scholarly journals Applying the auxin-inducible degradation (AID) system for rapid protein depletion in mammalian cells

2017 ◽  
Author(s):  
Bramwell G. Lambrus ◽  
Tyler C. Moyer ◽  
Andrew J. Holland
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Successful Implementation ◽  
Protein Depletion ◽  
Cellular Processes ◽  
Ubiquitin Ligase Complex ◽  
Short Period ◽  
Subsequent Degradation ◽  
Genomic Locations

AbstractThe ability to deplete a protein of interest is critical for dissecting cellular processes. Traditional methods of protein depletion are often slow-acting, which can be problematic when characterizing a cellular process that occurs within a short period of time. Furthermore, these methods are usually not reversible. Recent advances to achieve protein depletion function by inducibly trafficking proteins of interest to an endogenous E3 ubiquitin ligase complex to promote ubiquitination and subsequent degradation by the proteasome. One of these systems, the auxin-inducible degron (AID) system, has been shown to permit rapid and inducible degradation of AID-tagged target proteins in mammalian cells. The AID system can control the abundance of a diverse set of cellular proteins, including those contained within protein complexes, and is active in all phases of the cell cycle. Here we discuss considerations for the successful implementation of the AID system and describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of an AID degron in human cells. This method can also be adapted to insert other tags, such as fluorescent proteins, at defined genomic locations.

scholarly journals Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

2020 ◽  
Vol 21 (14) ◽  
pp. 5004
Author(s):  
Ekaterina O. Serebrovskaya ◽  
Nadezda M. Podvalnaya ◽  
Varvara V. Dudenkova ◽  
Anna S. Efremova ◽  
Nadya G. Gurskaya ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Hydrogen Peroxide Treatment ◽  
Damage Response

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

scholarly journals Post-translational modifications of Hsp90 and translating the chaperone code

2020 ◽  
Vol 295 (32) ◽  
pp. 11099-11117 ◽  
Author(s):  
Sarah J. Backe ◽  
Rebecca A. Sager ◽  
Mark R. Woodford ◽  
Alan M. Makedon ◽  
Mehdi Mollapour
Keyword(s):  
Protein Complexes ◽  
Cell Cycle Control ◽  
Heat Shock Protein 90 ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Chaperone Function ◽  
Short Period ◽  
Evolutionarily Conserved ◽  
Hsp90 Chaperone ◽  
The Stability

Cells have a remarkable ability to synthesize large amounts of protein in a very short period of time. Under these conditions, many hydrophobic surfaces on proteins may be transiently exposed, and the likelihood of deleterious interactions is quite high. To counter this threat to cell viability, molecular chaperones have evolved to help nascent polypeptides fold correctly and multimeric protein complexes assemble productively, while minimizing the danger of protein aggregation. Heat shock protein 90 (Hsp90) is an evolutionarily conserved molecular chaperone that is involved in the stability and activation of at least 300 proteins, also known as clients, under normal cellular conditions. The Hsp90 clients participate in the full breadth of cellular processes, including cell growth and cell cycle control, signal transduction, DNA repair, transcription, and many others. Hsp90 chaperone function is coupled to its ability to bind and hydrolyze ATP, which is tightly regulated both by co-chaperone proteins and post-translational modifications (PTMs). Many reported PTMs of Hsp90 alter chaperone function and consequently affect myriad cellular processes. Here, we review the contributions of PTMs, such as phosphorylation, acetylation, SUMOylation, methylation, O-GlcNAcylation, ubiquitination, and others, toward regulation of Hsp90 function. We also discuss how the Hsp90 modification state affects cellular sensitivity to Hsp90-targeted therapeutics that specifically bind and inhibit its chaperone activity. The ultimate challenge is to decipher the comprehensive and combinatorial array of PTMs that modulate Hsp90 chaperone function, a phenomenon termed the “chaperone code.”

scholarly journals Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters

2019 ◽  
Author(s):  
Felix Sigmund ◽  
Susanne Pettinger ◽  
Massimo Kube ◽  
Fabian Schneider ◽  
Martina Schifferer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Network Architecture ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Iron Core ◽  
Icosahedral Symmetry ◽  
Iron Storage ◽  
Cellular Processes ◽  
Ferroxidase Activity ◽  
Gene Reporters

Multi-colored gene reporters such as fluorescent proteins are indispensable for biomedical research, but equivalent tools for electron microscopy (EM), a gold standard for deciphering mechanistic details of cellular processes1,2and uncovering the network architecture of cell-circuits3,4, are still sparse and not easily multiplexable. Semi-genetic EM reporters are based on the precipitation of exogenous chemicals5–9which may limit spatial precision and tissue penetration and can affect ultrastructure due to fixation and permeabilization. The latter technical constraints also affect EM immunolabeling techniques10–13which may furthermore be complicated by limited epitope accessibility. The fully genetic iron storage protein ferritin generates contrast via its electron-dense iron core14–16, but its small size complicates differentiation of individual ferritin particles from cellular structures. To enable multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM), we here introduce the encapsulin system ofQuasibacillus thermotolerans(Qt) as a fully genetic iron-biomineralizing nanocompartment. We reveal by cryo-electron reconstructions that the Qt monomers (QtEnc) self-assemble to nanospheres with T=4 icosahedral symmetry and an ~44 nm diameter harboring two putative pore regions at the fivefold and threefold axes. We furthermore show that the native cargo (QtlMEF) auto-targets to the inner surface of QtEnc and exhibits ferroxidase activity leading to efficient iron sequestration inside mammalian cells. We then demonstrate that QtEnc can be robustly differentiated from the non-intermixing encapsulin ofMyxococcus xanthus17(Mx, ~32 nm) via a deep-learning model, thus enabling automated multiplexed EM gene reporter imaging in mammalian cells.

scholarly journals Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

2019 ◽  
Vol 20 (14) ◽  
pp. 3376 ◽  
Author(s):  
Kimberly Cramer ◽  
Anna-Lena Bolender ◽  
Iris Stockmar ◽  
Ralf Jungmann ◽  
Robert Kasper ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Bacterial Cells ◽  
Sted Microscopy ◽  
E Coli ◽  
Cellular Processes

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

scholarly journals Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice

2021 ◽  
Author(s):  
Ihsan Dereli ◽  
Marcello Stanzione ◽  
Fabrizio Olmeda ◽  
Frantzeskos Papanikos ◽  
Marek Baumann ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Protein Complexes ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Damage Response ◽  
Auxiliary Protein ◽  
Genomic Locations ◽  
Spatiotemporal Control

Abstract In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

scholarly journals Tafazzins from Drosophila and mammalian cells assemble in large protein complexes with a short half-life

Mitochondrion ◽  
2015 ◽  
Vol 21 ◽  
pp. 27-32 ◽  
Author(s):  
Yang Xu ◽  
Ashim Malhotra ◽  
Steven M. Claypool ◽  
Mindong Ren ◽  
Michael Schlame
Keyword(s):  
Half Life ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Large Protein ◽  
Short Half Life

scholarly journals Quotas and Gender Competence: Independent or Complementary Approaches to Gender Equality?

2021 ◽  
Vol 6 ◽  
Author(s):  
Angela Wroblewski
Keyword(s):  
Higher Education ◽  
Gender Equality ◽  
Cultural Change ◽  
Critical Discussion ◽  
Successful Implementation ◽  
Ministry Of Education ◽  
Female Representation ◽  
Paradoxical Situation ◽  
Short Period ◽  
And Gender

Austrian gender equality policy in higher education is characterized by the successful implementation of a comprehensive set of gender equality policies and persistent gender imbalances. After the introduction of a legal quota for university bodies, for instance, female representation in decision-making bodies increased significantly within a short period of time. However, this did not lead to a cultural change or the abolishment of barriers to women’s careers. Research has attributed this paradoxical situation to a lack of reflexivity because the current gender equality policies do not force institutions or individuals to challenge traditional practices, which are perceived to be merit-based and therefore gender neutral. To overcome this paradox, the Austrian Federal Ministry of Education, Science, and Research launched a policy process aimed at strengthening gender competence in all higher education processes—management, administration, teaching, and research. This paper provides a critical discussion of the Austrian quota regulation and its implementation. It also introduces the concept of gender competence and outlines the underlying assumptions as to why the new policy is expected to contribute to change. Following a critical reflection on these assumptions, the paper also discusses how existing steering instruments have to be adapted to support individual and institutional reflexivity.

scholarly journals Computational design of symmetrical eight-bladed β-propeller proteins

IUCrJ ◽  
2019 ◽  
Vol 6 (1) ◽  
pp. 46-55 ◽  
Author(s):  
Hiroki Noguchi ◽  
Christine Addy ◽  
David Simoncini ◽  
Staf Wouters ◽  
Bram Mylemans ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Evolutionary History ◽  
Protein Complexes ◽  
Protein A ◽  
Protein Structures ◽  
Computational Design ◽  
Computational Approach ◽  
Modular Architecture ◽  
Cellular Processes ◽  
History Of

β-Propeller proteins form one of the largest families of protein structures, with a pseudo-symmetrical fold made up of subdomains called blades. They are not only abundant but are also involved in a wide variety of cellular processes, often by acting as a platform for the assembly of protein complexes. WD40 proteins are a subfamily of propeller proteins with no intrinsic enzymatic activity, but their stable, modular architecture and versatile surface have allowed evolution to adapt them to many vital roles. By computationally reverse-engineering the duplication, fusion and diversification events in the evolutionary history of a WD40 protein, a perfectly symmetrical homologue called Tako8 was made. If two or four blades of Tako8 are expressed as single polypeptides, they do not self-assemble to complete the eight-bladed architecture, which may be owing to the closely spaced negative charges inside the ring. A different computational approach was employed to redesign Tako8 to create Ika8, a fourfold-symmetrical protein in which neighbouring blades carry compensating charges. Ika2 and Ika4, carrying two or four blades per subunit, respectively, were found to assemble spontaneously into a complete eight-bladed ring in solution. These artificial eight-bladed rings may find applications in bionanotechnology and as models to study the folding and evolution of WD40 proteins.

scholarly journals Miga-mediated endoplasmic reticulum–mitochondria contact sites regulate neuronal homeostasis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lingna Xu ◽  
Xi Wang ◽  
Jia Zhou ◽  
Yunyi Qiu ◽  
Weina Shang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Mitochondrial Dynamics ◽  
Molecular Organization ◽  
Lipid Transport ◽  
Cellular Processes ◽  
Contact Sites ◽  
Neuronal Homeostasis ◽  
Higher Eukaryotes ◽  
Lateral Sclerosis

Endoplasmic reticulum (ER)–mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. However, the molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. We found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells. We propose a model that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration.

Sign in / Sign up

Export Citation Format

Share Document