Biology subjects

Remans, K.

Publications and source records attributed to Remans, K..

4 recordsLinked to original sources

bioRxiv2021

Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Decreased susceptibilities of Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of Kelch13, which is the homologue of the redox sensor Keap1 in vertebrates. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of Kelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of Kelch13 resulted in ring-stage survival rates around 40%. While the loss of a potential disulfide bond between residues C580 and C532 had no effect on the artemisinin suceptibility, the thiol group of C473 could not be replaced. We also established a protocol for the production of recombinant Kelch13. In contrast to cysteine-to-serine replacements, common field mutations resulted in misfolded and insoluble protein. In summary, not the redox properties but impaired folding of Kelch13, resulting in a decreased Kelch13 abundance, is the central parameter for mutant selection.

biochemistry↗

bioRxiv2021

Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 {degrees}C, respectively. This process is significantly slowed down at 4 {degrees}C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.

biochemistry↗

bioRxiv2021

Extensive OMICS resource for Sf21 and Tni cell lines

Insect-derived cell lines, from Spodoptera frugiperda (Sf21) and from Trichoplusia ni (Tni), are the two most widely used cell lines for recombinant protein expression in combination with the Baculoviral Expression Vector System (BEVS). Genomic sequences and annotations are still incomplete for Sf21 and absent for Tni. In this study, we present an approach using different sequencing data types, including short-read sequencing, long synthetic and Oxford Nanopore reads, to build genomes. The Sf21 and Tni assemblies contain 4,020 scaffolds of 463 Mb in size with N50 of 364 Kb and 2,954 scaffolds of 332 Mb in size with N50 of 326 Kb, respectively. Furthermore, we built a new gene prediction workflow, which integrates transcriptome and proteome information using pre-existing tools. Using this approach, we predicted 21,506 Sf21 and 14,159 Tni genes, generated and integrated proteomic datasets to validate predicted genes and could identify 5577 and 4919 proteins in the Sf21 and Tni cell lines respectively. This integrative approach could be theoretically applied to any uncharacterized genome and result in valuable new resources. With this information available, Sf21 and Tni cells will become even better tools for protein expression and could be used in a wider range of applications, from promoter identification to genome engineering and editing.

genomics↗

bioRxiv2020

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 {micro}g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the up ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

biophysics↗