Quantification of nuclear transport inhibition by SARS-CoV-2 ORF6 using a broadly applicable live-cell dose-response pipeline

Mapping Intimacies ◽

10.1101/2021.12.10.472151 ◽

2021 ◽

Author(s):

Tae Yeon Yoo ◽

Timothy Mitchison

Keyword(s):

Dose Response ◽

Quantitative Measurement ◽

Nuclear Transport ◽

Terminal Region ◽

Live Cell ◽

Nuclear Pore ◽

Antiviral Responses ◽

Inhibitory Function ◽

Binding Function

SARS coronavirus ORF6 inhibits the classical nuclear import pathway to antagonize host antiviral responses. Several models were proposed to explain its inhibitory function, but quantitative measurement is needed for model evaluation and refinement. We report a broadly applicable live-cell method for calibrated dose-response characterization of the nuclear transport alteration by a protein of interest. Using this method, we found that SARS-CoV-2 ORF6 is ~5 times more potent than SARS-CoV-1 ORF6 in inhibiting bidirectional nuclear transport, due to differences in the NUP98-binding C-terminal region that is required for the inhibition. The N-terminal region was also required, but its membrane binding function was dispensable, since loss of the inhibitory function due to N-terminal truncation was rescued by forced oligomerization using a soluble construct. Based on these data, we propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the FG domains of NUP98 at the nuclear pore complex.

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Importin β contains a COOH-terminal nucleoporin binding region important for nuclear transport

The Journal of Cell Biology ◽

10.1083/jcb.200303085 ◽

2003 ◽

Vol 162 (3) ◽

pp. 391-401 ◽

Cited By ~ 97

Author(s):

Janna Bednenko ◽

Gino Cingolani ◽

Larry Gerace

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Binding Sites ◽

Nuclear Transport ◽

Terminal Region ◽

Nuclear Pore ◽

Binding Region ◽

Similar Extent ◽

Importin Α ◽

Import Receptor

Proteins containing a classical NLS are transported into the nucleus by the import receptor importin β, which binds to cargoes via the adaptor importin α. The import complex is translocated through the nuclear pore complex by interactions of importin β with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin β. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin β to a similar extent (∼50%). An importin β mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin β possesses two nucleoporin binding sites, both of which are important for its nuclear import function.

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Structural characterization of human importin alpha 7 in its cargo-free form at 2.5 Å resolution

Scientific Reports ◽

10.1038/s41598-021-03729-3 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

S. Tsimbalyuk ◽

C. M. Donnelly ◽

J. K. Forwood

Keyword(s):

Nuclear Transport ◽

Free Form ◽

Nuclear Pore ◽

Importin Alpha ◽

Localization Signal ◽

Health And Disease ◽

Isoform Specificity ◽

Alpha 7 ◽

Structural Insights

AbstractShuttling of macromolecules between nucleus and cytoplasm is a tightly regulated process mediated through specific interactions between cargo and nuclear transport proteins. In the classical nuclear import pathway, importin alpha recognizes cargo exhibiting a nuclear localization signal, and this complex is transported through the nuclear pore complex by importin beta. Humans possess seven importin alpha isoforms that can be grouped into three subfamilies, with many cargoes displaying specificity towards these importin alpha isoforms. The cargo binding sites within importin alpha isoforms are highly conserved in sequence, suggesting that specificity potentially relies on structural differences. Structures of some importin alpha isoforms, both in cargo-bound and free states, have been previously solved. However, there are currently no known structures of cargo free importin alpha isoforms within subfamily 3 (importin alpha 5, 6, 7). Here, we present the first crystal structure of human importin alpha 7 lacking the IBB domain solved at 2.5 Å resolution. The structure reveals a typical importin alpha architecture comprised of ten armadillo repeats and is most structurally conserved with importin alpha 5. Very little difference in structure was observed between the cargo-bound and free states, implying that importin alpha 7 does not undergo conformational change when binding cargo. These structural insights provide a strong platform for further evaluation of structure–function relationships and understanding how isoform specificity within the importin alpha family plays a role in nuclear transport in health and disease.

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Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719398115 ◽

2018 ◽

Vol 115 (17) ◽

pp. E3969-E3977 ◽

Cited By ~ 36

Author(s):

Sasikumar Rajoo ◽

Pascal Vallotton ◽

Evgeny Onischenko ◽

Karsten Weis

Keyword(s):

Nuclear Pore Complex ◽

Yeast Cells ◽

Nuclear Pore ◽

Altered Expression ◽

Copy Numbers ◽

Quantitative Image ◽

Multiple Copies ◽

Almost All ◽

High Degree

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

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Optically transparent vertical silicon nanowire arrays for live-cell imaging

Journal of Nanobiotechnology ◽

10.1186/s12951-021-00795-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Roey Elnathan ◽

Andrew W. Holle ◽

Jennifer Young ◽

Marina A. George ◽

Omri Heifler ◽

...

Keyword(s):

High Efficiency ◽

Silicon Nanowire ◽

Live Cell ◽

Si Nanowires ◽

Contrast Imaging ◽

Silicon Nanowire Arrays ◽

Transparent Substrate ◽

Optically Transparent ◽

Vertically Aligned

AbstractProgrammable nano-bio interfaces driven by tuneable vertically configured nanostructures have recently emerged as a powerful tool for cellular manipulations and interrogations. Such interfaces have strong potential for ground-breaking advances, particularly in cellular nanobiotechnology and mechanobiology. However, the opaque nature of many nanostructured surfaces makes non-destructive, live-cell characterization of cellular behavior on vertically aligned nanostructures challenging to observe. Here, a new nanofabrication route is proposed that enables harvesting of vertically aligned silicon (Si) nanowires and their subsequent transfer onto an optically transparent substrate, with high efficiency and without artefacts. We demonstrate the potential of this route for efficient live-cell phase contrast imaging and subsequent characterization of cells growing on vertically aligned Si nanowires. This approach provides the first opportunity to understand dynamic cellular responses to a cell-nanowire interface, and thus has the potential to inform the design of future nanoscale cellular manipulation technologies.

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KCTD proteins as Cullin3 E3 Ligase adapters

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314096946 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C305-C305

Author(s):

Alan Ji ◽

Gilbert Privé

Keyword(s):

Crystal Structure ◽

Structural Characterization ◽

Dissociation Constants ◽

E3 Ligase ◽

Terminal Region ◽

Btb Domain ◽

Substrate Protein ◽

Ubiquitin E3 Ligase ◽

Ubiquitin Moiety

Cullin3 (Cul3) is an ubiquitin E3 ligase responsible for catalyzing the transfer of an ubiquitin moiety from an E2 enzyme to a target substrate protein. The C-terminal region of Cul3 binds RBX1/E2-ubiquitin, while, the N-terminal region interacts with various BTB domain proteins which serve as substrate adaptors. Previously, our group determined the crystal structures of the homodimeric BTB proteins SPOP and KLHL3 in complex with the N-terminal domain of Cul3, revealing the determinants responsible for the BTB/Cul3 interaction [1, 2]. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors but these do not share many of the previously determined features for Cul3 binding. Furthermore, KCTD proteins form homotetramers and homopentamers via BTB oligomerization rather than the previously described homodimers. Despite these differences, many KCTD proteins interact with Cul3 with dissociation constants of approximately 50 nM. While the target substrates for many of the KCTD/Cul3 E3 ligase complexes are unknown, recent studies have implicated the GABAβ2 receptor as an interactor of KCTD 8, 12, 12b and 16. Here, we report the pentameric crystal structure of the KCTD9 BTB domain and our progress on the structural characterization of Cul3/KCTD/substrate complexes.

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Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

Journal of Bacteriology ◽

10.1128/jb.187.15.5067-5074.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5067-5074 ◽

Cited By ~ 51

Author(s):

Daisuke Kasai ◽

Eiji Masai ◽

Keisuke Miyauchi ◽

Yoshihiro Katayama ◽

Masao Fukuda

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Terminal Region ◽

Ring Cleavage ◽

Sphingomonas Paucimobilis ◽

Acid Protein ◽

Demethylation Pathway ◽

Dioxygenase Gene ◽

Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

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Microwave Atomic Force Microscopy: Quantitative Measurement and Characterization of Electrical Properties on the Nanometer Scale

Applied Physics Express ◽

10.1143/apex.5.016602 ◽

2011 ◽

Vol 5 (1) ◽

pp. 016602 ◽

Cited By ~ 8

Author(s):

Lan Zhang ◽

Yang Ju ◽

Atsushi Hosoi ◽

Akifumi Fujimoto

Keyword(s):

Atomic Force Microscopy ◽

Electrical Properties ◽

Quantitative Measurement ◽

Nanometer Scale ◽

Force Microscopy ◽

Atomic Force

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Characterization of antral gastrin cells with region-specific antisera.

Journal of Histochemistry & Cytochemistry ◽

10.1177/25.12.925341 ◽

1977 ◽

Vol 25 (12) ◽

pp. 1317-1321 ◽

Cited By ~ 61

Author(s):

L I Larsson ◽

J F Rehfeld

Keyword(s):

Cross Reactivity ◽

Terminal Region ◽

Gastrin Cells ◽

Related Peptide ◽

Specific Antisera

A number of gastrin antisera, which in radioimmunoassay systems showed no or negligible cross-reactivity towards the structurally and functionally related peptide cholecystokinin were found to react with both gastrin and cholecystokinin cells when used for immunocytochemistry. This discrepancy was shown to be due either to reactivity against a COOH-terminal region common to gastrin and cholecystokinin or to the occurrence of heterogenous antibody populations in the antisera. By differential absorptions the latter type of antisera could be rendered specific for gastrin. Antisera reactive against the NH2-terminal, middle or COOH-terminal regions of human heptadecapeptide gastrin were prepared and together with a specific cholecystokinin antiserum used for the characterization of antral gastrin cells of different species. The results indicate that only the COOH-terminal region of gastrin is conserved during evolution.

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