Sequence-specific protein aggregation generates defined protein knockdowns in plants

Abstract

Protein aggregation is determined by short (5-15 amino acids) aggregation-prone regions (APRs) of the polypeptide sequence that self-associate in a specific manner to form b-structured inclusions. Here, we demonstrate that the sequence specificity of APRs can be exploited to selectively knock down proteins with different localization and function in plants. Synthetic aggregation-prone peptides derived from the APRs of either the negative regulators of the brassinosteroid (BR) signaling, the glycogen synthase kinase 3/Arabidopsis SHAGGY-like kinases (GSK3/ASKs), or the starch-degrading enzyme a-glucan water dikinase were designed. Stable expression of the APRs in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) induced aggregation of the target proteins, giving rise to plants displaying constitutive BR responses and increased starch content, respectively. Overall, we show that the sequence specificity of APRs can be harnessed to generate aggregation-associated phenotypes in a targeted manner in different subcellular compartments. This study points toward the potential application of induced targeted aggregation as a useful tool to knock down protein functions in plants and, especially, to generate beneficial traits in crops.

Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis

“…In order to generate efficient aggregation seeds, here, we employed a tandem repeat design previously validated 14 , 15 , in which the redundant APR is incorporated as a tandem repeat separated by a linker constituted by a single proline. In order to increase the colloidal stability of these aggregating peptides, the APRs are flanked by aggregation gatekeepers, a class of residues that was previously shown to reduce aggregation kinetics 8 , 16 , 17 .…”

“…In other words, when a protein aggregates, it will generally only aggregate with identical proteins. We previously exploited the fact that aggregation is sequence specific and that most aggregating sequences are sparse in a proteome to induce specific protein knockdown of target proteins in plants 15 and mammalian cells 23 , or to achieve protein detection in western blot using protein-specific APRs 7 . During these exercises, we realized, however, that a minority of aggregation-prone sequences are found within several and sometimes many proteins.…”

De novo design of a biologically active amyloid

“…Moreover, the two homologs share 84% sequence identity overall and 90% in the TANGO-regions. To derive peptide sequences that are likely to form amyloid structure in isolation, but also have a high potential for forming soluble oligomers (29), we devised a strategy (30,31) that makes use of a sequence feature of functional amyloids and yeast prions, which often contain several Aggregation Prone Regions (APR) (32) closely connected by disordered regions (33). Hence we placed two APRs in a peptide, separated by a rigid proline-proline linker, mimicking these repeat patterns.…”

Rational Design of Amyloid‐Like Fibrillary Structures for Tailoring Food Protein Techno‐Functionality and Their Potential Health Implications

“…Incubation of ovalbumin with the N ‐terminal region pN 1‐22 under acidic conditions resulted in straight fibrils that were distinct from the semi‐flexible fibrils formed from intact ovalbumin incubated under the same conditions without such peptide (Kawachi et al., ). Interestingly, peptide‐based seeding of protein aggregation also happens in living cells and organisms, as demonstrated in transgenic plants (Betti et al., ), in mammalian cells (Gallardo et al., ) and in bacteria (Bednarska et al., ).…”