Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.
- MeSH
- COVID-19 * MeSH
- glykoprotein S, koronavirus genetika MeSH
- lidé MeSH
- mutace MeSH
- SARS-CoV-2 genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.
- Publikační typ
- časopisecké články MeSH
Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.
In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.
- MeSH
- COVID-19 * MeSH
- fylogeneze MeSH
- glykoprotein S, koronavirus genetika MeSH
- křečci praví MeSH
- lidé MeSH
- rekombinace genetická MeSH
- SARS-CoV-2 genetika MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.
Acid-β-glucosidase (GCase, EC3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme® ) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step toward developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild-type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine learning-based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single-nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.
- MeSH
- celulasy * genetika MeSH
- Gaucherova nemoc * farmakoterapie genetika MeSH
- heterozygot MeSH
- lidé MeSH
- mutace MeSH
- Parkinsonova nemoc * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- COVID-19 * MeSH
- lidé MeSH
- SARS-CoV-2 * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
We hereby describe the process of design and selection of nonantibody protein binders mimicking cytokine signaling. We chose to mimic signaling of IFN-λ1, type 3 interferon (also known as IL-29) for its novelty and the importance of its biological functions. All four known interferons λ signal through binding to the extracellular domains of IL-28 receptor 1 (IL-28R1) and IL-10 receptor 2 (IL-10R2). Our binders were therefore trained to bind both receptors simultaneously. The bifunctional binder molecules were developed by yeast display, a method of directed evolution. The signaling capacity of the bivalent binders was tested by measuring phosphorylation of the JAK/STAT signaling pathway and production of mRNA of six selected genes naturally induced by IFN- λ1 in human cell lines. The newly developed bivalent binders offer opportunities to study cytokine-related biological functions and modulation of the cell behavior by receptor activation on the cell surfaces alternative to the use of natural IFN-λ.
- MeSH
- antivirové látky metabolismus MeSH
- cytokiny metabolismus MeSH
- interferony * metabolismus MeSH
- interleukiny * metabolismus MeSH
- lidé MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viruses rapidly co-evolve with their hosts. The 9 million sequenced SARS-CoV-2 genomes by March 2022 provide a detailed account of viral evolution, showing that all amino acids have been mutated many times. However, only a few became prominent in the viral population. Here, we investigated the emergence of the same mutations in unrelated parallel lineages and the extent of such convergent evolution on the molecular level in the spike (S) protein. We found that during the first phase of the pandemic (until mid 2021, before mass vaccination) 31 mutations evolved independently ≥3-times within separated lineages. These included all the key mutations in SARS-CoV-2 variants of concern (VOC) at that time, indicating their fundamental adaptive advantage. The omicron added many more mutations not frequently seen before, which can be attributed to the synergistic nature of these mutations, which is more difficult to evolve. The great majority (24/31) of S-protein mutations under convergent evolution tightly cluster in three functional domains; N-terminal domain, receptor-binding domain, and Furin cleavage site. Furthermore, among the S-protein receptor-binding motif mutations, ACE2 affinity-improving substitutions are favoured. Next, we determined the mutation space in the S protein that has been covered by SARS-CoV-2. We found that all amino acids that are reachable by single nucleotide changes have been probed multiple times in early 2021. The substitutions requiring two nucleotide changes have recently (late 2021) gained momentum and their numbers are increasing rapidly. These provide a large mutation landscape for SARS-CoV-2 future evolution, on which research should focus now.
Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC.
- MeSH
- buněčná membrána metabolismus MeSH
- CHO buňky MeSH
- cholesterol metabolismus MeSH
- Cricetulus MeSH
- endozomy metabolismus MeSH
- fenotyp MeSH
- lyzozomy metabolismus MeSH
- Niemannova-Pickova nemoc typu C metabolismus patologie MeSH
- sfingolipidy metabolismus MeSH
- sfingosin metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
