bims-indpro 2022-09-25 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022‒09‒25
seventeen papers selected by
Sara Mingu
Johannes Gutenberg University

Folding and self-assembly of short intrinsically disordered peptides and protein regions.
AlphaFold and structural mass spectrometry enable interrogations on the intrinsically disordered regions in cyanobacterial light-harvesting complex phycobilisome.
Convergent Views on Disordered Protein Dynamics From NMR and Computational Approaches.
Functional benefit of structural disorder for the replication of measles, Nipah and Hendra viruses.
pH-Dependent Compaction of the Intrinsically Disordered Poly-E Motif in Titin.
The Role of Intrinsically Disordered Proteins in Liquid-Liquid Phase Separation during Calcium Carbonate Biomineralization.
Intrinsically disordered regions of tristetraprolin and DCP2 directly interact to mediate decay of ARE-mRNA.
The Ni(II)-Binding Activity of the Intrinsically Disordered Region of Human NDRG1, a Protein Involved in Cancer Development.
The Role of Disordered Regions in Orchestrating the Properties of Multidomain Proteins: The SARS-CoV-2 Nucleocapsid Protein and Its Interaction with Enoxaparin.
Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase.
Analysis of the Contribution of Intrinsic Disorder in Shaping Potyvirus Genetic Diversity.
A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion.
Tuning the Dynamics of Viral Factories-Inspired Compartments Formed by Peptide-RNA Liquid-Liquid Phase Separation.
Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo.
Molecular interactions of FG nucleoporin repeats at high resolution.
Distinct neurotoxic TDP-43 fibril polymorphs are generated by heterotypic interactions with α-Synuclein.
Disorder in the human Skp1 structure is the key to its adaptability to bind many different proteins in the SCF complex assembly.

Nanoscale Adv. 2021 Apr 06. 3(7): 1789-1812

Folding and self-assembly of short intrinsically disordered peptides and protein regions.

Pablo G Argudo, Juan J Giner-Casares.

Proteins and peptide fragments are highly relevant building blocks in self-assembly for nanostructures with plenty of applications. Intrinsically disordered proteins (IDPs) and protein regions (IDRs) are defined by the absence of a well-defined secondary structure, yet IDPs/IDRs show a significant biological activity. Experimental techniques and computational modelling procedures for the characterization of IDPs/IDRs are discussed. Directed self-assembly of IDPs/IDRs allows reaching a large variety of nanostructures. Hybrid materials based on the derivatives of IDPs/IDRs show a promising performance as alternative biocides and nanodrugs. Cell mimicking, in vivo compartmentalization, and bone regeneration are demonstrated for IDPs/IDRs in biotechnological applications. The exciting possibilities of IDPs/IDRs in nanotechnology with relevant biological applications are shown.

DOI: https://doi.org/10.1039/d0na00941e
J Mol Biol. 2022 Sep 15. pii: S0022-2836(22)00451-X. [Epub ahead of print] 167831

AlphaFold and structural mass spectrometry enable interrogations on the intrinsically disordered regions in cyanobacterial light-harvesting complex phycobilisome.

Haijun Liu.

  Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of proteins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for interrogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS). Here, a joint platform is built to probe such structural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor complex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers.

Keywords:  AlphaFold; ApcG; PB-loop; chemical cross-linking; mass spectrometry; phycobilisome

DOI:  https://doi.org/10.1016/j.jmb.2022.167831
Biophys J. 2022 Sep 20. pii: S0006-3495(22)00766-4. [Epub ahead of print]

Convergent Views on Disordered Protein Dynamics From NMR and Computational Approaches.

Martin Blackledge, Fabien Ferrage, Pavel Kadeřávek, Nicola Salvi, Vojtěch Zapletal, Zuzana Jaseňáková, Milan Zachrdla, Petr Padrta, Subhash Narasimhan, Thorsten Marquardsen, Jean-Max Tyburn, Lukáš Žídek.

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDR) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. Nuclear magnetic resonance (NMR) together with molecular dynamic (MD) simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and MD simulations. In order to extend an informational content of NMR data to lower frequencies, more sensitive to slower motions, we combined the standard (high-field) NMR relaxation experiments with high resolution relaxometry. Altogether we collected data reporting the relaxation at 12 different magnetic fields resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the time scale of tens of nanosecond with the help of relaxometry data is discussed. Interestingly, in our case it appears to be mostly relevant for a region involved in formation of temporary contacts within the disordered region which was previously proven to be biologically important.

DOI: https://doi.org/10.1016/j.bpj.2022.09.016
Essays Biochem. 2022 Sep 23. pii: EBC20220045. [Epub ahead of print]

Functional benefit of structural disorder for the replication of measles, Nipah and Hendra viruses.

Frank Gondelaud, Giulia Pesce, Juliet F Nilsson, Christophe Bignon, Denis Ptchelkine, Denis Gerlier, Cyrille Mathieu, Sonia Longhi.

  Measles, Nipah and Hendra viruses are severe human pathogens within the Paramyxoviridae family. Their non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the viral RNA-dependent-RNA-polymerase (RpRd) for transcription and replication. The RpRd is a complex made of the large protein (L) and of the phosphoprotein (P), the latter serving as an obligate polymerase cofactor and as a chaperon for N. Both the N and P proteins are enriched in intrinsically disordered regions (IDRs), i.e. regions devoid of stable secondary and tertiary structure. N possesses a C-terminal IDR (NTAIL), while P consists of a large, intrinsically disordered N-terminal domain (NTD) and a C-terminal domain (CTD) encompassing alternating disordered and ordered regions. The V and W proteins, two non-structural proteins that are encoded by the P gene via a mechanism of co-transcriptional edition of the P mRNA, are prevalently disordered too, sharing with P the disordered NTD. They are key players in the evasion of the host antiviral response and were shown to phase separate and to form amyloid-like fibrils in vitro. In this review, we summarize the available information on IDRs within the N, P, V and W proteins from these three model paramyxoviruses and describe their molecular partnership. We discuss the functional benefit of disorder to virus replication in light of the critical role of IDRs in affording promiscuity, multifunctionality, fine regulation of interaction strength, scaffolding functions and in promoting liquid-liquid phase separation and fibrillation.

Keywords:  amyloid; intrinsically disordered proteins; liquid-liquid phase separations; paramyxoviruses; phase transitions

DOI:  https://doi.org/10.1042/EBC20220045
Biology (Basel). 2022 Sep 01. pii: 1302. [Epub ahead of print]11(9):

pH-Dependent Compaction of the Intrinsically Disordered Poly-E Motif in Titin.

Sophia Manukian, Gerrick E Lindberg, Emily Punch, Sudarshi Premawardhana Dassanayake Mudiyanselage, Matthew J Gage.

  The conformational sensitivity of intrinsically disordered proteins to shifts in pH due to their high degree of charged residues has been recognized for well over a decade. However, the role of the non-ionizable residues in this pH sensitivity remains poorly understood. Our lab has been investigating the pH sensitivity of the poly-E motifs of the PEVK region of the muscle protein titin, which provides an ideal model system to explore this question. Using a series of 15-amino acid peptides derived from one of the poly-E motif sequences, we have investigated the role of side-chain chemistry in the conformational flexibility of this region. Our results demonstrate that aromatic side chains and proline content are the two variables that most influence pH sensitivity. The introduction of aromatic side chains resulted in a more collapsed structure, even at pH 7, while the removal of prolines resulted in a higher degree of pH sensitivity. These results highlight the importance of considering the impact of non-ionizable residues on IDP function, especially when considering the impact of pH on conformational flexibility.

Keywords:  PEVK; circular dichroism; conformational change; intrinsically disordered proteins; titin

DOI:  https://doi.org/10.3390/biology11091302
Biomolecules. 2022 Sep 09. pii: 1266. [Epub ahead of print]12(9):

The Role of Intrinsically Disordered Proteins in Liquid-Liquid Phase Separation during Calcium Carbonate Biomineralization.

Aneta Tarczewska, Klaudia Bielak, Anna Zoglowek, Katarzyna Sołtys, Piotr Dobryszycki, Andrzej Ożyhar, Mirosława Różycka.

  Some animal organs contain mineralized tissues. These so-called hard tissues are mostly deposits of calcium salts, usually in the form of calcium phosphate or calcium carbonate. Examples of this include fish otoliths and mammalian otoconia, which are found in the inner ear, and they are an essential part of the sensory system that maintains body balance. The composition of ear stones is quite well known, but the role of individual components in the nucleation and growth of these biominerals is enigmatic. It is sure that intrinsically disordered proteins (IDPs) play an important role in this aspect. They have an impact on the shape and size of otoliths. It seems probable that IDPs, with their inherent ability to phase separate, also play a role in nucleation processes. This review discusses the major theories on the mechanisms of biomineral nucleation with a focus on the importance of protein-driven liquid-liquid phase separation (LLPS). It also presents the current understanding of the role of IDPs in the formation of calcium carbonate biominerals and predicts their potential ability to drive LLPS.

Keywords:  biomineralization; calcium carbonate; intrinsically disordered proteins; liquid–liquid phase separation; nucleation pathways; otoliths

DOI:  https://doi.org/10.3390/biom12091266
Nucleic Acids Res. 2022 Sep 21. pii: gkac797. [Epub ahead of print]

Intrinsically disordered regions of tristetraprolin and DCP2 directly interact to mediate decay of ARE-mRNA.

Vincent D Maciej, Nevena Mateva, Juliane Schwarz, Theresa Dittmers, Megha Mallick, Henning Urlaub, Sutapa Chakrabarti.

The RNA-binding protein tristetraprolin (TTP) is a potent activator of mRNA decay, specifically for transcripts bearing AU-rich elements (AREs) in their 3'-untranslated regions. TTP functions as a mediator for mRNA decay by interacting with the decay machinery and recruiting it to the target ARE-mRNA. In this study, we report a weak, but direct interaction between TTP and the human decapping enzyme DCP2, which impacts the stability of ARE transcripts. The TTP-DCP2 interaction is unusual as it involves intrinsically disordered regions (IDRs) of both binding partners. We show that the IDR of DCP2 has a propensity for oligomerization and liquid-liquid phase separation in vitro. Binding of TTP to DCP2 leads to its partitioning into phase-separated droplets formed by DCP2, suggesting that molecular crowding might facilitate the weak interaction between the two proteins and enable assembly of a decapping-competent mRNA-protein complex on TTP-bound transcripts in cells. Our studies underline the role of weak interactions in the cellular interaction network and their contribution towards cellular functionality.

DOI: https://doi.org/10.1093/nar/gkac797
Biomolecules. 2022 Sep 09. pii: 1272. [Epub ahead of print]12(9):

The Ni(II)-Binding Activity of the Intrinsically Disordered Region of Human NDRG1, a Protein Involved in Cancer Development.

Ylenia Beniamino, Vittoria Cenni, Mario Piccioli, Stefano Ciurli, Barbara Zambelli.

  Nickel exposure is associated with tumors of the respiratory tract such as lung and nasal cancers, acting through still-uncharacterized mechanisms. Understanding the molecular basis of nickel-induced carcinogenesis requires unraveling the mode and the effects of Ni(II) binding to its intracellular targets. A possible Ni(II)-binding protein and a potential focus for cancer treatment is hNDRG1, a protein induced by Ni(II) through the hypoxia response pathway, whose expression correlates with higher cancer aggressiveness and resistance to chemotherapy in lung tissue. The protein sequence contains a unique C-terminal sequence of 83 residues (hNDRG1*C), featuring a three-times-repeated decapeptide, involved in metal binding, lipid interaction and post-translational phosphorylation. In the present work, the biochemical and biophysical characterization of unmodified hNDRG1*C was performed. Bioinformatic analysis assigned it to the family of the intrinsically disordered regions and the absence of secondary and tertiary structure was experimentally proven by circular dichroism and NMR. Isothermal titration calorimetry revealed the occurrence of a Ni(II)-binding event with micromolar affinity. Detailed information on the Ni(II)-binding site and on the residues involved was obtained in an extensive NMR study, revealing an octahedral paramagnetic metal coordination that does not cause any major change of the protein backbone, which is coherent with CD analysis. hNDRG1*C was found in a monomeric form by light-scattering experiments, while the full-length hNDRG1 monomer was found in equilibrium between the dimer and tetramer, both in solution and in human cell lines. The results are the first essential step for understanding the cellular function of hNDRG1*C at the molecular level, with potential future applications to clarify its role and the role of Ni(II) in cancer development.

Keywords:  circular dichroism; intrinsically disordered regions; isothermal titration calorimetry; light scattering; lung cancer; nickel; nmr

DOI:  https://doi.org/10.3390/biom12091272
Biomolecules. 2022 Sep 15. pii: 1302. [Epub ahead of print]12(9):

The Role of Disordered Regions in Orchestrating the Properties of Multidomain Proteins: The SARS-CoV-2 Nucleocapsid Protein and Its Interaction with Enoxaparin.

Marco Schiavina, Letizia Pontoriero, Giuseppe Tagliaferro, Roberta Pierattelli, Isabella C Felli.

  Novel and efficient strategies need to be developed to interfere with the SARS-CoV-2 virus. One of the most promising pharmaceutical targets is the nucleocapsid protein (N), responsible for genomic RNA packaging. N is composed of two folded domains and three intrinsically disordered regions (IDRs). The globular RNA binding domain (NTD) and the tethered IDRs are rich in positively charged residues. The study of the interaction of N with polyanions can thus help to elucidate one of the key driving forces responsible for its function, i.e., electrostatics. Heparin, one of the most negatively charged natural polyanions, has been used to contrast serious cases of COVID-19 infection, and we decided to study its interaction with N at the molecular level. We focused on the NTR construct, which comprises the NTD and two flanking IDRs, and on the NTD construct in isolation. We characterized this interaction using different nuclear magnetic resonance approaches and isothermal titration calorimetry. With these tools, we were able to identify an extended surface of NTD involved in the interaction. Moreover, we assessed the importance of the IDRs in increasing the affinity for heparin, highlighting how different tracts of these flexible regions modulate the interaction.

Keywords:  COVID-19; IDP; NMR; SARS-CoV-2; enoxaparin; viral proteins

DOI:  https://doi.org/10.3390/biom12091302
Int J Mol Sci. 2022 Sep 07. pii: 10317. [Epub ahead of print]23(18):

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase.

Gerda Wachtl, Éva Schád, Krisztina Huszár, Antonio Palazzo, Zoltán Ivics, Ágnes Tantos, Tamás I Orbán.

  The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transposase are well described. There is an RNase H-like fold containing the DDD motif responsible for the catalytic DNA cleavage and joining reactions and a C-terminal cysteine-rich domain important for interaction with the transposon DNA. However, the protein also contains a ~100 amino acid long N-terminal disordered region (NTDR) whose function is currently unknown. Here we show that deletion of the NTDR significantly impairs piggyBac transposition, although the extent of decrease is strongly cell-type specific. Moreover, replacing the NTDR with scrambled but similarly disordered sequences did not rescue transposase activity, indicating the importance of sequence conservation. Cell-based transposon excision and integration assays reveal that the excision step is more severely affected by NTDR deletion. Finally, bioinformatic analyses indicated that the NTDR is specific for the piggyBac superfamily and is also present in domesticated, transposase-derived proteins incapable of catalyzing transposition. Our results indicate an essential role of the NTDR in the "fine-tuning" of transposition and its significance in the functions of piggyBac-originated co-opted genes.

Keywords:  DNA transposon; PGBD; intrinsically disordered protein; piggyBac

DOI:  https://doi.org/10.3390/ijms231810317
Viruses. 2022 Sep 03. pii: 1959. [Epub ahead of print]14(9):

Analysis of the Contribution of Intrinsic Disorder in Shaping Potyvirus Genetic Diversity.

Guillaume Lafforgue, Thierry Michon, Justine Charon.

  Intrinsically disordered regions (IDRs) are abundant in the proteome of RNA viruses. The multifunctional properties of these regions are widely documented and their structural flexibility is associated with the low constraint in their amino acid positions. Therefore, from an evolutionary stand point, these regions could have a greater propensity to accumulate non-synonymous mutations (NS) than highly structured regions (ORs, or 'ordered regions'). To address this hypothesis, we compared the distribution of non-synonymous mutations (NS), which we relate here to mutational robustness, in IDRs and ORs in the genome of potyviruses, a major genus of plant viruses. For this purpose, a simulation model was built and used to distinguish a possible selection phenomenon in the biological datasets from randomly generated mutations. We analyzed several short-term experimental evolution datasets. An analysis was also performed on the natural diversity of three different species of potyviruses reflecting their long-term evolution. We observed that the mutational robustness of IDRs is significantly higher than that of ORs. Moreover, the substitutions in the ORs are very constrained by the conservation of the physico-chemical properties of the amino acids. This feature is not found in the IDRs where the substitutions tend to be more random. This reflects the weak structural constraints in these regions, wherein an amino acid polymorphism is naturally conserved. In the course of evolution, potyvirus IDRs and ORs follow different evolutive paths with respect to their mutational robustness. These results have forced the authors to consider the hypothesis that IDRs and their associated amino acid polymorphism could constitute a potential adaptive reservoir.

Keywords:  diversity; mutational robustness; potyvirus; protein intrinsic disorder

DOI:  https://doi.org/10.3390/v14091959
Nucleic Acids Res. 2022 Sep 22. pii: gkac761. [Epub ahead of print]

A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion.

Mamadou Amadou Diallo, Sébastien Pirotte, Yunlong Hu, Léa Morvan, Krzysztof Rakus, Nicolás M Suárez, Lee PoTsang, Hisao Saneyoshi, Yan Xu, Andrew J Davison, Peter Tompa, Joel L Sussman, Alain Vanderplasschen.

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.

DOI: https://doi.org/10.1093/nar/gkac761
Adv Mater. 2022 Sep 22. e2206371

Tuning the Dynamics of Viral Factories-Inspired Compartments Formed by Peptide-RNA Liquid-Liquid Phase Separation.

Itai Katzir, Elvira Haimov, Ayala Lampel.

  Viral factories are intracellular microcompartments formed by mammalian viruses in their host cells, and contain necessary machinery for viral genome replication, capsid assembly and maturation, thus serve as "factories" for formation of new viral particles. Recent evidence suggest that these compartments are formed by liquid-liquid phase separation (LLPS) of viral proteins and nucleic acids and present dynamic properties. Inspired by the remarkable functionalities of viral factories we designed dynamic compartments that are formed by complexation between a minimalistic, disordered peptide and RNA. By systematic studies using sequence variants we show that the material properties of the compartments can be modulated by changes to the peptide sequence, at a single amino acid level. Moreover, by taking this approach to the next step, we developed liquid compartments with light-induced tunable dynamics. Our results demonstrate that the material properties of liquid droplets can be temporally regulated by increasing peptide polarity and charge, and that these changes can be further utilized for controlled partitioning and release of payloads from the compartments. This article is protected by copyright. All rights reserved.

Keywords:  coacervates; liquid droplets; liquid-liquid phase separation; membraneless organelles; peptides; viral factories

DOI:  https://doi.org/10.1002/adma.202206371
Acta Neuropathol. 2022 Sep 19.

Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo.

Rachel E Lackie, Aline S de Miranda, Mei Peng Lim, Vladislav Novikov, Nimrod Madrer, Nadun C Karunatilleke, Benjamin S Rutledge, Stephanie Tullo, Anne Brickenden, Matthew E R Maitland, David Greenberg, Daniel Gallino, Wen Luo, Anoosha Attaran, Irina Shlaifer, Esther Del Cid Pellitero, Caroline Schild-Poulter, Thomas M Durcan, Edward A Fon, Martin Duennwald, Flavio H Beraldo, M Mallar Chakravarty, Timothy J Bussey, Lisa M Saksida, Hermona Soreq, Wing-Yiu Choy, Vania F Prado, Marco A M Prado.

  The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.

Keywords:  A53T; Chaperone; HOP; Hsp70; Hsp90; Lewy body; Neuropathology; Parkinson’s; Pre-formed fibrils; STIP1; Touchscreens; α-Synuclein

DOI:  https://doi.org/10.1007/s00401-022-02491-8
Nat Chem. 2022 Sep 22.

Molecular interactions of FG nucleoporin repeats at high resolution.

Alain Ibáñez de Opakua, James A Geraets, Benedikt Frieg, Christian Dienemann, Adriana Savastano, Marija Rankovic, Maria-Sol Cima-Omori, Gunnar F Schröder, Markus Zweckstetter.

Proteins that contain repeat phenylalanine-glycine (FG) residues phase separate into oncogenic transcription factor condensates in malignant leukaemias, form the permeability barrier of the nuclear pore complex and mislocalize in neurodegenerative diseases. Insights into the molecular interactions of FG-repeat nucleoporins have, however, remained largely elusive. Using a combination of NMR spectroscopy and cryoelectron microscopy, we have identified uniformly spaced segments of transient β-structure and a stable preformed α-helix recognized by messenger RNA export factors in the FG-repeat domain of human nucleoporin 98 (Nup98). In addition, we have determined at high resolution the molecular organization of reversible FG-FG interactions in amyloid fibrils formed by a highly aggregation-prone segment in Nup98. We have further demonstrated that amyloid-like aggregates of the FG-repeat domain of Nup98 have low stability and are reversible. Our results provide critical insights into the molecular interactions underlying the self-association and phase separation of FG-repeat nucleoporins in physiological and pathological cell activities.

DOI: https://doi.org/10.1038/s41557-022-01035-7
J Biol Chem. 2022 Sep 15. pii: S0021-9258(22)00941-3. [Epub ahead of print] 102498

Distinct neurotoxic TDP-43 fibril polymorphs are generated by heterotypic interactions with α-Synuclein.

Shailendra Dhakal, Alicia S Robang, Nemil Bhatt, Nicha Puangmalai, Leiana Fung, Rakez Kayed, Anant K Paravastu, Vijayaraghavan Rangachari.

  Amyloid aggregates of specific proteins form important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show co-morbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by co-localizations of more than one amyloid protein, prompting the question of whether direct interactions between different amyloid proteins could generate heterotypic amyloids. To answer this question, we investigated the effect of α-synuclein (αS) on DNA-binding protein TDP-43 aggregation inspired by their co-existence in pathologies such as Lewy body dementia and limbic predominant age-related TDP-43 encephalopathy. We previously showed αS and prion-like C-terminal domain (PrLD) of TDP-43 synergistically interact to generate toxic heterotypic aggregates. Here, we extend these studies to investigate whether αS induces structurally and functionally distinct polymorphs of PrLD aggregates. Using αS -PrLD heterotypic aggregates generated in two different stoichiometric proportions, we show αS can affect PrLD fibril forms. The fibril samples have distinctive residue-level structural signatures in NMR spectra, dye-binding capability, proteinase K (PK) stability, and SDS-sensitive thermal stability. By gold nanoparticle labeling and transmission electron microscopy, we show the presence of both αS and PrLD proteins within the same fibrils, confirming the existence of heterotypic hybrid fibrils. We also observe αS and PrLD co-localize in the cytosol of neuroblastoma cells and show that the heterotypic PrLD fibrils selectively induce synaptic dysfunction in primary neurons. These findings establish the existence of heterotypic amyloid and provide a molecular basis for the observed overlap between synucleinopathies and TDP-43 proteinopathies.

Keywords:  PrLD; TDP-43; amyloids; co-pathologies; heterotypic aggregates; neurodegeneration; polymorphs; α-synuclein

DOI:  https://doi.org/10.1016/j.jbc.2022.102498
J Mol Biol. 2022 Sep 15. pii: S0022-2836(22)00450-8. [Epub ahead of print] 167830

Disorder in the human Skp1 structure is the key to its adaptability to bind many different proteins in the SCF complex assembly.

Amrita Bhattacharya, Vaibhav Kumar Shukla, Nitin Kachariya, Preeti, Parveen Sehrawat, Ashutosh Kumar.

  Skp1(S-phase kinase-associated protein 1 - Homo sapiens) is an adapter protein of the SCF(Skp1-Cullin1-Fbox) complex, which links the constant components (Cul1-RBX) and the variable receptor (F-box proteins) in Ubiquitin E3 ligase. It is intriguing how Skp1 can recognise and bind to a variety of structurally different F-box proteins. For practical reasons, previous efforts have used truncated Skp1, and thus it has not been possible to track the crucial aspects of the substrate recognition process. In this background, we report the solution structure of the full-length Skp1 protein determined by NMR spectroscopy for the first time and investigate the sequence-dependent dynamics in the protein. The solution structure reveals that Skp1 has an architecture: β1-β2-H1-H2-L1-H3-L2-H4-H5-H6-H7(partially formed) and a long tail-like disordered C-terminus. Structural analysis using DALI (Distance Matrix Alignment) reveals conserved domain structure across species for Skp1. Backbone dynamics investigated using NMR relaxation suggest substantial variation in the motional timescales along the length of the protein. The loops and the C-terminal residues are highly flexible, and the (R2/R1) data suggests μs-ms timescale motions in the helices as well. Further, the dependence of amide proton chemical shift on temperature and curved profiles of their residuals indicate that the residues undergo transitions between native state and excited state. The curved profiles for several residues across the length of the protein suggest that there are native-like low-lying excited states, particularly for several C-terminal residues. Our results provide a rationale for how the protein can adapt itself, bind, and get functionally associated with other proteins in the SCF complex by utilising its flexibility and conformational sub-states.

Keywords:  NMR Dynamics; Protein Structure; SCF complex; Skp1; Structural adaptability

DOI:  https://doi.org/10.1016/j.jmb.2022.167830