bims-indpro 2022-04-17 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–04–17
seventeen papers selected by
Sara Mingu, Johannes Gutenberg University

Multivalent Interactions with Intrinsically Disordered Proteins Probed by Surface Plasmon Resonance.
Investigating the disordered regions (MoRFs, SLiMs and LCRs) and functions of mimicry proteins/peptides in silico.
AN ENANTIOMERIC FRAGMENT PAIR (EFP) APPROACH FOR THE STUDY OF CELLULAR UPTAKE OF INTRINSICALLY DISORDERED PROTEINS.
Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region.
Dimerization Activity of a Disordered N-Terminal Domain from Drosophila CLAMP Protein.
Folding or Holding? - Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease.
Short Linear Motifs (SLiMs) in "Core" RxLR Effectors of Phytophthora parasitica var. nicotianae: a Case of PpRxLR1 Effector.
Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98.
Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.
Targeting intrinsically disordered regions facilitates discovery of CaV3.2 inhibitory peptides for AAV-mediated peripheral analgesia.
Bioinformatic identification of TRK-fused gene protein (TFG) as a previously unrecognized amyloidogenic protein.
Artificial neural networks for solution scattering data analysis.
Impact of porous nanomaterials on inhibiting protein aggregation behaviour.
Time-resolved detection of SDS-induced conformational changes in α-synuclein by a micro-stopped-flow system.
In Silico Characterisation of the Late Embryogenesis Abundant (LEA) Protein Families and Their Role in Desiccation Tolerance in Ramonda serbica Panc.
Bacterial hydrophilins promote pathogen desiccation tolerance.
Zinc enhances liquid-liquid phase separation of Tau protein and aggravates mitochondrial damages in cells.

Methods Mol Biol. 2022 ;2502 311-328

Multivalent Interactions with Intrinsically Disordered Proteins Probed by Surface Plasmon Resonance.

Larisa E Kapinos, Roderick Y H Lim.

  Multivalent interactions underpin associations between intrinsically disordered proteins (IDPs) and their binding partners. This is a subject of considerable interest and governs how nuclear transport receptors (NTRs) orchestrate the nucleocytoplasmic transport (NCT) of signal-specific cargoes through nuclear pore complexes (NPCs) in eukaryotic cells. Specifically, IDPs termed phenylalanine-glycine nucleoporins (FG Nups) exert multivalent interactions with NTRs to facilitate their transport selectivity and speed through the NPC. Here, we document the use of surface plasmon resonance (SPR) to quantify the affinity and kinetics of NTR-FG Nup binding as a function of FG Nup surface density. Moreover, we describe an in situ method that measures conformational height changes that occur in a FG Nup layer following NTR-binding. Protocols by which the as-obtained SPR results are treated with respect to mass transport limitations are further described. Overall, the SPR methodology described here can be applied to studying multivalent interactions and the role of avidity in diverse biological and biointerfacial systems.

Keywords:  Biological interface; Exportin; FG Nucleoporins; Importin; Intrinsically disordered proteins; Karyopherin; Multivalent interactions; Nuclear pore complex; Steady state; Surface plasmon resonance

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_21
PLoS One. 2022 ;17(4): e0265657

Investigating the disordered regions (MoRFs, SLiMs and LCRs) and functions of mimicry proteins/peptides in silico.

Anjali Garg, Govinda Rao Dabburu, Neelja Singhal, Manish Kumar.

Microbial mimicry of the host proteins/peptides can elicit host auto-reactive T- or B-cells resulting in autoimmune disease(s). Since intrinsically disordered protein regions (IDPRs) are involved in several host cell signaling and PPI networks, molecular mimicry of the IDPRs can help the pathogens in substituting their own proteins in the host cell-signaling and PPI networks and, ultimately hijacking the host cellular machinery. Thus, the present study was conducted to discern the structural disorder and intrinsically disordered protein regions (IDPRs) like, molecular recognition features (MoRFs), short linear motifs (SLiMs), and low complexity regions (LCRs) in the experimentally verified mimicry proteins and peptides (mimitopes) of bacteria, viruses and host. Also, functional characteristics of the mimicry proteins were studied in silico. Our results indicated that 78% of the bacterial host mimicry proteins and 45% of the bacterial host mimitopes were moderately/highly disordered while, 73% of the viral host mimicry proteins and 31% of the viral host mimitopes were moderately/highly disordered. Among the pathogens, 27% of the bacterial mimicry proteins and 13% of the bacterial mimitopes were moderately/highly disordered while, 53% of the viral mimicry proteins and 21% of the viral mimitopes were moderately/highly disordered. Though IDPR were frequent in host, bacterial and viral mimicry proteins, only a few mimitopes overlapped with the IDPRs like, MoRFs, SLiMs and LCRs. This suggests that most of the microbes cannot use molecular mimicry to modulate the host PPIs and hijack the host cell machinery. Functional analyses indicated that most of the pathogens exhibited mimicry with the host proteins involved in ion binding and signaling pathways. This is the first report on the disordered regions and functional aspects of experimentally proven host and microbial mimicry proteins.

DOI: https://doi.org/10.1371/journal.pone.0265657
Chembiochem. 2022 Apr 13.

AN ENANTIOMERIC FRAGMENT PAIR (EFP) APPROACH FOR THE STUDY OF CELLULAR UPTAKE OF INTRINSICALLY DISORDERED PROTEINS.

Alejandro R Foley, Jevgenij Raskatov.

  The study of intrinsically disordered and amyloidogenic proteins poses a major challenge to researchers: the propensity of the system to aggregate and to form amyloid fibrils and deposits . This intrinsic nature limits the way amyloids can be studied and increases the level of complexity of the techniques needed to study the system of interest. Recent reports suggest that cellular recognition and internalization of pre-fibrillary species of amyloidogenic peptides and proteins may initiate some of its toxic actions. Therefore, developing novels tools to facilitate the understanding and determination of the interactions between intrinsically disordered proteins and the cellular membrane is becoming increasingly valuable. Here, we present and propose an approach for the study of the interactions of intrinsically disordered proteins with the cellular surface based on the use of enantiomeric fragment pairs (EFPs). By following a stepwise methodology in which the amyloidogenic peptide or protein is fragmented into specific segments, we show how this approach can be exploited to differentiate between different types of cellular uptake, to determine the degree of receptor-mediated cellular internalization of intrinsically disordered peptides and proteins, and to pinpoint the specific regions within the amino acid sequence responsible for the cellular recognition. Adopting this approach overcomes aggregation-related challenges and offers a particularly well-suited platform for the elucidation of receptor-intermediated recognition, uptake, and toxicity.

Keywords:  Enantiomeric peptides, mirror image, cellular uptake, amyloid, aggregation

DOI:  https://doi.org/10.1002/cbic.202200146
Elife. 2022 Apr 13. pii: e75047. [Epub ahead of print]11

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region.

Sandhya Balasubramanian, Matteo Andreani, Júlia Goncalves Andrade, Tannishtha Saha, Devakumar Sundaravinayagam, Javier Garzón, Wenzhu Zhang, Oliver Popp, Shin-Ichiro Hiraga, Ali Rahjouei, Daniel B Rosen, Philipp Mertins, Brian T Chait, Anne D Donaldson, Michela Di Virgilio.

  RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

Keywords:  DNA replication fork protection; DSB resection inhibition; RIF1; SQ motifs; cancer biology; genetics; genomics; intrinsically disordered region; mouse

DOI:  https://doi.org/10.7554/eLife.75047
Int J Mol Sci. 2022 Mar 31. pii: 3862. [Epub ahead of print]23(7):

Dimerization Activity of a Disordered N-Terminal Domain from Drosophila CLAMP Protein.

Evgeniya Tikhonova, Sofia Mariasina, Olga Arkova, Oksana Maksimenko, Pavel Georgiev, Artem Bonchuk.

  In Drosophila melanogaster, CLAMP is an essential zinc-finger transcription factor that is involved in chromosome architecture and functions as an adaptor for the dosage compensation complex. Most of the known Drosophila architectural proteins have structural N-terminal homodimerization domains that facilitate distance interactions. Because CLAMP performs architectural functions, we tested its N-terminal region for the presence of a homodimerization domain. We used a yeast two-hybrid assay and biochemical studies to demonstrate that the adjacent N-terminal region between 46 and 86 amino acids is capable of forming homodimers. This region is conserved in CLAMP orthologs from most insects, except Hymenopterans. Biophysical techniques, including nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS), suggested that this domain lacks secondary structure and has features of intrinsically disordered regions despite the fact that the protein structure prediction algorithms suggested the presence of beta-sheets. The dimerization domain is essential for CLAMP functions in vivo because its deletion results in lethality. Thus, CLAMP is the second architectural protein after CTCF that contains an unstructured N-terminal dimerization domain.

Keywords:  C2H2 protein; NMR; architectural protein; dimerization; dosage compensation; intrinsically disordered protein; small-angle X-ray scattering

DOI:  https://doi.org/10.3390/ijms23073862
J Biol Chem. 2022 Apr 06. pii: S0021-9258(22)00345-3. [Epub ahead of print] 101905

Folding or Holding? - Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease.

Benjamin S Rutledge, Wing-Yiu Choy, Martin L Duennwald.

  The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as Hsp70 and Hsp90, defend cells against the toxic consequences of protein misfolding remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions the toxic misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the toxic accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the underlying mechanisms and how they modulate neurodegeneration. In his review, we compare recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and TDP-43. We propose that Hsp90 and to some extent Hsp70 interact with these proteins through an extended and dynamic interface that span the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90-client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multi-domain interactions allows polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more energy efficient by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.

Keywords:  Hsp90; TDP-43; alpha-synuclein; intrinsically disordered proteins; molecular chaperones; tau

DOI:  https://doi.org/10.1016/j.jbc.2022.101905
Microbiol Spectr. 2022 Apr 11. e0177421

Short Linear Motifs (SLiMs) in "Core" RxLR Effectors of Phytophthora parasitica var. nicotianae: a Case of PpRxLR1 Effector.

Jane Chepsergon, Celiwe Innocentia Nxumalo, Brenda S C Salasini, Aquillah M Kanzi, Lucy Novungayo Moleleki.

  Oomycetes of the genus Phytophthora encompass several of the most successful plant pathogens described to date. The success of infection by Phytophthora species is attributed to the pathogens' ability to secrete effector proteins that alter the host's physiological processes. Structural analyses of effector proteins mainly from bacterial and viral pathogens have revealed the presence of intrinsically disordered regions that host short linear motifs (SLiMs). These motifs play important biological roles by facilitating protein-protein interactions as well as protein translocation. Nonetheless, SLiMs in Phytophthora species RxLR effectors have not been investigated previously and their roles remain unknown. Using a bioinformatics pipeline, we identified 333 candidate RxLR effectors in the strain INRA 310 of Phytophthora parasitica. Of these, 71 (21%) were also found to be present in 10 other genomes of P. parasitica, and hence, these were designated core RxLR effectors (CREs). Within the CRE sequences, the N terminus exhibited enrichment in intrinsically disordered regions compared to the C terminus, suggesting a potential role of disorder in effector translocation. Although the disorder content was reduced in the C-terminal regions, it is important to mention that most SLiMs were in this terminus. PpRxLR1 is one of the 71 CREs identified in this study, and its genes encode a 6-amino acid (aa)-long SLiM at the C terminus. We showed that PpRxLR1 interacts with several host proteins that are implicated in defense. Structural analysis of this effector using homology modeling revealed the presence of potential ligand-binding sites. Among key residues that were predicted to be crucial for ligand binding, L102 and Y106 were of interest since they form part of the 6-aa-long PpRxLR1 SLiM. In silico substitution of these two residues to alanine was predicted to have a significant effect on both the function and the structure of PpRxLR1 effector. Molecular docking simulations revealed possible interactions between PpRxLR1 effector and ubiquitin-associated proteins. The ubiquitin-like SLiM carried in this effector was shown to be a potential mediator of these interactions. Further studies are required to validate and elucidate the underlying molecular mechanism of action. IMPORTANCE The continuous gain and loss of RxLR effectors makes the control of Phytophthora spp. difficult. Therefore, in this study, we endeavored to identify RxLR effectors that are highly conserved among species, also known as "core" RxLR effectors (CREs). We reason that these highly conserved effectors target conserved proteins or processes; thus, they can be harnessed in breeding for durable resistance in plants. To further understand the mechanisms of action of CREs, structural dissection of these proteins is crucial. Intrinsically disordered regions (IDRs) that do not adopt a fixed, three-dimensional fold carry short linear motifs (SLiMs) that mediate biological functions of proteins. The presence and potential role of these SLiMs in CREs of Phytophthora spp. have been overlooked. To our knowledge, we have effectively identified CREs as well as SLiMs with the potential of promoting effector virulence. Together, this work has advanced our comprehension of Phytophthora RxLR effector function and may facilitate the development of innovative and effective control strategies.

Keywords:  Phytophthora; Phytophthora spp.; SLiMs; plant immunity; “core” RxLR effectors

DOI:  https://doi.org/10.1128/spectrum.01774-21
Int J Mol Sci. 2022 Mar 23. pii: 3495. [Epub ahead of print]23(7):

Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98.

Lisa Diez, Larisa E Kapinos, Janine Hochmair, Sabrina Huebschmann, Alvaro Dominguez-Baquero, Amelie Vogt, Marija Rankovic, Markus Zweckstetter, Roderick Y H Lim, Susanne Wegmann.

  Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer's disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.

Keywords:  FG-Nups; MAPT; nuclear pore complex; posttranslational modifications

DOI:  https://doi.org/10.3390/ijms23073495
J Chem Phys. 2022 Apr 14. 156(14): 141501

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.

Anders Barth, Oleg Opanasyuk, Thomas-Otavio Peulen, Suren Felekyan, Stanislav Kalinin, Hugo Sanabria, Claus A M Seidel.

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τD and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E-τD diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines.

DOI: https://doi.org/10.1063/5.0089134
Pain. 2022 Apr 14.

Targeting intrinsically disordered regions facilitates discovery of CaV3.2 inhibitory peptides for AAV-mediated peripheral analgesia.

Seung Min Shin, Justas Lauzadis, Brandon Itson-Zoske, Yongsong Cai, Fan Fan, Gayathri Natarajan, Wai-Meng Kwok, Michelino Puopolo, Quinn H Hogan, Hongwei Yu.

ABSTRACT: Ample data support a prominent role of peripheral T-type calcium channels 3.2 (CaV3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of CaV3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from the natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. Here, we report the engineering of the potent and selective iPAs of CaV3.2 from the intrinsically disordered regions (IDR) of CaV3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in CaV3.2 protein and identified several CaV3.2iPA candidates that significantly reduced CaV3.2 current in HEK293 cells stably expressing human wide-type CaV3.2. Two prototype CaV3.2iPAs (iPA1 and iPA2) derived from the IDRs of CaV3.2 intracellular loop 2 and 3 respectively were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by CaV3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain following tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated CaV3.2iPA expression suppressed neuronal excitability, suggesting that CaV3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that CaV3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral CaV3.2-targeting strategy for clinical treatment of pain.

DOI: https://doi.org/10.1097/j.pain.0000000000002650
J Biol Chem. 2022 Apr 08. pii: S0021-9258(22)00360-X. [Epub ahead of print] 101920

Bioinformatic identification of TRK-fused gene protein (TFG) as a previously unrecognized amyloidogenic protein.

Gregory M Rosenberg, Kevin A Murray, Lukasz Salwinski, Michael P Hughes, Romany Abskharon, David S Eisenberg.

  Low-complexity domains of proteins have been shown to self-associate, and pathogenic mutations within these domains often drive the proteins into amyloid aggregation associated with disease. These domains may be especially susceptible to amyloidogenic mutations because they are commonly intrinsically disordered and function in self-association. The question therefore arises whether a search for pathogenic mutations in low-complexity domains of the human proteome can lead to identification of other proteins associated with amyloid disease. Here, we take a computational approach to identify documented pathogenic mutations within low-complexity domains which may favor amyloid formation. Using this approach, we identify numerous known amyloidogenic mutations, including several such mutations within proteins previously unidentified as amyloidogenic. Among the latter group, we focus on two mutations within the TRK-fused gene protein (TFG), known to play roles in protein secretion and innate immunity, which are associated with two different peripheral neuropathies. We show that both mutations increase the propensity of TFG to form amyloid fibrils. We therefore conclude that TFG is a novel amyloid protein and propose that the diseases associated with its mutant forms may be amyloidoses.

Keywords:  Charcot-Marie-Tooth disease (CMT); amyloid; electron microscopy (EM); intrinsically disordered protein; low-complexity domain; protein structure

DOI:  https://doi.org/10.1016/j.jbc.2022.101920
Structure. 2022 Mar 30. pii: S0969-2126(22)00092-2. [Epub ahead of print]

Artificial neural networks for solution scattering data analysis.

Dmitry S Molodenskiy, Dmitri I Svergun, Alexey G Kikhney.

  Small-angle X-ray scattering (SAXS) experiments are widely used for the characterization of biological macromolecules in solution. SAXS patterns contain information on the size and shape of dissolved particles in nanometer resolution. Here we propose a method for primary SAXS data analysis based on the application of artificial neural networks (NNs). Trained on synthetic SAXS data, the feedforward NNs are able to reliably predict molecular weight and maximum intraparticle distance (Dmax) directly from the experimental data. The method is applicable to data from monodisperse solutions of folded proteins, intrinsically disordered proteins, and nucleic acids. Extensive tests on synthetic SAXS data generated in various angular ranges with varying levels of noise demonstrated a higher accuracy and better robustness of the NN approach compared to the existing methods.

Keywords:  SAXS; artificial intelligence; machine learning; neural networks

DOI:  https://doi.org/10.1016/j.str.2022.03.011
RSC Adv. 2021 Jan 14. 11(6): 3354-3362

Impact of porous nanomaterials on inhibiting protein aggregation behaviour.

Munmun Bardhan, Sandip Dolui, Siddhi Chaudhuri, Uttam Paul, Gaurav Bhattacharjee, Manorama Ghosal, Nakul C Maiti, Debashis Mukhopadhyay, Dulal Senapati.

Aggregation of intrinsically disordered as well as the ordered proteins under certain premises or physiological conditions leads to pathological disorder. Here we have presented a detailed investigation on the effect of a porous metallic (Au) and a non-metallic (Si) nanomaterial on the formation of ordered (fiber-like/amyloid) and disordered (amorphous) aggregates of proteins. Porous nanogold (PNG) was found to reduce the amyloid aggregation of insulin but does not have much impact on the lag phase in the aggregation kinetics, whereas porous nano-silica (PNS) was found both to decrease the amount of aggregation as well as prolong the lag phase of amyloid fiber formation from insulin. On the other hand, both the porous nanoparticles are found to decrease the extent of amorphous aggregation (with slight improvement for PNS) of pathogenic huntingtin (Htt) protein in Huntington's disease cell model. This is a noted direct observation in controlling and understanding protein aggregation diseases which may help us to formulate nanotherapeutic drugs for future clinical applications.

DOI: https://doi.org/10.1039/d0ra10927d
RSC Adv. 2020 Dec 24. 11(2): 1086-1097

Time-resolved detection of SDS-induced conformational changes in α-synuclein by a micro-stopped-flow system.

Shunki Takaramoto, Yusuke Nakasone, Kei Sadakane, Shinsaku Maruta, Masahide Terazima.

An intrinsically disordered protein, α-synuclein (αSyn), binds to negatively charged phospholipid membranes and adopts an α-helical structure. This conformational change is also induced by interaction with sodium dodecyl sulfate (SDS), which is an anionic surfactant used in previous studies to mimic membrane binding. However, while the structure of the αSyn and SDS complex has been studied widely by various static measurements, the process of structural change from the denatured state to the folded state remains unclear. In this study, the interaction dynamics between αSyn and SDS micelles was investigated using time-resolved measurements with a micro-stopped-flow system, which has been recently developed. In particular, the time-resolved diffusion based on the transient grating technique in combination with a micro-stopped-flow system revealed the gradual change in diffusion triggered by the presence of SDS micelles. This change is induced not only by binding to SDS micelles, but also by an intramolecular conformational change. It was interesting to find that the diffusion coefficient decreased in an intermediate state and then increased to the final state in the binding reaction. We also carried out stopped-flow-kinetic measurements of circular dichroism and intramolecular fluorescence resonance energy transfer, and the D change was assigned to the formation of a compact structure derived from the helix bending on the micelle.

DOI: https://doi.org/10.1039/d0ra09614h
Int J Mol Sci. 2022 Mar 24. pii: 3547. [Epub ahead of print]23(7):

In Silico Characterisation of the Late Embryogenesis Abundant (LEA) Protein Families and Their Role in Desiccation Tolerance in Ramonda serbica Panc.

Ana Pantelić, Strahinja Stevanović, Sonja Milić Komić, Nataša Kilibarda, Marija Vidović.

  Ramonda serbica Panc. is an ancient resurrection plant able to survive a long desiccation period and recover metabolic functions upon watering. The accumulation of protective late embryogenesis abundant proteins (LEAPs) is a desiccation tolerance hallmark. To propose their role in R. serbica desiccation tolerance, we structurally characterised LEAPs and evaluated LEA gene expression levels in hydrated and desiccated leaves. By integrating de novo transcriptomics and homologues LEAP domains, 318 R. serbica LEAPs were identified and classified according to their conserved motifs and phylogeny. The in silico analysis revealed that hydrophilic LEA4 proteins exhibited an exceptionally high tendency to form amphipathic α-helices. The most abundant, atypical LEA2 group contained more hydrophobic proteins predicted to fold into the defined globular domains. Within the desiccation-upregulated LEA genes, the majority encoded highly disordered DEH1, LEA1, LEA4.2, and LEA4.3 proteins, while the greatest portion of downregulated genes encoded LEA2.3 and LEA2.5 proteins. While dehydrins might chelate metals and bind DNA under water deficit, other intrinsically disordered LEAPs might participate in forming intracellular proteinaceous condensates or adopt amphipathic α-helical conformation, enabling them to stabilise desiccation-sensitive proteins and membranes. This comprehensive LEAPs structural characterisation is essential to understanding their function and regulation during desiccation aiming at crop drought tolerance improvement.

Keywords:  3D protein structure modelling; de novo transcriptome assembly; differentially expressed gene analysis; drought; intrinsically disordered proteins; liquid–liquid phase separation; resurrection plants; secondary structure prediction

DOI:  https://doi.org/10.3390/ijms23073547
Cell Host Microbe. 2022 Apr 05. pii: S1931-3128(22)00149-4. [Epub ahead of print]

Bacterial hydrophilins promote pathogen desiccation tolerance.

Erin R Green, Joseph N Fakhoury, Andrew J Monteith, Hualiang Pi, David P Giedroc, Eric P Skaar.

  Acinetobacter baumannii is a leading cause of hospital-acquired infections, where outbreaks are driven by its ability to persist on surfaces in a desiccated state. Here, we show that A. baumannii causes more virulent pneumonia following desiccation and profile the genetic requirements for desiccation. We find that desiccation tolerance is enhanced upon the disruption of Lon protease, which targets unfolded and aggregated proteins for degradation. Notably, two bacterial hydrophilins, DtpA and DtpB, are transcriptionally upregulated in Δlon via the two-component regulator, BfmR. These proteins, both hydrophilic and intrinsically disordered, promote desiccation tolerance in A. baumannii. Additionally, recombinant DtpA protects purified enzymes from inactivation and improves the desiccation tolerance of a probiotic bacterium when heterologously expressed. These results demonstrate a connection between environmental persistence and pathogenicity in A. baumannii, provide insight into the mechanisms of extreme desiccation tolerance, and reveal potential applications for bacterial hydrophilins in the preservation of protein- and live bacteria-based pharmaceuticals.

Keywords:  Acinetobacter baumannii; BfmR; DtpA; DtpB; IDP; Lon; desiccation; hydrophilin

DOI:  https://doi.org/10.1016/j.chom.2022.03.019
Int J Biol Macromol. 2022 Apr 08. pii: S0141-8130(22)00732-2. [Epub ahead of print]

Zinc enhances liquid-liquid phase separation of Tau protein and aggravates mitochondrial damages in cells.

Ying-Ying Gao, Tao Zhong, Li-Qiang Wang, Na Zhang, Yan Zeng, Ji-Ying Hu, Hai-Bin Dang, Jie Chen, Yi Liang.

  Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer's disease. Liquid-liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregation can then be enhanced by zinc. However, it is unclear whether zinc modulates the formation of Tau stress granules in cells. We herein report that zinc promotes the formation of stress granules containing a pathological mutant ΔK280 of full-length human Tau. Furthermore, zinc promotes LLPS of ΔK280 of full-length Tau, shifting the equilibrium phase boundary to a lower protein concentration, and modulates the liquid nature of droplets formed by this pathological mutation. Zinc also promotes pathological phosphorylation of ΔK280 in neuronal cells, and aggravates mitochondrial damage and elevates reactive oxygen species production induced by Tau aggregation. Importantly, we show that treatment of cells with zinc increases the interaction between full-length Tau and G3BP1 inside stress granules to promote the formation of Tau filaments and increase Tau toxicity in neuronal cells. Collectively, these results demonstrate how Tau condensation and mitochondrial damages induced by Tau aggregation are enhanced by zinc to deteriorate the pathogenesis of Alzheimer's disease, bridging the gap between Tau LLPS and aggregation in neuronal cells.

Keywords:  Alzheimer's disease; Protein aggregation; Protein liquid-liquid phase separation; Tau protein; Tau toxicity; Zinc

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.04.034