Abstract
Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a peripheral membrane protein located on the cytosolic leaflet of the Golgi apparatus in mammalian cells. GAPR-1 belongs to the CAP (cysteine-rich secretory proteins, antigen 5, pathogenesis-related-1) superfamily of proteins, harboring predicted amyloidogenic regions, especially in the CAP1 and CAP2 motifs. The presence
... read more
of these predicted amyloidogenic regions within the CAP domain implies that the amyloid-like aggregation pathway might be a shared characteristic of the CAP superfamily. Consistent with this, GAPR-1 has been shown to interact with an amyloid oligomer-specific antibody and form amyloid-like fibrils in vitro.
In recent years, Saccharomyces cerevisiae has become a favored model organism for exploring reversible protein aggregation, including amyloid-like aggregation. A recent trend involves the development of yeast cell-based assays, or "humanized yeast systems," enabling the study of human protein functions in yeast. The induction of phase-separated protein droplets (condensates) in yeast by overexpressing amyloid-prone proteins provides insight into the mechanisms of protein phase separation and its potential link to the formation of functional/pathological amyloids in the human body. Here, we delve into the amyloidogenic properties of GAPR-1 in S. cerevisiae. The study illustrates that GAPR-1-GFP expression meets the criteria of a protein with amyloidogenic properties, leading to the formation of reversible intracellular protein condensates. The thesis investigates factors influencing GAPR-1 oligomerization in yeast, particularly the role of lipid bilayers and metal ions. Additionally, we explore the role of GAPR-1 as a negative regulator of autophagy and its potential interaction with Beclin 1, a key initiator of autophagy. Building on previous findings, the study suggests that the oligomerization of GAPR-1 may be instrumental in its interaction with components of the autophagic machinery, thereby influencing the regulation of autophagy.
Furthermore, we investigate the role of phosphorylation in regulating the oligomeric states of GAPR-1 and Beclin 1 in a yeast model system, with a focus on understanding how phosphorylation influences their interaction. At the end, we introduces a novel assay system designed to explore protein-protein interactions, based on the formation of biomolecular condensates in S. cerevisiae. Expanding on earlier observations of mutual interference in condensate formation during the co-expression of GAPR-1 and Beclin 1, our assay system is extended to investigate various protein-protein interactions. This innovative assay system offers a versatile platform for investigating diverse protein-protein interactions and their dynamics within cellular condensates. We explores protein-protein interactions involving GAPR-1, extending beyond its known interaction with Beclin 1 and its inhibitory effect on Aβ peptide (1-40) amyloid formation. The investigation reveals that GAPR-1 forms heterotypic interactions not only with Beclin 1 but also with the Aβ peptide (1-40) and Huntington protein. These findings suggest a potential role of GAPR-1 in modulating interactions with other amyloidogenic proteins, with potential implications for cellular processes related to autophagy and protein aggregation. In conclusion, the discussion chapter explores the concept of cross-seeding among amyloid proteins, highlighting the significance of interactions between misfolded proteins with different sequences or structural motifs, leading to hybrid or mixed amyloid structures.The potential therapeutic opportunities arising from targeting these interactions to modulate the formation and progression of amyloid aggregates are also discussed.
show less
