Intermolecular interactions underlie protein/peptide phase separation irrespective of sequence and structure at crowded milieu.

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological phenomenon underlying the sequestration of macromolecules (such as proteins and nucleic acids) into membraneless organelles in cells. Unstructured and intrinsically disordered domains are known to facilitate multivalent interactions driving protein LLPS. We hypothesized that LLPS could be an intrinsic property of proteins/polypeptides but with distinct phase regimes irrespective of their sequence and structure. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study in the presence and absence of molecular crowder, polyethylene glycol (PEG-8000). We showed that all proteins and even highly charged polypeptides (under study) can undergo liquid condensate formation, however with different phase regimes and intermolecular interactions. We further demonstrated that electrostatic, hydrophobic, and H-bonding or a combination of such intermolecular interactions plays a crucial role in individual protein/peptide LLPS.

Amyloid fibril-based thixotropic hydrogels for modeling of tumor spheroids in vitro.

Biomaterials mimicking extracellular matrices (ECM) for three-dimensional (3D) cultures have gained immense interest in tumor modeling and in vitro organ development. Here, we introduce a new class of amyloid fibril-based peptide hydrogels as a versatile biomimetic ECM scaffold for 3D cell culture and homogenous tumor spheroid modeling. We show that these amyloid fibril-based hydrogels are thixotropic and allow cancer cell adhesion, proliferation, and migration. All seven designed hydrogels support 3D cell culture with five different cancer cell lines forming spheroid with necrotic core and upregulation of the cancer biomarkers. We further developed the homogenous, single spheroid using the drop cast method and the data suggest that all hydrogels support the tumor spheroid formation but with different necrotic core diameters. The detailed gene expression analysis of MCF7 spheroid by microarray suggested the involvement of pro-oncogenes and significant regulatory pathways responsible for tumor spheroid formation. Further, using breast tumor tissue from a mouse xenograft model, we show that selected amyloid hydrogels support the formation of tumor spheroids with a well-defined necrotic core, cancer-associated gene expression, higher drug resistance, and tumor heterogeneity reminiscent of the original tumor. Altogether, we have developed an easy-to-use, rapid, cost-effective, and scalable platform for generating in vitro cancer models for the screening of anti-cancer therapeutics and developing personalized medicine.

Intermediates of α-synuclein aggregation: Implications in Parkinson's disease pathogenesis.

Cytoplasmic deposition of aberrantly misfolded α-synuclein (α-Syn) is a common feature of synucleinopathies, including Parkinson's disease (PD). However, the precise pathogenic mechanism of α-Syn in synucleinopathies remains elusive. Emerging evidence has suggested that α-Syn may contribute to PD pathogenesis in several ways; wherein the contribution of fibrillar species, for exerting toxicity and disease transmission, cannot be neglected. Further, the oligomeric species could be the most plausible neurotoxic species causing neuronal cell death. However, understanding the structural and molecular insights of these oligomers are very challenging due to the heterogeneity and transient nature of the species. In this review, we discuss the recent advancements in understanding the formation and role of α-Syn oligomers in PD pathogenesis. We also summarize the different types of α-Syn oligomeric species and potential mechanisms to exert neurotoxicity. Finally, we address the possible ways to target α-Syn as a promising approach against PD and the possible future directions.