bims-indpro 2022-02-27 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–02–27
nineteen papers selected by
Sara Mingu, Johannes Gutenberg University

Intrinsically Disordered Proteins: Perspective on COVID-19 Infection and Drug Discovery.
Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection.
The Disordered Dehydrin and Its Role in Plant Protection: A Biochemical Perspective.
The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling.
Translocation, Rejection and Trapping of Polyampholytes.
Regulatory Roles of the N-Terminal Intrinsically Disordered Region of Modular Src.
Protein Dynamics to Define and Refine Disordered Protein Ensembles.
CRL4Cdt2 Ubiquitin Ligase, A Genome Caretaker Controlled by Cdt2 Binding to PCNA and DNA.
On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains.
A FRET-Based Biosensor for the Src N-Terminal Regulatory Element.
Liquid-Liquid Phase Separation of TDP-43 and FUS in Physiology and Pathology of Neurodegenerative Diseases.
The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.
The epigenetic dimension of protein structure.
Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1.
Casein kinase 1 and disordered clock proteins form functionally equivalent, phospho-based circadian modules in fungi and mammals.
Functional organization of RNA polymerase II in nuclear subcompartments.
Identification of intrinsically disorder regions in non-structural proteins of SARS-CoV-2: New insights into drug and vaccine resistance.
EGCG Promotes FUS Condensate Formation in a Methylation-Dependent Manner.
Poly(ADP-ribose) drives condensation of FUS via a transient interaction.

ACS Infect Dis. 2022 Feb 23.

Intrinsically Disordered Proteins: Perspective on COVID-19 Infection and Drug Discovery.

Rumiana Tenchov, Qiongqiong Angela Zhou.

  Since the beginning of the COVID-19 pandemic caused by SARS-CoV-2, millions of patients have been diagnosed and many of them have died from the disease worldwide. The identification of novel therapeutic targets are of utmost significance for prevention and treatment of COVID-19. SARS-CoV-2 is a single-stranded RNA virus with a 30 kb genome packaged into a membrane-enveloped virion, transcribing several tens of proteins. The belief that the amino acid sequence of proteins determines their 3D structure which, in turn, determines their function has been a central principle of molecular biology for a long time. Recently, it has been increasingly realized, however, that there is a large group of proteins that lack a fixed or ordered 3D structure, yet they exhibit important biological activities─so-called intrinsically disordered proteins and protein regions (IDPs/IDRs). Disordered regions in viral proteins are generally associated with viral infectivity and pathogenicity because they endow the viral proteins the ability to easily and promiscuously bind to host proteins; therefore, the proteome of SARS-CoV-2 has been thoroughly examined for intrinsic disorder. It has been recognized that, in fact, the SARS-CoV-2 proteome exhibits significant levels of structural order, with only the nucleocapsid (N) structural protein and two of the nonstructural proteins being highly disordered. The spike (S) protein of SARS-CoV-2 exhibits significant levels of structural order, yet its predicted percentage of intrinsic disorder is still higher than that of the spike protein of SARS-CoV. Noteworthy, however, even though IDPs/IDRs are not common in the SARS-CoV-2 proteome, the existing ones play major roles in the functioning and virulence of the virus and are thus promising drug targets for rational antiviral drug design. Presented here is a COVID-19 perspective on the intrinsically disordered proteins, summarizing recent results on the SARS-CoV-2 proteome disorder features, their physiological and pathological relevance, and their prominence as prospective drug target sites.

Keywords:  COVID-19; SARS-CoV-2; drug design; intrinsically disordered protein; nucleocapsid protein; proteome; spike protein

DOI:  https://doi.org/10.1021/acsinfecdis.2c00031
Elife. 2022 Feb 22. pii: e74884. [Epub ahead of print]11

Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection.

Xiao-Dong Fang, Qiang Gao, Ying Zang, Ji-Hui Qiao, Dong-Min Gao, Wen-Ya Xu, Ying Wang, Dawei Li, Xian-Bing Wang.

  Liquid-liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infection is largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and the 5' trailer of genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host casein kinase 1 (CK1)-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted viroplasm formation and virus replication. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state for virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of the viral P protein and virus infection.

Keywords:  plant biology; viruses

DOI:  https://doi.org/10.7554/eLife.74884
Biomolecules. 2022 Feb 11. pii: 294. [Epub ahead of print]12(2):

The Disordered Dehydrin and Its Role in Plant Protection: A Biochemical Perspective.

Margaret A Smith, Steffen P Graether.

  Dehydrins are intrinsically disordered proteins composed of several well conserved sequence motifs known as the Y-, S-, F-, and K-segments, the latter of which is a defining feature of all dehydrins. These segments are interspersed by regions of low sequence conservation and are organized modularly, which results in seven different architectures: Kn, SKn, YnSKn, YnKn, KnS, FnK and FnSKn. Dehydrins are expressed ubiquitously throughout the plant kingdom during periods of low intracellular water content, and are capable of improving desiccation tolerance in plants. In vitro evidence of dehydrins shows that they are involved in the protection of membranes, proteins and DNA from abiotic stresses. However, the molecular mechanisms by which these actions are achieved are as of yet somewhat unclear. With regards to macromolecule cryoprotection, there is evidence to suggest that a molecular shield-like protective effect is primarily influenced by the hydrodynamic radius of the dehydrin and to a lesser extent by the charge and hydrophobicity. The interaction between dehydrins and membranes is thought to be a surface-level, charge-based interaction that may help to lower the transition temperature, allowing membranes to maintain fluidity at low temperatures and preventing membrane fusion. In addition, dehydrins are able to protect DNA from damage, showing that these abiotic stress protection proteins have multiple roles.

Keywords:  abiotic stress; cryoprotection; dehydration; dehydrins; intrinsically disordered; structure

DOI:  https://doi.org/10.3390/biom12020294
Nucleic Acids Res. 2022 Feb 21. pii: gkac087. [Epub ahead of print]

The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling.

Alexandra Bergfort, Tarek Hilal, Benno Kuropka, İbrahim Avşar Ilik, Gert Weber, Tuğçe Aktaş, Christian Freund, Markus C Wahl.

Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling.

DOI: https://doi.org/10.1093/nar/gkac087
Polymers (Basel). 2022 Feb 18. pii: 797. [Epub ahead of print]14(4):

Translocation, Rejection and Trapping of Polyampholytes.

Yeong-Beom Kim, Min-Kyung Chae, Jeong-Man Park, Albert Johner, Nam-Kyung Lee.

  Polyampholytes (PA) are a special class of polymers comprising both positive and negative monomers along their sequence. Most proteins have positive and negative residues and are PAs. Proteins have a well-defined sequence while synthetic PAs have a random charge sequence. We investigated the translocation behavior of random polyampholyte chains through a pore under the action of an electric field by means of Monte Carlo simulations. The simulations incorporated a realistic translocation potential profile along an extended asymmetric pore and translocation was studied for both directions of engagement. The study was conducted from the perspective of statistics for disordered systems. The translocation behavior (translocation vs. rejection) was recorded for all 220 sequences comprised of N = 20 charged monomers. The results were compared with those for 107 random sequences of N = 40 to better demonstrate asymptotic laws. At early times, rejection was mainly controlled by the charge sequence of the head part, but late translocation/rejection was governed by the escape from a trapped state over an antagonistic barrier built up along the sequence. The probability distribution of translocation times from all successful attempts revealed a power-law tail. At finite times, there was a population of trapped sequences that relaxed very slowly (logarithmically) with time. If a subensemble of sequences with prescribed net charge was considered the power-law decay was steeper for a more favorable net charge. Our findings were rationalized by theoretical arguments developed for long chains. We also provided operational criteria for the translocation behavior of a sequence, explaining the selection by the translocation process. From the perspective of protein translocation, our findings can help rationalize the behavior of intrinsically disordered proteins (IDPs), which can be modeled as polyampholytes. Most IDP sequences have a strong net charge favoring translocation. Even for sequences with those large net charges, the translocation times remained very dispersed and the translocation was highly sequence-selective.

Keywords:  Monte Carlo simulation; drift-diffusion; polyampholytes; probability distribution function; translocation

DOI:  https://doi.org/10.3390/polym14040797
Int J Mol Sci. 2022 Feb 17. pii: 2241. [Epub ahead of print]23(4):

Regulatory Roles of the N-Terminal Intrinsically Disordered Region of Modular Src.

Goro Kato.

  Src, the prototype of Src family kinases (SFKs), is a modular protein consisting of SH4 (SH4) and unique (UD) domains in an N-terminal intrinsically disordered region (IDR), and SH3, SH2, and kinase (KD) folded domains conserved among SFKs. Src functions as a pleiotropic signaling hub in proliferating and post-mitotic cells, and it is related to cancer and neurological diseases. However, its regulatory mechanism is unclear because the existing canonical model is derived from crystallographic analyses of folded constructs lacking the IDR. This work reviews nuclear magnetic resonance analyses of partially structured lipid-binding segments in the flexible UD and the fuzzy intramolecular complex (FIMC) comprising IDR and SH3 domains, which interacts with lipid membranes and proteins. Furthermore, recently determined IDR-related Src characteristics are discussed, including dimerization, SH4/KD intramolecular fastener bundling of folded domains, and the sorting of adhesive structures. Finally, the modulatory roles of IDR phosphorylation in Src activities involving the FIMC are explored. The new regulatory roles of IDRs are integrated with the canonical model to elucidate the functions of full-length Src. This review presents new aspects of Src regulation, and provides a future direction for studies on the structure and function of Src, and their implications for pathological processes.

Keywords:  SH3 domain; SH4 domain; Src; canonical model; fuzzy intramolecular complex (FIMC); intrinsically disordered region (IDR); kinase domain; nuclear magnetic resonance (NMR); phosphorylation; unique domain (UD)

DOI:  https://doi.org/10.3390/ijms23042241
J Phys Chem B. 2022 Feb 25.

Protein Dynamics to Define and Refine Disordered Protein Ensembles.

Pavithra M Naullage, Mojtaba Haghighatlari, Ashley Namini, João M C Teixeira, Jie Li, Oufan Zhang, Claudiu C Gradinaru, Julie D Forman-Kay, Teresa Head-Gordon.

Intrinsically disordered proteins and unfolded proteins have fluctuating conformational ensembles that are fundamental to their biological function and impact protein folding, stability, and misfolding. Despite the importance of protein dynamics and conformational sampling, time-dependent data types are not fully exploited when defining and refining disordered protein ensembles. Here we introduce a computational framework using an elastic network model and normal-mode displacements to generate a dynamic disordered ensemble consistent with NMR-derived dynamics parameters, including transverse R2 relaxation rates and Lipari-Szabo order parameters (S2 values). We illustrate our approach using the unfolded state of the drkN SH3 domain to show that the dynamical ensembles give better agreement than a static ensemble for a wide range of experimental validation data including NMR chemical shifts, J-couplings, nuclear Overhauser effects, paramagnetic relaxation enhancements, residual dipolar couplings, hydrodynamic radii, single-molecule fluorescence Förster resonance energy transfer, and small-angle X-ray scattering.

DOI: https://doi.org/10.1021/acs.jpcb.1c10925
Genes (Basel). 2022 Jan 29. pii: 266. [Epub ahead of print]13(2):

CRL4Cdt2 Ubiquitin Ligase, A Genome Caretaker Controlled by Cdt2 Binding to PCNA and DNA.

Muadz Ahmad Mazian, Kumpei Yamanishi, Mohd Zulhilmi Abdul Rahman, Menega Ganasen, Hideo Nishitani.

  The ubiquitin ligase CRL4Cdt2 plays a vital role in preserving genomic integrity by regulating essential proteins during S phase and after DNA damage. Deregulation of CRL4Cdt2 during the cell cycle can cause DNA re-replication, which correlates with malignant transformation and tumor growth. CRL4Cdt2 regulates a broad spectrum of cell cycle substrates for ubiquitination and proteolysis, including Cdc10-dependent transcript 1 or Chromatin licensing and DNA replication factor 1 (Cdt1), histone H4K20 mono-methyltransferase (Set8) and cyclin-dependent kinase inhibitor 1 (p21), which regulate DNA replication. However, the mechanism it operates via its substrate receptor, Cdc10-dependent transcript 2 (Cdt2), is not fully understood. This review describes the essential features of the N-terminal and C-terminal parts of Cdt2 that regulate CRL4 ubiquitination activity, including the substrate recognition domain, intrinsically disordered region (IDR), phosphorylation sites, the PCNA-interacting protein-box (PIP) box motif and the DNA binding domain. Drugs targeting these specific domains of Cdt2 could have potential for the treatment of cancer.

Keywords:  CDK phosphorylation; CRL4Cdt2; DNA binding domain; IDR; PIP box; cell cycle; intrinsically disordered region; ubiquitination

DOI:  https://doi.org/10.3390/genes13020266
Biomolecules. 2022 Jan 26. pii: 209. [Epub ahead of print]12(2):

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains.

Francesca Malagrinò, Valeria Pennacchietti, Daniele Santorelli, Livia Pagano, Caterina Nardella, Awa Diop, Angelo Toto, Stefano Gianni.

  The vast majority of our current knowledge about the biochemical and biophysical properties of proteins derives from in vitro studies conducted on isolated globular domains. However, a very large fraction of the proteins expressed in the eukaryotic cell are structurally more complex. In particular, the discovery that up to 40% of the eukaryotic proteins are intrinsically disordered, or possess intrinsically disordered regions, and are highly dynamic entities lacking a well-defined three-dimensional structure, revolutionized the structure-function paradigm and our understanding of proteins. Moreover, proteins are mostly characterized by the presence of multiple domains, influencing each other by intramolecular interactions. Furthermore, proteins exert their function in a crowded intracellular milieu, transiently interacting with a myriad of other macromolecules. In this review we summarize the literature tackling these themes from both the theoretical and experimental perspectives, highlighting the effects on protein folding and function that are played by (i) flanking disordered tails; (ii) contiguous protein domains; (iii) interactions with the cellular environment, defined as quinary structures. We show that, in many cases, both the folding and function of protein domains is remarkably perturbed by the presence of these interactions, pinpointing the importance to increase the level of complexity of the experimental work and to extend the efforts to characterize protein domains in more complex contexts.

Keywords:  IDPs; crowding; multidomain proteins; protein–protein interactions

DOI:  https://doi.org/10.3390/biom12020209
Biosensors (Basel). 2022 Feb 04. pii: 96. [Epub ahead of print]12(2):

A FRET-Based Biosensor for the Src N-Terminal Regulatory Element.

Guillermo Iruela, Alejandro Fernández, Amin Sagar, Francisco Javier Carvajal, Pau Bernadó, Miquel Pons.

  In signaling proteins, intrinsically disordered regions often represent regulatory elements, which are sensitive to environmental effects, ligand binding, and post-translational modifications. The conformational space sampled by disordered regions can be affected by environmental stimuli and these changes trigger, vis a vis effector domain, downstream processes. The disordered nature of these regulatory elements enables signal integration and graded responses but prevents the application of classical approaches for drug screening based on the existence of a fixed three-dimensional structure. We have designed a genetically encodable biosensor for the N-terminal regulatory element of the c-Src kinase, the first discovered protooncogene and lead representative of the Src family of kinases. The biosensor is formed by two fluorescent proteins forming a FRET pair fused at the two extremes of a construct including the SH4, unique and SH3 domains of Src. An internal control is provided by an engineered proteolytic site allowing the generation of an identical mixture of the disconnected fluorophores. We show FRET variations induced by ligand binding. The biosensor has been used for a high-throughput screening of a library of 1669 compounds with seven hits confirmed by NMR.

Keywords:  NMR; c-Src; fluorescence; fuzzy complexes; high-throughput screening; intrinsically disordered proteins (IDP)

DOI:  https://doi.org/10.3390/bios12020096
Front Mol Biosci. 2022 ;9 826719

Liquid-Liquid Phase Separation of TDP-43 and FUS in Physiology and Pathology of Neurodegenerative Diseases.

Jenny L Carey, Lin Guo.

  Liquid-liquid phase separation of RNA-binding proteins mediates the formation of numerous membraneless organelles with essential cellular function. However, aberrant phase transition of these proteins leads to the formation of insoluble protein aggregates, which are pathological hallmarks of neurodegenerative diseases including ALS and FTD. TDP-43 and FUS are two such RNA-binding proteins that mislocalize and aggregate in patients of ALS and FTD. They have similar domain structures that provide multivalent interactions driving their phase separation in vitro and in the cellular environment. In this article, we review the factors that mediate and regulate phase separation of TDP-43 and FUS. We also review evidences that connect the phase separation property of TDP-43 and FUS to their functional roles in cells. Aberrant phase transition of TDP-43 and FUS leads to protein aggregation and disrupts their regular cell function. Therefore, restoration of functional protein phase of TDP-43 and FUS could be beneficial for neuronal cells. We discuss possible mechanisms for TDP-43 and FUS aberrant phase transition and aggregation while reviewing the methods that are currently being explored as potential therapeutic strategies to mitigate aberrant phase transition and aggregation of TDP-43 and FUS.

Keywords:  ALS; FUS; TDP-43; liquid-liquid phase separation; stress granules

DOI:  https://doi.org/10.3389/fmolb.2022.826719
Int J Mol Sci. 2022 Feb 19. pii: 2315. [Epub ahead of print]23(4):

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.

Carlos Gil-Durán, Romina V Sepúlveda, Maximiliano Rojas, Víctor Castro-Fernández, Victoria Guixé, Inmaculada Vaca, Gloria Levicán, Fernando D González-Nilo, María-Cristina Ravanal, Renato Chávez.

  Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (β/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-β-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (β/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (β/α)8-barrel domain.

Keywords:  (β/α)8-barrel domain; GH10 endoxylanase; intrinsically disordered region; new activities; protein dynamics

DOI:  https://doi.org/10.3390/ijms23042315
Biomol Concepts. 2022 Feb 21. 13(1): 55-60

The epigenetic dimension of protein structure.

Fodil Azzaz, Jacques Fantini.

  Accurate prediction of protein structure is one of the most challenging goals of biology. The most recent achievement is AlphaFold, a machine learning method that has claimed to have solved the structure of almost all human proteins. This technological breakthrough has been compared to the sequencing of the human genome. However, this triumphal statement should be treated with caution, as we identified serious flaws in some AlphaFold models. Disordered regions are often represented by large loops that clash with the overall protein geometry, leading to unrealistic structures, especially for membrane proteins. In fact, AlphaFold comes up against the notion that protein folding is not solely determined by genomic information. We suggest that all parameters controlling the structure of a protein without being strictly encoded in its amino acid sequence should be coined "epigenetic dimension of protein structure." Such parameters include for instance protein solvation by membrane lipids, or the structuration of disordered proteins upon ligand binding, but exclude sequence-encoded sites of post-translational modifications such as glycosylation. In our view, this paradigm is necessary to reconcile two opposite properties of living systems: beyond rigorous biological coding, evolution has given way to a certain level of uncertainty and anarchy.

Keywords:  AlphaFold; Robetta; intrinsically disordered proteins; lipid; membrane; protein structure

DOI:  https://doi.org/10.1515/bmc-2022-0006
Antioxidants (Basel). 2022 Jan 27. pii: 243. [Epub ahead of print]11(2):

Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1.

Vy Ngo, Nadun C Karunatilleke, Anne Brickenden, Wing-Yiu Choy, Martin L Duennwald.

  Cells that experience high levels of oxidative stress respond by inducing antioxidant proteins through activation of the protein transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is negatively regulated by the E3 ubiquitin ligase Kelch-like ECH-associated protein 1 (Keap1), which binds to Nrf2 to facilitate its ubiquitination and ensuing proteasomal degradation under basal conditions. Here, we studied protein folding and misfolding in Nrf2 and Keap1 in yeast, mammalian cells, and purified proteins under oxidative stress conditions. Both Nrf2 and Keap1 are susceptible to protein misfolding and inclusion formation upon oxidative stress. We propose that the intrinsically disordered regions within Nrf2 and the high cysteine content of Keap1 contribute to their oxidation and the ensuing misfolding. Our work reveals previously unexplored aspects of Nrf2 and Keap1 regulation and/or dysregulation by oxidation-induced protein misfolding.

Keywords:  Keap1; Nrf2; oxidation; oxidative stress; protein misfolding

DOI:  https://doi.org/10.3390/antiox11020243
Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2118286119. [Epub ahead of print]119(9):

Casein kinase 1 and disordered clock proteins form functionally equivalent, phospho-based circadian modules in fungi and mammals.

Daniela Marzoll, Fidel E Serrano, Anton Shostak, Carolin Schunke, Axel C R Diernfellner, Michael Brunner.

  Circadian clocks are timing systems that rhythmically adjust physiology and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks are based on transcriptional-translational feedback loops (TTFLs). Yet TTFL-core components such as Frequency (FRQ) in Neurospora and Periods (PERs) in animals are not conserved, leaving unclear how a 24-h period is measured on the molecular level. Here, we show that CK1 is sufficient to promote FRQ and mouse PER2 (mPER2) hyperphosphorylation on a circadian timescale by targeting a large number of low-affinity phosphorylation sites. Slow phosphorylation kinetics rely on site-specific recruitment of Casein Kinase 1 (CK1) and access of intrinsically disordered segments of FRQ or mPER2 to bound CK1 and on CK1 autoinhibition. Compromising CK1 activity and substrate binding affects the circadian clock in Neurospora and mammalian cells, respectively. We propose that CK1 and the clock proteins FRQ and PERs form functionally equivalent, phospho-based timing modules in the core of the circadian clocks of fungi and animals.

Keywords:  CK1; FRQ; PER; circadian clock; intrinsically disordered

DOI:  https://doi.org/10.1073/pnas.2118286119
Curr Opin Cell Biol. 2022 Feb 22. pii: S0955-0674(22)00006-0. [Epub ahead of print]74 88-96

Functional organization of RNA polymerase II in nuclear subcompartments.

Karsten Rippe, Argyris Papantonis.

  Distinct clusters of RNA polymerase II are responsible for gene transcription inside eukaryotic cell nuclei. Despite the functional implications of such subnuclear organization, the attributes of these clusters and the mechanisms underlying their formation remain only partially understood. Recently, the concept of proteins and RNA phase-separating into liquid-like droplets was proposed to drive the formation of transcriptionally-active subcompartments. Here, we attempt to reconcile previous with more recent findings, and discuss how the different ways of assembling the active RNA polymerase II transcriptional machinery relate to nuclear compartmentalization.

Keywords:  Binding kinetics; Chromatin binding; Intrinsically disordered region; Looping; Nuclear organization; Phase separation; Transcription factor; Transcription factory; Transcriptional activation

DOI:  https://doi.org/10.1016/j.ceb.2022.01.005
Mol Cell Biochem. 2022 Feb 24.

Identification of intrinsically disorder regions in non-structural proteins of SARS-CoV-2: New insights into drug and vaccine resistance.

Farah Anjum, Taj Mohammad, Purva Asrani, Alaa Shafie, Shailza Singh, Dharmendra Kumar Yadav, Vladimir N Uversky, Md Imtaiyaz Hassan.

  The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 2019 and caused coronavirus disease 2019 (COVID-19), which causes pneumonia and severe acute respiratory distress syndrome. It is a highly infectious pathogen that promptly spread. Like other beta coronaviruses, SARS-CoV-2 encodes some non-structural proteins (NSPs), playing crucial roles in viral transcription and replication. NSPs likely have essential roles in viral pathogenesis by manipulating many cellular processes. We performed a sequence-based analysis of NSPs to get insights into their intrinsic disorders, and their functions in viral replication were annotated and discussed in detail. Here, we provide newer insights into the structurally disordered regions of SARS-CoV-2 NSPs. Our analysis reveals that the SARS-CoV-2 proteome has a chunk of the disordered region that might be responsible for increasing its virulence. In addition, mutations in these regions are presumably responsible for drug and vaccine resistance. These findings suggested that the structurally disordered regions of SARS-CoV-2 NSPs might be invulnerable in COVID-19.

Keywords:  COVID-19; COVID-19 therapeutics; Intrinsically disordered proteins; Molecular pathogenesis; SARS-CoV-2; Vaccine development

DOI:  https://doi.org/10.1007/s11010-022-04393-5
Cells. 2022 Feb 09. pii: 592. [Epub ahead of print]11(4):

EGCG Promotes FUS Condensate Formation in a Methylation-Dependent Manner.

Aneta J Lenard, Qishun Zhou, Corina Madreiter-Sokolowski, Benjamin Bourgeois, Hermann Habacher, Yukti Khanna, Tobias Madl.

  Millions of people worldwide are affected by neurodegenerative diseases (NDs), and to date, no effective treatment has been reported. The hallmark of these diseases is the formation of pathological aggregates and fibrils in neural cells. Many studies have reported that catechins, polyphenolic compounds found in a variety of plants, can directly interact with amyloidogenic proteins, prevent the formation of toxic aggregates, and in turn play neuroprotective roles. Besides harboring amyloidogenic domains, several proteins involved in NDs possess arginine-glycine/arginine-glycine-glycine (RG/RGG) regions that contribute to the formation of protein condensates. Here, we aimed to assess whether epigallocatechin gallate (EGCG) can play a role in neuroprotection via direct interaction with such RG/RGG regions. We show that EGCG directly binds to the RG/RGG region of fused in sarcoma (FUS) and that arginine methylation enhances this interaction. Unexpectedly, we found that low micromolar amounts of EGCG were sufficient to restore RNA-dependent condensate formation of methylated FUS, whereas, in the absence of EGCG, no phase separation could be observed. Our data provide new mechanistic roles of EGCG in the regulation of phase separation of RG/RGG-containing proteins, which will promote understanding of the intricate function of EGCG in cells.

Keywords:  EGCG; FUS; LLPS; RG/RGG; arginine methylation; neurodegenerative diseases

DOI:  https://doi.org/10.3390/cells11040592
Mol Cell. 2022 Feb 12. pii: S1097-2765(22)00061-2. [Epub ahead of print]

Poly(ADP-ribose) drives condensation of FUS via a transient interaction.

Kevin Rhine, Morgan Dasovich, Joseph Yoniles, Mohsen Badiee, Sophie Skanchy, Laura R Ganser, Yingda Ge, Charlotte M Fare, James Shorter, Anthony K L Leung, Sua Myong.

  Poly(ADP-ribose) (PAR) is an RNA-like polymer that regulates an increasing number of biological processes. Dysregulation of PAR is implicated in neurodegenerative diseases characterized by abnormal protein aggregation, including amyotrophic lateral sclerosis (ALS). PAR forms condensates with FUS, an RNA-binding protein linked with ALS, through an unknown mechanism. Here, we demonstrate that a strikingly low concentration of PAR (1 nM) is sufficient to trigger condensation of FUS near its physiological concentration (1 μM), which is three orders of magnitude lower than the concentration at which RNA induces condensation (1 μM). Unlike RNA, which associates with FUS stably, PAR interacts with FUS transiently, triggering FUS to oligomerize into condensates. Moreover, inhibition of a major PAR-synthesizing enzyme, PARP5a, diminishes FUS condensation in cells. Despite their structural similarity, PAR and RNA co-condense with FUS, driven by disparate modes of interaction with FUS. Thus, we uncover a mechanism by which PAR potently seeds FUS condensation.

Keywords:  FUS; LLPS; PARP5a; PARylation; RNA; condensation; length dependence; poly(ADP-ribose); stress response; transient interaction

DOI:  https://doi.org/10.1016/j.molcel.2022.01.018