Biology subjects

Fujisawa, R.

Publications and source records attributed to Fujisawa, R..

10 recordsLinked to original sources

bioRxiv2024

Wasted efforts impair random search efficiency and reduce choosiness in mate-pairing termites

Random search theories predict that animals employ movement patterns that optimize encounter rates with target resources. However, animals are not always able to achieve the best search strategy. Energy depletion, for example, limits searchers movement activities, forcing them to adjust their behaviors before and after encounters. Here, we quantified the cost of mate search in a termite, Reticulitermes speratus, and revealed that the searching cost reduces the selectivity of mating partners. After a dispersal flight, termites search for a mating partner with limited reserved energy. We found that their movement activity and diffusiveness progressively declined over extended mate search. Our data-based simulations qualitatively confirmed that the reduced movement diffusiveness decreased the searching efficiency. Also, prolonged search periods reduced colony foundation success and the number of offspring. Thus, mate search imposes doubled costs on termites. Finally, we found that termites with an extended mate search reduced the selectivity of mating partners, where males immediately paired with any encountering females. Thus, termites dramatically changed their mate search behavior depending on their physiological conditions. Our finding highlights that accounting for the searchers internal states is essential to fill the gap between random search theories and empirical behavioral observations.

ecology↗

bioRxiv2023

DONSON is required for CMG helicase assembly in the mammalian cell cycle

DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier-Gorlin Syndrome. The other 12 encode components of the CDC45-MCM-GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45-MCM-GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45-MCM-GINS during S-phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45-MCM-GINS helicase from S-phase cells and our data indicate that DONSON is dispensable for loading of the MCM2-7 helicase core onto chromatin during G1-phase, but instead is essential for CDC45-MCM-GINS assembly during S-phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier-Gorlin syndrome.

biochemistry↗

bioRxiv2022

Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

The unfolding of ubiquitylated proteins by the p97 / Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 is essential in many areas of eukaryotic cell biology. Previous studies showed that yeast Cdc48-Ufd1-Npl4 is governed by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here we show that substrate processing by mammalian p97-UFD1-NPL4 involves a complex interplay between ubiquitin chain length and additional p97 cofactors. Using disassembly of the ubiquitylated CMG helicase as a model in vitro system, we find that reconstituted p97-UFD1-NPL4 only unfolds substrates with very long ubiquitin chains. However, this high ubiquitin threshold is greatly reduced, to a level resembling yeast Cdc48-Ufd1-Npl4, by the UBXN7, FAF1 or FAF2 partners of mammalian p97-UFD1-NPL4. Stimulation by UBXN7/FAF1/FAF2 requires the UBX domain that connects each factor to p97, together with the ubiquitin-binding UBA domain of UBXN7 and a previously uncharacterised coiled-coil domain in FAF1/FAF2. Furthermore, we show that deletion of the UBXN7 and FAF1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

molecular biology↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

biochemistry↗

bioRxiv2021

Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. In order to identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play a key role in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5,000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified 4 compounds as potential inhibitors of nsp14, all of which also show antiviral capacity in a cell based model of SARS-CoV-2 infection. Three of the 4 compounds also exhibited synergistic effects on viral replication with remdesivir.

bioengineering↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp15 Endoribonuclease

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered the economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp14/nsp10 Exoribonuclease

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

biochemistry↗

bioRxiv2019

Dose old age lead to gluttony? Effects of aging on predation and locomotion in the assassin bug Amphibolus venator.

Animal behaviors are often affected by aging. In many insect species, locomotor activity decreases with aging. Foraging ability may also decrease with aging. However, few studies have investigated the effects of aging on both locomotor activity and foraging ability. In the present study, we tested the aging effect on locomotor activity and foraging ability in the assassin bug Amphibolus venator. The present results showed that locomotor activity decreased with age, similar to findings in many other animal species. However, foraging ability increased with age. Namely, the decline in locomotor activity with age did not lead to a decline in foraging ability. The positive relationship between foraging ability and age may be related to the type of predation, sit-and-wait, used by A. venator via alterations in investment in reproductive traits with age.

animal behavior and cognition↗

bioRxiv2019

DNA replication stress induced by trifluridine determines tumor cell fate according to p53 status

DNA replication stress is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analog-type chemotherapeutic drugs introduce DNA damage and exacerbate DNA replication stress in tumor cells. However, the mechanisms underlying tumor cytotoxicity triggered by the drugs are not fully understood. Here, we show that the fluorinated thymidine analog trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases. FTD acted as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand. At the cellular level, FTD decreased thymidine triphosphate in the dNTP pool and induced FTD triphosphate accumulation, resulting in replication fork stalling caused by FTD incorporation into DNA. DNA lesions involving single-stranded DNA were generated as a result of replication fork stalling, and the p53-p21 pathway was activated. Although FTD suppressed tumor cell growth irrespective of p53 status, tumor cell fate diverged at the G2/M phase transition according to p53 status; tumor cells with wild-type p53 underwent cellular senescence via mitosis skip, whereas tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results suggest that DNA replication stress induced by a nucleoside analog-type chemotherapeutic drug triggers tumor cytotoxicity by determining tumor cell fate according to p53 status.\n\nSignificanceThis study identified a unique type of DNA replication stress induced by trifluridine, which directs tumor cell fate either toward cellular senescence or apoptotic cell death according to p53 status.

cancer biology↗

bioRxiv2018

Regulatory mechanism predates the evolution of ant-like swarm intelligence in simulated robots

The evolution of complexity is one of the prime features of life on Earth. Although well accepted as the product of adaptation, the dynamics underlying the evolutionary build-up of complex adaptive systems remains poorly resolved. Using simulated robot swarms that exhibit ant-like group foraging with trail pheromones, we show that their swarm intelligence paradoxically involves regulatory behavior that arises in advance. We focused on a \"traffic rule\" on their foraging trail as a regulatory trait. We allowed the simulated robot swarms to evolve pheromone responsiveness and behaviors simultaneously. In most cases, the traffic rule, initially arising as selectively neutral component behaviors, assisted the group foraging system to bypass a fitness valley caused by overcrowding on the trail. Our study reveals a hitherto underappreciated role of regulatory mechanisms in the origin of swarm intelligence, as well as highlights the importance of embodiment in the study of their evolution.

evolutionary biology↗