scholarly journals Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus

2021 ◽  
Vol 22 (7) ◽  
pp. 3310
Author(s):  
Hareth A. Al-Wassiti ◽  
David R. Thomas ◽  
Kylie M. Wagstaff ◽  
Stewart A. Fabb ◽  
David A. Jans ◽  
...  
Keyword(s):  
Confocal Imaging ◽  
Nuclear Localisation Signal ◽  
Nuclear Entry ◽  
Terminal Protein ◽  
Nuclear Localisation ◽  
Host Interactions ◽  
Nuclear Uptake ◽  
Covalently Linked ◽  
Nuclear Shuttling ◽  
Future Work

Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.

scholarly journals Targeting novel LSD1-dependent ACE2 demethylation domains inhibits SARS-CoV-2 replication

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Wen Juan Tu ◽  
Robert D. McCuaig ◽  
Michelle Melino ◽  
Daniel J. Rawle ◽  
Thuy T. Le ◽  
...  
Keyword(s):  
Viral Replication ◽  
Treatment Options ◽  
Critical Role ◽  
Post Exposure Prophylaxis ◽  
Nuclear Entry ◽  
Importin Α ◽  
Active Transcription ◽  
Nuclear Shuttling ◽  
Asymptomatic Phase ◽  
Exposure Prophylaxis

AbstractTreatment options for COVID-19 remain limited, especially during the early or asymptomatic phase. Here, we report a novel SARS-CoV-2 viral replication mechanism mediated by interactions between ACE2 and the epigenetic eraser enzyme LSD1, and its interplay with the nuclear shuttling importin pathway. Recent studies have shown a critical role for the importin pathway in SARS-CoV-2 infection, and many RNA viruses hijack this axis to re-direct host cell transcription. LSD1 colocalized with ACE2 at the cell surface to maintain demethylated SARS-CoV-2 spike receptor-binding domain lysine 31 to promote virus–ACE2 interactions. Two newly developed peptide inhibitors competitively inhibited virus–ACE2 interactions, and demethylase access to significantly inhibit viral replication. Similar to some other predominantly plasma membrane proteins, ACE2 had a novel nuclear function: its cytoplasmic domain harbors a nuclear shuttling domain, which when demethylated by LSD1 promoted importin-α-dependent nuclear ACE2 entry following infection to regulate active transcription. A novel, cell permeable ACE2 peptide inhibitor prevented ACE2 nuclear entry, significantly inhibiting viral replication in SARS-CoV-2-infected cell lines, outperforming other LSD1 inhibitors. These data raise the prospect of post-exposure prophylaxis for SARS-CoV-2, either through repurposed LSD1 inhibitors or new, nuclear-specific ACE2 inhibitors.

scholarly journals Targeted cell imaging properties of a deep red luminescent iridium(iii) complex conjugated with a c-Myc signal peptide

2020 ◽  
Vol 11 (6) ◽  
pp. 1599-1606 ◽  
Author(s):  
Adam H. Day ◽  
Martin H. Übler ◽  
Hannah L. Best ◽  
Emyr Lloyd-Evans ◽  
Robert J. Mart ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Cell Imaging ◽  
Organometallic Complex ◽  
Nuclear Localisation Signal ◽  
Nuclear Localisation ◽  
Imaging Properties

Visualising a c-Myc nuclear localisation signal peptide using an organometallic complex.

scholarly journals Low-intensity vibration restores nuclear YAP levels and acute YAP nuclear shuttling in mesenchymal stem cells subjected to simulated microgravity

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Matthew Thompson ◽  
Kali Woods ◽  
Joshua Newberg ◽  
Julia Thom Oxford ◽  
Gunes Uzer
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Simulated Microgravity ◽  
Mechanical Forces ◽  
Cell Level ◽  
Nuclear Entry ◽  
Low Intensity ◽  
Nuclear Shuttling ◽  
Low Intensity Vibration ◽  
Daily Application

AbstractReducing the musculoskeletal deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of otherwise rigorous and time-expensive exercise regimens in space. The ability of low-intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC), which maintain bone-making osteoblasts, is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein), which is shuttled into the nucleus in response to cyto-mechanical forces. Here, using YAP nuclear shuttling as a measurement outcome, we tested the effect of 72 h of clinostat-induced simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied after the 72 h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry would be alleviated by the daily application of LIVDT. Results showed that while both acute LIVAT and LPA treatments increased nuclear YAP entry by 50 and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over SMG only, counterparts. These cell-level observations suggest that daily LIV treatments are a feasible countermeasure for restoring basal nuclear YAP levels and increasing the YAP nuclear shuttling in MSCs under SMG.

scholarly journals A functional bipartite nuclear localisation signal in the cytokine interleukin-5

FEBS Letters ◽  
1997 ◽  
Vol 406 (3) ◽  
pp. 315-320 ◽  
Author(s):  
David A. Jans ◽  
Lyndall J. Briggs ◽  
Sonja E. Gustin ◽  
Patricia Jans ◽  
Sally Ford ◽  
...  
Keyword(s):  
Nuclear Localisation Signal ◽  
Nuclear Localisation ◽  
Interleukin 5

scholarly journals Low Intensity Vibration Restores Nuclear YAP Levels and Acute YAP Nuclear Shuttling in Mesenchymal Stem Cells Subjected to Simulated Microgravity

2020 ◽  
Author(s):  
M Thompson ◽  
K Woods ◽  
J Newberg ◽  
JT Oxford ◽  
G Uzer
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Simulated Microgravity ◽  
Mechanical Forces ◽  
Cell Level ◽  
Nuclear Entry ◽  
Low Intensity ◽  
Nuclear Shuttling ◽  
Low Intensity Vibration ◽  
Daily Application

AbstractReducing the bone deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of rigorous and time-expensive exercise regimens under microgravity. The ability of low intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC) which maintain bone-making osteoblasts is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein) which is shuttled into the nucleus in response cyto-mechanical forces. Here, using YAP nuclear shuttling as a measure of MSC mechanoresponse, we tested the effect of 72 hours of simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied at the end of the 72h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry will be alleviated by daily application of LIVDT. Results showed that while both acute LIVAT and LPA treatments increased nuclear YAP entry by 50% and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. Daily dosing of LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over the non-LIVDT treated, SMG only, counterparts. These cell level observations suggest that utilizing daily LIV treatments is a feasible countermeasure for increasing the YAP-mediated anabolic responsiveness of MSCs to subsequent mechanical challenge under SMG.

scholarly journals Small molecule ERK5 kinase inhibitors paradoxically activate ERK5 signalling: be careful what you wish for…

2020 ◽  
Vol 48 (5) ◽  
pp. 1859-1875
Author(s):  
Simon J. Cook ◽  
Julie A. Tucker ◽  
Pamela A. Lochhead
Keyword(s):  
Protein Kinase ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Kinase Domain ◽  
Transactivation Domain ◽  
Nuclear Localisation Signal ◽  
Nuclear Localisation ◽  
Cancer And Inflammation

ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities (kinase and transcriptional) in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors.

Metabolic Processes Are Differentially Regulated During Wild-Type and Attenuated Dengue Virus Infection in Aedes aegypti

2022 ◽  
Author(s):  
Tanamas Siriphanitchakorn ◽  
Cassandra Modahl ◽  
R. Manjunatha Kini ◽  
Eng Eong Ooi ◽  
Milly Choy
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Denv Infection ◽  
Critical Event ◽  
Mosquito Vectors ◽  
Wild Type ◽  
Metabolic Processes ◽  
Transcriptomic Response ◽  
Host Interactions ◽  
Future Work

Successful completion of the dengue virus (DENV) life cycle in its mosquito vectors is important for efficient human–mosquito–human cycle of transmission, but the virus–mosquito interactions that underpin this critical event are poorly defined. To understand the virus–host interactions that determine viral infection by Aedes aegypti, the principal DENV vector, the authors compared transcriptomic changes in the head/thorax of the mosquito after intrathoracic infection with the wild-type DENV2 16681 strain and its attenuated derivative, PDK53. Using high-throughput RNA-sequencing, the authors identified 1,629 differentially expressed genes (DEGs) during 16681 infection, compared with only 22 DEGs identified during PDK53 infection, indicating that 16681 infection triggers a more robust host transcriptomic response compared with PDK53 infection. The authors further found that 16681 infection, but not PDK53 infection, altered metabolism in these heads/thoraces. Altogether, our findings reveal differential regulation of metabolic processes during wild-type and attenuated DENV infection, and suggest the need for future work to study the role of metabolic processes in determining DENV infection and replication in its mosquito vectors.

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