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
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
Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.
- MeSH
- algoritmy * MeSH
- dánio pruhované MeSH
- Escherichia coli metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- molekulární modely MeSH
- proteiny chemie metabolismus MeSH
- rozpustnost MeSH
- rychlé screeningové testy MeSH
- stabilita proteinů * MeSH
- teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interleukin 24 (IL-24) is a cytokine with the potential to be an effective treatment for autoimmune diseases and cancer. However, its instability and difficulties in its production have hampered detailed biological and biophysical studies. We approached the challenges of IL-24 production by using the PROSS algorithm to design more stable variants of IL-24. We used homology models built from the sequences and known structures of IL-20 and IL-19 and predicted and produced several extensively mutated IL-24 variants that were highly stable and produced in large yields; one of them was crystallized (IL-24B, PDB ID 6GG1; 3D Interactive at http://proteopedia.org/w/Journal: FEBS_Journal:1). The mutated variants, however, lost most of their binding capacity to the extracellular parts of cognate receptors. While the affinity to the receptor 2 (IL-20R2) was preserved, the variants lost affinity to IL-20R1 and IL-22R1 (shared receptors 1). Back engineering of the variants revealed that reintroduction of a single IL-24 wild-type residue (T198) to the patch interacting with receptors 1 restored 80% of the binding affinity and signaling capacity, accompanied by an acceptable drop in the protein stability by 9 °C. Multiple sequence alignment explains the stabilizing effect of the mutated residues in the IL-24 variants by their presence in the related and more stable cytokines IL-20 and IL-19. Our homology-based approach can enhance existing methods for protein engineering and represents a viable alternative to study and produce difficult proteins for which only in silico structural information is available, estimated as >40% of all important drug targets.
