bims-nucpor 2021-10-31 papers

bims-nucpor

Biomed News

on Nuclear pore complex and nucleoporins in stress, aging and disease

Issue of 2021–10–31
seven papers selected by
Sara Mingu, Johannes Gutenberg University

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time.
Design of Multifunctional Nanopore Using Polyampholyte Brush with Composition Gradient.
Role of karyopherin nuclear transport receptors in nuclear transport by nuclear trafficking peptide.
Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking.
Upstream open reading frame with NOTCH2NLC GGC expansion generates polyglycine aggregates and disrupts nucleocytoplasmic transport: implications for polyglycine diseases.
Core Protein-Directed Antivirals and Importin β Can Synergistically Disrupt HBV Capsids.
Nuclear import of BCL11B is mediated by a classical nuclear localization signal and not the Krüppel-like zinc fingers.

Nat Commun. 2021 Oct 27. 12(1): 6211

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time.

Jan Andreas Ruland, Annika Marie Krüger, Kerstin Dörner, Rohan Bhatia, Sabine Wirths, Daniel Poetes, Ulrike Kutay, Jan Peter Siebrasse, Ulrich Kubitscheck.

Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s-1 in vivo.

DOI: https://doi.org/10.1038/s41467-021-26323-7
ACS Nano. 2021 Oct 28.

Design of Multifunctional Nanopore Using Polyampholyte Brush with Composition Gradient.

Shiyi Qin, Kai Huang, Igal Szleifer.

  Molecular organizations and charge patterns inside biological nanopores are optimized by evolution to enhance ionic and molecular transport. Inspired by the nuclear pore complex that employs asymmetrically arranged disordered proteins for its gating, we here design an artificial nanopore coated by an asymmetric polyampholyte brush as a model system to study the asymmetric mass transport under nanoconfinement. A nonequilibrium steady-state molecular theory is developed to account for the intricate charge regulation effect of the weak polyampholyte and to address the coupling between the polymer conformation and the external electric field. On the basis of this state-of-the-art theoretical method, we present a comprehensive theoretical description of the stimuli-responsive structural behaviors and transport properties inside the nanopore with all molecular details considered. Our model demonstrates that by incorporating a gradient of pH sensitivity into the polymer coatings of the nanopore, a variety of asymmetric charge patterns and functional structures can be achieved, in a pH-responsive manner that allows for multiple functions to be implemented into the designed system. The asymmetric charge pattern inside the nanopore leads to an electrostatic trap for major current carriers, which turns the nanopore into an ionic rectifier with a rectification factor above 1000 at optimized pH and salt concentration. Our theory further predicts that the nanopore design behaves like a double-gated nanofluidic device with pH-triggered opening of the gates, which can serve as an ion pump and pH-responsive molecular filter. These results deepen our understanding of asymmetric transport in nanoconfined systems and provide guidelines for designing polymer-coated smart nanopores.

Keywords:  asymmetry; composition gradient; molecular theory; nanopore; weak polyampholyte

DOI:  https://doi.org/10.1021/acsnano.1c05543
Exp Cell Res. 2021 Oct 22. pii: S0014-4827(21)00447-X. [Epub ahead of print]409(1): 112893

Role of karyopherin nuclear transport receptors in nuclear transport by nuclear trafficking peptide.

Yoichi Teratake, Yayoi Kimura, Yukihito Ishizaka.

  Nuclear trafficking peptide (NTP), a cell-penetrating peptide (CPP) composed of 10 amino acids (aa) (RIFIHFRIGC), has potent nuclear trafficking activity. Recently, we established a protein-based cell engineering system by using NTP, but it remained elusive how NTP functions as a CPP with nuclear orientation. In the present study, we identified importin subunit β1 (IMB1) and transportin 1 (TNPO1) as cellular proteins underlying the activity of NTP. These karyopherin nuclear transport receptors were identified as candidate molecules by liquid chromatography/mass spectrometry analysis, and downregulation of each protein by small interfering RNA significantly reduced NTP activity (P < 0.01). Biochemical analyses revealed that NTP bound directly to both molecules, and the forced expression of an IMB1 fragment (296-516 aa) or TNPO1 fragment (1-297 aa), which both contain binding sites to NTP, reduced nuclear NTP-green fluorescent protein (GFP) levels when it was added to cell culture medium. NTP is derived from viral protein R (Vpr) of human immunodeficiency virus-1, and Vpr enters the nucleus and exerts pleiotropic functions. Notably, Vpr bound directly to IMB1 and TNPO1, and its function was significantly impaired by the forced expression of the 296-516-aa fragment of IMB1 and 1-297-aa fragment of TNPO1. Interestingly, NTP completely blocked the physical association of Vpr with IMB1 and TNPO1. Although the nuclear localization mechanism of Vpr remains unknown, our data suggest that NTP functions as a novel nuclear localization signal of Vpr.

Keywords:  HIV-1; Importin b1; Nuclear cargo; Nuclear trafficking peptide; Transportin 1; Vpr

DOI:  https://doi.org/10.1016/j.yexcr.2021.112893
J Virol. 2021 Oct 27. JVI0162921

Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking.

Tingyu Peng, Xusheng Qiu, Lei Tan, Shengqing Yu, Binghuan Yang, Jun Dai, Xiaowen Liu, Yingjie Sun, Cuiping Song, Weiwei Liu, Chunchun Meng, Ying Liao, Weifeng Yuan, Tao Ren, Xiufan Liu, Chan Ding.

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M's nuclear-cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were deeply analyzed. Here, two types of combined NLS and NES signals were identified within NDV-M. The Herts/33-type M was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M was mostly retained in the nuclei and showed retarded VLP production. Two critical residues, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, the modification of which regulates the nuclear-cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued rLaSota strains rLaSota-R247K, -S263R, and -DM (double mutation) showed about twofold higher HA titers and 10-fold higher EID50 titers than wild-type (wt) rLaSota. Further, the MDT and ICPI values of those recombinant viruses were slightly higher than that of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV, and even those of all other paramyxoviruses. It is beneficial for the development of vaccines and therapies for paramyxoviruses. Importance Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked ND as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and opens up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach to improving paramyxovirus vaccines.

DOI: https://doi.org/10.1128/JVI.01629-21
Acta Neuropathol. 2021 Oct 25.

Upstream open reading frame with NOTCH2NLC GGC expansion generates polyglycine aggregates and disrupts nucleocytoplasmic transport: implications for polyglycine diseases.

Shaoping Zhong, Yangye Lian, Wenyi Luo, Rongkui Luo, Xiaoling Wu, Jun Ji, Yuan Ji, Jing Ding, Xin Wang.

  Neuronal intranuclear inclusion disease (NIID) is neurodegenerative disease characterized by widespread inclusions. Despite the identification of GGC repeat expansion in 5'UTR of NOTCH2NLC gene in adult-onset NIIDs, its pathogenic mechanism remains unclear. Gain-of-function poly-amino-acid proteins generated by unconventional translation have been revealed in nucleotide repeat expansion disorders, inspiring us to explore the possibility of unconventional translation in NIID. Here we demonstrated that NOTCH2NLC 5'UTR triggers the translation of a polyglycine (polyG)-containing protein, N2NLCpolyG. N2NLCpolyG accumulates in p62-positive inclusions in cultured cells, mouse models, and NIID patient tissues with NOTCH2NLC GGC expansion. Translation of N2NLCpolyG is initiated by an upstream open reading frame (uORF) embedding the GGC repeats. N2NLCpolyG tends to aggregate with the increase of GGC repeat units, and displays phase separation properties. N2NLCpolyG aggregation impairs nuclear lamina and nucleocytoplasmic transport but does not necessarily cause acute death on neuronal cells. Our study suggests a similarity of pathogenic mechanisms between NIID and another GGC-repeat disease, fragile X-associated tremor ataxia syndrome. These findings expand our knowledge of protein gain-of-function in NIID, and further highlight evidence for a novel spectrum of diseases caused by aberrant polyG protein aggregation, namely the polyG diseases.

Keywords:  GGC repeat expansion; NOTCH2NLC; Neuronal intranuclear inclusion disease; Nucleocytoplasmic transport; Polyglycine; Upstream open reading frame

DOI:  https://doi.org/10.1007/s00401-021-02375-3
J Virol. 2021 Oct 27. JVI0139521

Core Protein-Directed Antivirals and Importin β Can Synergistically Disrupt HBV Capsids.

Christine Kim, Lauren F Barnes, Christopher J Schlicksup, Angela J Patterson, Brian B Bothner, Martin F Jarrold, Che-Yen Joseph Wang, Adam Zlotnick.

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Impβ) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Impβ and trypsin in vitro. We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Impβ synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Impβ. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.

DOI: https://doi.org/10.1128/JVI.01395-21
J Cell Sci. 2021 Oct 29. pii: jcs.258655. [Epub ahead of print]

Nuclear import of BCL11B is mediated by a classical nuclear localization signal and not the Krüppel-like zinc fingers.

Piotr Grabarczyk, Martin Delin, Dorota Rogińska, Lukas Schulig, Hannes Forkel, Maren Depke, Andreas Link, Bogusław Machaliński, Christian Andreas Schmidt.

  The Krüppel-like transcription factor BCL11B is characterized by wide tissue distribution and crucial functions in key developmental and cellular processes and various pathologies including cancer or HIV infection. Although basics of BCL11B activity and relevant interactions with other proteins were uncovered, how this exclusively nuclear protein localizes to its compartment remained unclear. Here, we demonstrate that unlike other KLFs, BCL11B does not require the C-terminal DNA-binding domain to pass through the nuclear envelope but encodes an independent, previously unidentified nuclear localization signal (NLS) which is located distantly from the zinc finger domains and fulfills the essential criteria of an autonomous NLS. First, it can redirect a heterologous cytoplasmic protein to the nucleus. Second, its mutations cause aberrant localization of the protein of origin. Finally, we provide experimental and in silico evidences of the direct interaction with importin alpha. The relative conservation of this motif allows formulating a consensus sequence (K/R)K-X13-14-KR+K++ which can be found in all BCL11B orthologues among vertebrates and in the closely related protein BCL11A.

Keywords:  BCL11B; Krüppel-like transcription factors; Nuclear localization signal

DOI:  https://doi.org/10.1242/jcs.258655