP-2 stimulation of PR activity; the DBD and AF-1 are required together. JDP-2 lacks an intrinsic activation domain and makes direct protein interactions with other coactivators, including CBP and p300 CBP-associated factor (pCAF), but not with SRCs. These results indicate that JDP-2 stimulates AF-1 activity by the novel mechanism of docking to the DBD and recruiting or stabilizing N-terminal PR interactions with other general coactivators. JDP-2 has preferential activity on PR among the nuclear receptors tested and is expressed in progesterone target cells and tissues, suggesting that it has a physiological role in PR function.

scholarly journals Protein Interactions of the MLL PHD Fingers Modulate MLL Target Gene Regulation in Human Cells

2001 ◽  
Vol 21 (10) ◽  
pp. 3589-3597 ◽  
Author(s):  
Keri Fair ◽  
Melanie Anderson ◽  
Elena Bulanova ◽  
Huaifeng Mi ◽  
Maximilian Tropschug ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Target Gene ◽  
Target Genes ◽  
Leukemia Cells ◽  
Altered Expression ◽  
Amino Acid Sequence Conservation

ABSTRACT The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression ofHOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.

scholarly journals Evidence that TAF-TATA Box-Binding Protein Interactions Are Required for Activated Transcription in Mammalian Cells

2002 ◽  
Vol 22 (8) ◽  
pp. 2788-2798 ◽  
Author(s):  
Lisa S. Martel ◽  
Helen J. Brown ◽  
Arnold J. Berk
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Convex Surface ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Surface ◽  
Substitution Mutations ◽  
Residue Substitution ◽  
Tata Box Binding Protein

ABSTRACT Surfaces of human TATA box-binding protein (hsTBP) required for activated transcription in vivo were defined by constructing a library of surface residue substitution mutations and assaying them for their ability to support activated transcription in transient-transfection assays. In earlier work, three regions were identified where mutations inhibited activated transcription without interfering with TATA box DNA binding. One region is on the upstream surface of the N-terminal TBP repeat with respect to the direction of transcription and corresponds to the TBP surface that interacts with TFIIA. A second region on the stirrup of the C-terminal TBP repeat corresponds to the TFIIB-binding surface. Here we report that the third region where mutations inhibit activated transcription in mammalian cells, the convex surface of the N-terminal repeat, corresponds to a surface on TBP that interacts with hsTAF1, the major scaffold subunit of TFIID. Since mutations at the center of the hsTAF1-interacting region inhibit the ability of the protein to support activated transcription in vivo, these results are consistent with the conclusion that an interaction between hsTBP and TAFIIs is required for activated transcription in mammalian cells.

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