intrinsically disordered regions Dotaz Zobrazit nápovědu
Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are now recognised as major determinants in cellular regulation. This white paper presents a roadmap for future e-infrastructure developments in the field of IDP research within the ELIXIR framework. The goal of these developments is to drive the creation of high-quality tools and resources to support the identification, analysis and functional characterisation of IDPs. The roadmap is the result of a workshop titled "An intrinsically disordered protein user community proposal for ELIXIR" held at the University of Padua. The workshop, and further consultation with the members of the wider IDP community, identified the key priority areas for the roadmap including the development of standards for data annotation, storage and dissemination; integration of IDP data into the ELIXIR Core Data Resources; and the creation of benchmarking criteria for IDP-related software. Here, we discuss these areas of priority, how they can be implemented in cooperation with the ELIXIR platforms, and their connections to existing ELIXIR Communities and international consortia. The article provides a preliminary blueprint for an IDP Community in ELIXIR and is an appeal to identify and involve new stakeholders.
The self-processing module (SPM) is an internal segment of the FrpC protein (P415-F591) secreted by the pathogenic Gram-negative bacterium Neisseria meningitidis during meningococcal infection of human upper respiratory tract. SPM mediates 'protein trans-splicing', a unique natural mechanism for editing of proteins, which involves a calcium-dependent autocatalytic cleavage of the peptide bond between D414 and P415 and covalent linkage of the cleaved fragment through its carboxy-terminal group of D414 to [Formula: see text]-amino group of lysine residue within a neighboring polypeptide chain. We present an NMR resonance assignment of the calcium-free SPM, which displays characteristic features of intrinsically disordered proteins. Non-uniformly sampled 5D HN(CA)CONH, 4D HCBCACON, and HCBCANCO spectra were recorded to resolve poorly dispersed resonance frequencies of the disordered protein and 91 % of SPM residues were unambiguously assigned. Analysis of the chemical shifts revealed that two regions of the intrinsically disordered SPM (A95-S101 and R120-I127) have a tendency to form a helical structure, whereas the residues P1-D7 and G36-A40 have the propensity to adopt a [Formula: see text]-structure.
- MeSH
- bakteriální proteiny chemie MeSH
- membránové proteiny chemie MeSH
- Neisseria meningitidis metabolismus MeSH
- nukleární magnetická rezonance biomolekulární * MeSH
- sekundární struktura proteinů MeSH
- vnitřně neuspořádané proteiny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The pre-tetramerization loop (PTL) of the human tumor suppressor protein p53 is an intrinsically disordered region (IDR) necessary for the tetramerization process, and its flexibility contributes to the essential conformational changes needed. Although the IDR can be accurately simulated in the traditional manner of molecular dynamics (MD) with the end-to-end distance (EEdist) unhindered, we sought to explore the effects of restraining the EEdist to the values predicted by electron microscopy (EM) and other distances. Simulating the PTL trajectory with a restrained EEdist , we found an increased agreement of nuclear magnetic resonance (NMR) chemical shifts with experiments. Additionally, we observed a plethora of secondary structures and contacts that only appear when the trajectory is restrained. Our findings expand the understanding of the tetramerization of p53 and provide insight into how mutations could make the protein impotent. In particular, our findings demonstrate the importance of restraining the EEdist in studying IDRs and how their conformations change under different conditions. Our results provide a better understanding of the PTL and the conformational dynamics of IDRs in general, which are useful for further studies regarding mutations and their effects on the activity of p53.
- MeSH
- konformace proteinů MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- nádorový supresorový protein p53 chemie MeSH
- sekundární struktura proteinů MeSH
- simulace molekulární dynamiky * MeSH
- vnitřně neuspořádané proteiny * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to be involved in different interaction networks with different consequences, ranging from regulatory signalling networks to the formation of membrane-less organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDPs with a subsequent purification procedure that allows for the production of modified IDP. The robustness of our protocol is demonstrated using a challenging system: RNA polymerase II C-terminal domain (CTD); that is, a low-complexity repetitive region with multiple phosphorylation sites. In vitro phosphorylation approaches fail to yield multiple-site phosphorylated CTD, whereas our in vivo protocol allows the rapid production of near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies.
- MeSH
- Escherichia coli genetika MeSH
- exprese genu MeSH
- fosforylace MeSH
- lidé MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- posttranslační úpravy proteinů MeSH
- proteinové domény MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- transformace genetická MeSH
- tyrosin analýza genetika MeSH
- vnitřně neuspořádané proteiny chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli, and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation, and ubiquitination sites. Our results show for the first time that the RDPA protein Bromodomain-containing protein 4 (BRD4) associates with PIP2 in the RNA-dependent manner via electrostatic interactions, and that altered PIP2 levels affect the number of nuclear foci of BRD4 protein. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to form foci presumably via phase separation. This suggests the pivotal role of PIP2 in the establishment of a functional nuclear architecture competent for gene expression.
- MeSH
- buněčné jádro * metabolismus genetika MeSH
- fosfatidylinositol-4,5-difosfát * metabolismus MeSH
- fosforylace MeSH
- jaderné proteiny * metabolismus genetika MeSH
- lidé MeSH
- proteiny buněčného cyklu metabolismus genetika MeSH
- proteiny obsahující bromodoménu MeSH
- RNA metabolismus genetika MeSH
- transkripční faktory * metabolismus genetika MeSH
- vazba proteinů MeSH
- vnitřně neuspořádané proteiny * metabolismus genetika chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.
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- cirkulární dichroismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- exony * MeSH
- gelová chromatografie MeSH
- konformace proteinů MeSH
- mikroskopie atomárních sil MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proteiny zubní skloviny chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Various host-guest peptide series are used by experimentalists as reference conformational states. One such use is as a baseline for random-coil NMR chemical shifts. Comparison to this random-coil baseline, through secondary chemical shifts, is used to infer protein secondary structure. The use of these random-coil data sets rests on the perception that the reference chemical shifts arise from states where there is little or no conformational bias. However, there is growing evidence that the conformational composition of natively and nonnatively unfolded proteins fail to approach anything that can be construed as random coil. Here, we use molecular dynamics simulations of an alanine-based host-guest peptide series (AAXAA) as a model of unfolded and denatured states to examine the intrinsic propensities of the amino acids. We produced ensembles that are in good agreement with the experimental NMR chemical shifts and confirm that the sampling of the 20 natural amino acids in this peptide series is be far from random. Preferences toward certain regions of conformational space were both present and dependent upon the environment when compared under conditions typically used to denature proteins, i.e., thermal and chemical denaturation. Moreover, the simulations allowed us to examine the conformational makeup of the underlying ensembles giving rise to the ensemble-averaged chemical shifts. We present these data as an intrinsic backbone propensity library that forms part of our Structural Library of Intrinsic Residue Propensities to inform model building, to aid in interpretation of experiment, and for structure prediction of natively and nonnatively unfolded states.
It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions that can both contribute to function. Although equally interesting, the disordered regions are difficult to study, because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. NMR spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, a task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe the structural properties of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. Ataxin-3 is a 42-kDa protein containing a globular N-terminal Josephin domain and a C-terminal tail that comprises 13 polyglutamine repeats within a low complexity region. We developed a strategy that allowed us to achieve 87% assignment of the NMR spectrum using a mixed protocol based on high-dimensionality, high-resolution experiments and different labeling schemes. Thanks to the almost complete spectral assignment, we proved that the C-terminal tail is flexible, with extended helical regions, and interacts only marginally with the rest of the protein. We could also, for the first time to our knowledge, observe the structural propensity of the polyglutamine repeats within the context of the full-length protein and show that its structure is stabilized by the preceding region.
Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.
- Publikační typ
- časopisecké články MeSH
Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.
- MeSH
- katalytická doména * MeSH
- sekundární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
