Protein delivery into plant cells: Toward in vivo structural biology

Understanding the biologically relevant structural and functional behavior of proteins inside living plant cells is only possible through the combination of structural biology and cell biology. The state-of-the-art structural biology techniques are typically applied to molecules that are isolated from their native context. Although most experimental conditions can be easily controlled while dealing with an isolated, purified protein, a serious shortcoming of such in vitro work is that we cannot mimic the extremely complex intracellular environment in which the protein exists and functions. Therefore, it is highly desirable to investigate proteins in their natural habitat, i.e., within live cells. This is the major ambition of in-cell NMR, which aims to approach structure-function relationship under true in vivo conditions following delivery of labeled proteins into cells under physiological conditions. With a multidisciplinary approach that includes recombinant protein production, confocal fluorescence microscopy, nuclear magnetic resonance (NMR) spectroscopy and different intracellular protein delivery strategies, we explore the possibility to develop in-cell NMR studies in living plant cells. While we provide a comprehensive framework to set-up in-cell NMR, we identified the efficient intracellular introduction of isotope-labeled proteins as the major bottleneck. Based on experiments with the paradigmatic intrinsically disordered proteins (IDPs) Early Response to Dehydration protein 10 and 14, we also established the subcellular localization of ERD14 under abiotic stress. © 2017 Cedeño, Pauwels and Tompa

Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin

Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies. © 2017 Nguyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Fructose-1,6-bisphosphate couples glycolytic flux to activation of Ras

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1Δ) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency. © 2017 The Author(s)

Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Reduction in Structural Disorder and Functional Complexity in the Thermal Adaptation of Prokaryotes

Genomic correlates of evolutionary adaptation to very low or very high optimal growth temperature (OGT) values have been the subject of many studies. Whereas these provided a protein-structural rationale of the activity and stability of globular proteins/enzymes, the point has been neglected that adaptation to extreme temperatures could also have resulted from an increased use of intrinsically disordered proteins (IDPs), which are resistant to these conditions in vitro. Contrary to these expectations, we found a conspicuously low level of structural disorder in bacteria of very high (and very low) OGT values. This paucity of disorder does not reflect phylogenetic relatedness, i.e. it is a result of genuine adaptation to extreme conditions. Because intrinsic disorder correlates with important regulatory functions, we asked how these bacteria could exist without IDPs by studying transcription factors, known to harbor a lot of function-related intrinsic disorder. Hyperthermophiles have much less transcription factors, which have reduced disorder compared to their mesophilic counterparts. On the other hand, we found by systematic categorization of proteins with long disordered regions that there are certain functions, such as translation and ribosome biogenesis that depend on structural disorder even in hyperthermophiles. In all, our observations suggest that adaptation to extreme conditions is achieved by a significant functional simplification, apparent at both the level of the genome and individual genes/proteins

High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using δ subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences

Self-assembly of Microcapsules via Colloidal Bond Hybridization and Anisotropy

Particles with directional interactions are promising building blocks for new functional materials and may serve as models for biological structures. Mutually attractive nanoparticles that are deformable due to flexible surface groups, for example, may spontaneously order themselves into strings, sheets and large vesicles. Furthermore, anisotropic colloids with attractive patches can self-assemble into open lattices and colloidal equivalents of molecules and micelles. However, model systems that combine mutual attraction, anisotropy, and deformability have---to the best of our knowledge---not been realized. Here, we synthesize colloidal particles that combine these three characteristics and obtain self-assembled microcapsules. We propose that mutual attraction and deformability induce directional interactions via colloidal bond hybridization. Our particles contain both mutually attractive and repulsive surface groups that are flexible. Analogous to the simplest chemical bond, where two isotropic orbitals hybridize into the molecular orbital of H2, these flexible groups redistribute upon binding. Via colloidal bond hybridization, isotropic spheres self-assemble into planar monolayers, while anisotropic snowman-like particles self-assemble into hollow monolayer microcapsules. A modest change of the building blocks thus results in a significant leap in the complexity of the self-assembled structures. In other words, these relatively simple building blocks self-assemble into dramatically more complex structures than similar particles that are isotropic or non-deformable