bims-indpro 2023-01-08 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2023–01–08
nine papers selected by
Sara Mingu, Johannes Gutenberg University

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates.
PQBP5/NOL10 maintains and anchors the nucleolus under physiological and osmotic stress conditions.
Functional partitioning of transcriptional regulators by patterned charge blocks.
Alpha-synuclein: a pathological factor with Aβ and tau and biomarker in Alzheimer's disease.
The effect of diphenylethane side-chain substituents on dibenzocyclohexadiene formation and their inhibition of α-synuclein aggregation in vitro.
Enrichment of intrinsically disordered residues in ohnologs facilitates abiotic stress resilience in Brassica rapa.
Crystal structure of a covalently linked Aurora-A-MYCN complex.
Distinct conformational changes occur within the intrinsically unstructured pro-domain of pro-Nerve Growth Factor in the presence of ATP and Mg2.
NMR Analysis of the Interactions and Conformational Plasticity of Dynein Intermediate Chain.

Proc Natl Acad Sci U S A. 2023 Jan 10. 120(2): e2216338120

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates.

Sandeep K Rai, Roopali Khanna, Anamika Avni, Samrat Mukhopadhyay.

  Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins, namely tau and prion, undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing symphony of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, and ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau:PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman results in conjunction with atomic force microscopy and multi-color fluorescence imaging reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader biological role of complex phase transitions in physiology and disease.

Keywords:  biological phase transitions; complex coacervation; intrinsically disordered proteins; membraneless organelles; protein aggregation

DOI:  https://doi.org/10.1073/pnas.2216338120
Nat Commun. 2023 Jan 04. 14(1): 9

PQBP5/NOL10 maintains and anchors the nucleolus under physiological and osmotic stress conditions.

Xiaocen Jin, Hikari Tanaka, Meihua Jin, Kyota Fujita, Hidenori Homma, Maiko Inotsume, Huang Yong, Kenichi Umeda, Noriyuki Kodera, Toshio Ando, Hitoshi Okazawa.

Polyglutamine binding protein 5 (PQBP5), also called nucleolar protein 10 (NOL10), binds to polyglutamine tract sequences and is expressed in the nucleolus. Using dynamic imaging of high-speed atomic force microscopy, we show that PQBP5/NOL10 is an intrinsically disordered protein. Super-resolution microscopy and correlative light and electron microscopy method show that PQBP5/NOL10 makes up the skeletal structure of the nucleolus, constituting the granule meshwork in the granular component area, which is distinct from other nucleolar substructures, such as the fibrillar center and dense fibrillar component. In contrast to other nucleolar proteins, which disperse to the nucleoplasm under osmotic stress conditions, PQBP5/NOL10 remains in the nucleolus and functions as an anchor for reassembly of other nucleolar proteins. Droplet and thermal shift assays show that the biophysical features of PQBP5/NOL10 remain stable under stress conditions, explaining the spatial role of this protein. PQBP5/NOL10 can be functionally depleted by sequestration with polyglutamine disease proteins in vitro and in vivo, leading to the pathological deformity or disappearance of the nucleolus. Taken together, these findings indicate that PQBP5/NOL10 is an essential protein needed to maintain the structure of the nucleolus.

DOI: https://doi.org/10.1038/s41467-022-35602-w
Cell. 2022 Dec 30. pii: S0092-8674(22)01526-4. [Epub ahead of print]

Functional partitioning of transcriptional regulators by patterned charge blocks.

Heankel Lyons, Reshma T Veettil, Prashant Pradhan, Christy Fornero, Nancy De La Cruz, Keiichi Ito, Mikayla Eppert, Robert G Roeder, Benjamin R Sabari.

  Components of transcriptional machinery are selectively partitioned into specific condensates, often mediated by protein disorder, yet we know little about how this specificity is achieved. Here, we show that condensates composed of the intrinsically disordered region (IDR) of MED1 selectively partition RNA polymerase II together with its positive allosteric regulators while excluding negative regulators. This selective compartmentalization is sufficient to activate transcription and is required for gene activation during a cell-state transition. The IDRs of partitioned proteins are necessary and sufficient for selective compartmentalization and require alternating blocks of charged amino acids. Disrupting this charge pattern prevents partitioning, whereas adding the pattern to proteins promotes partitioning with functional consequences for gene activation. IDRs with similar patterned charge blocks show similar partitioning and function. These findings demonstrate that disorder-mediated interactions can selectively compartmentalize specific functionally related proteins from a complex mixture of biomolecules, leading to regulation of a biochemical pathway.

Keywords:  biomolecular condensates; functional compartmentalization; gene activation; nuclear organization; phase separation; protein disorder; selective partitioning; transcription

DOI:  https://doi.org/10.1016/j.cell.2022.12.013
Alzheimers Res Ther. 2022 Dec 31. 14(1): 201

Alpha-synuclein: a pathological factor with Aβ and tau and biomarker in Alzheimer's disease.

Kyu Hwan Shim, Min Ju Kang, Young Chul Youn, Seong Soo A An, SangYun Kim.

   BACKGROUND: Alpha-synuclein (α-syn) is considered the main pathophysiological protein component of Lewy bodies in synucleinopathies. α-Syn is an intrinsically disordered protein (IDP), and several types of structural conformations have been reported, depending on environmental factors. Since IDPs may have distinctive functions depending on their structures, α-syn can play different roles and interact with several proteins, including amyloid-beta (Aβ) and tau, in Alzheimer's disease (AD) and other neurodegenerative disorders.
MAIN BODY: In previous studies, α-syn aggregates in AD brains suggested a close relationship between AD and α-syn. In addition, α-syn directly interacts with Aβ and tau, promoting mutual aggregation and exacerbating the cognitive decline. The interaction of α-syn with Aβ and tau presented different consequences depending on the structural forms of the proteins. In AD, α-syn and tau levels in CSF were both elevated and revealed a high positive correlation. Especially, the CSF α-syn concentration was significantly elevated in the early stages of AD. Therefore, it could be a diagnostic marker of AD and help distinguish AD from other neurodegenerative disorders by incorporating other biomarkers.
CONCLUSION: The overall physiological and pathophysiological functions, structures, and genetics of α-syn in AD are reviewed and summarized. The numerous associations of α-syn with Aβ and tau suggested the significance of α-syn, as a partner of the pathophysiological roles in AD. Understanding the involvements of α-syn in the pathology of Aβ and tau could help address the unresolved issues of AD. In particular, the current status of the CSF α-syn in AD recommends it as an additional biomarker in the panel for AD diagnosis.

Keywords:  Alzheimer’s disease; Amyloid-beta; Biomarker; Cerebrospinal fluid; Tau; α-Synuclein

DOI:  https://doi.org/10.1186/s13195-022-01150-0
Bioorg Med Chem. 2022 Dec 26. pii: S0968-0896(22)00540-5. [Epub ahead of print]78 117147

The effect of diphenylethane side-chain substituents on dibenzocyclohexadiene formation and their inhibition of α-synuclein aggregation in vitro.

Gabriel Bernardes, Omer Munir, Ed S Krol.

  The naturally-occurring di-catechol lignan nordihydroguaiaretic acid (NDGA) and an analog without methyl groups on the butyl linker both undergo intramolecular cyclization at pH 7.4 to form dibenzocyclooctadienes. Both NDGA and these dibenzocyclooctadienes have been shown to prevent in vitro aggregation of α-synuclein, an intrinsically disordered protein associated with Parkinson's disease. NDGA possesses two vicinal methyl groups on the butyl linker and the presence of these methyl groups attenuates the rate of intramolecular cyclization versus the unsubstituted analog, in opposition to the anticipated Thorpe-Ingold effect, likely due to steric repulsions during cyclization. Numerous 1,2-bis-ethane di-catechols are known to inhibit α-synuclein aggregation in vitro and we hypothesize that these compounds undergo a similar intramolecular cyclization and the cyclized products may be responsible for the activity. To test this hypothesis we prepared a series of 1,2-bis-ethane di-catechols with 0, 2 and 4 methyl substituents on the linker. We have confirmed that these compounds undergo intramolecular cyclization to form dibenzocyclohexadienes and that steric interactions between the methyl substituents leads to an increase in the rate of intramolecular cyclization, which is in contrast to what was observed for lignan di-catechols. The rate of cyclization to form six-membered rings is 10-30 times more rapid than formation of eight membered rings and the dibenzocyclohexadienes also prevent in vitro aggregation of α-synuclein.

Keywords:  Di-catechol diphenylethanes; Dibenzocyclohexadienes; Geminal dimethyl effect; In vitro alpha-synuclein aggregation; Intramolecular cyclization; Oxidative instability; Vicinal dimethyl effect

DOI:  https://doi.org/10.1016/j.bmc.2022.117147
J Plant Res. 2023 Jan 06.

Enrichment of intrinsically disordered residues in ohnologs facilitates abiotic stress resilience in Brassica rapa.

Shayani Das Laha, Deepyaman Das, Tapash Ghosh, Soumita Podder.

  Arabidopsis thaliana and Brassica rapa are in the same evolutionary lineage, although the latter experienced an additional whole genome triplication event. Therefore, it would be intriguing to investigate the traits that gene duplication imposes to mediate plant stress tolerance. Here, we noticed that B. rapa abiotic stress resistance (ASR) genes which code at least one stress responsive domain have a significantly higher number of paralogs than A. thaliana. Analysing the disordered content of the ASR genes in both species, we found that intrinsically disordered residues (IDR) are specifically enriched in whole genome duplication (WGD) derived paralogs. Subsequently, domain similarity analysis between WGD pairs of both species has revealed that majority of WGD pairs in B. rapa did not share domains with each other. Furthermore, domain enrichment analysis has shown that B. rapa paralogs contain 36 distinct stress responsive enriched domains, significantly higher than A. thaliana paralogs. Next, we performed MSA to investigate the domain conservation between orthologs and ohnologs pairs, we found that 80.13% of B. rapa ohnologs acquire new domains, depicting the fact that ohnologs play a significant role in stress-related behaviours. The average IDR content of the ohnologs enriching new domains after gene duplication in B. rapa (0.19), is also significantly higher than A. thaliana (0.04). Interestingly, we also found that all of these attributes i.e., exhibiting higher number of WGD paralogs and enhancement of IDR in ASR genes of B. rapa compared to A. thaliana is exclusive for ASR genes only. No such significant differences were observed in randomly selected non-ASR genes between the two species. Together these results provide strong support for the hypothesis that augmentation of IDR content followed by a whole genome duplication event imposes the stress resistance potentiality in B. rapa. This research will shed light on the mechanism of how B. rapa is able to successfully adapt to stress over the evolutionary timescale.

Keywords:  Abiotic stress; Domain; Intrinsically disordered regions; Ohnologs; Orthologs; Whole genome duplication

DOI:  https://doi.org/10.1007/s10265-022-01432-6
Acta Crystallogr D Struct Biol. 2023 Jan 01. 79(Pt 1): 1-9

Crystal structure of a covalently linked Aurora-A-MYCN complex.

Mathias Diebold, Lars Schönemann, Martin Eilers, Christoph Sotriffer, Hermann Schindelin.

  Formation of the Aurora-A-MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A-MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A-MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders.

Keywords:  Aurora-A; MYCN; cancer targets; cross-linking; neuroblastoma cells; oncogenic transcription factors

DOI:  https://doi.org/10.1107/S2059798322011433
Protein Sci. 2023 Jan 05. e4563

Distinct conformational changes occur within the intrinsically unstructured pro-domain of pro-Nerve Growth Factor in the presence of ATP and Mg2.

Francesca Paoletti, Sonia Covaceuszach, Alberto Cassetta, Antonio N Calabrese, Urban Novak, Petr Konarev, Jože Grdadolnik, Doriano Lamba, Simona Golič Grdadolnik.

  Nerve Growth Factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here we show by Isothermal Titration Calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+ , known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by Small Angle X-ray Scattering and Hydrogen Deuterium eXchange Mass Spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis. This article is protected by copyright. All rights reserved.

Keywords:  HDX-MS; NMR spectroscopy; SAXS; conformational rearrangement of the intrinsically unstructured domain (IUD); intermolecular interactions; proNGF

DOI:  https://doi.org/10.1002/pro.4563
Methods Mol Biol. 2023 ;2623 241-256

NMR Analysis of the Interactions and Conformational Plasticity of Dynein Intermediate Chain.

Kayla A Jara, Elisar J Barbar.

  Cytoplasmic dynein complexes play crucial roles in intracellular transport of cellular organelles. While the motor domain of dynein is well characterized by techniques such as X-ray crystallography and cryo-electron microscopy (Cryo-EM), structural representations of dynein usually include only the more packed and easily resolved regions and omit the long flexible and poorly structured regions. One such flexible region is the N-terminal half of the intermediate chain (IC), which contains almost 300 amino acids that are predicted to be disordered. This level of disorder makes IC impossible to study by X-ray crystallography and Cryo-EM, but amenable to study by solution nuclear magnetic resonance (NMR), a powerful technique that can elucidate residue-specific information in a dynamic ensemble of structures, and transient binding interactions of associated proteins. Here, we describe the methods we use to characterize flexible and disordered proteins including protein expression, purification, sample preparation, and NMR data acquisition and analysis.

Keywords:  Dynein intermediate chain; Intrinsically disordered proteins; NMR spectroscopy; Protein; Transient interactions; Transient structure; protein interactions

DOI:  https://doi.org/10.1007/978-1-0716-2958-1_15