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The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics.

Type I fimbriae are the main adhesive organelles of uropathogenic Escherichia coli (UPEC), consisting of four different subunits. Their component with the most important role in establishing bacterial infections is the FimH adhesin located at the fimbrial tip. This two-domain protein mediates adhesion to host epithelial cells through interaction with terminal mannoses on epithelial glycoproteins. Here, we propose that the amyloidogenic potential of FimH can be exploited for the development of therapeutic agents against Urinary Tract Infections (UTIs). Aggregation-prone regions (APRs) were identified via computational methods, and peptide-analogues corresponding to FimH lectin domain APRs were chemically synthesized and studied with the aid of both biophysical experimental techniques and molecular dynamic simulations. Our findings indicate that these peptide-analogues offer a promising set of antimicrobial candidate molecules since they can either interfere with the folding process of FimH or compete for the mannose-binding pocket.

Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF by engineering the aggregation hotspots. Initially, 21 mutants were designed based on the previously-identified aggregation-prone regions (APRs) and then four of them including mutants No. 4 (L91K, I119K), 7 (V13S, L91K), 14 (L91D, I119D), and 21 (A51E) were selected based on molecular dynamics (MD) simulations for further experimental studies. The recombinantly produced rhKGF and mutants were analyzed regarding secondary structure, thermal stability, aggregation propensity, and biological activity. Far-UV CD spectroscopy showed that the mutants have similar secondary structure with rhKGF. A51E mutant showed enhanced stability and decreased monomer loss under heat stress suggesting its reduced aggregation propensity compared to rhKGF. Mutant No. 14 showed higher stability and less aggregation tendency than mutant No. 4 indicating that only mutations decreasing pI of rhKGF are effective in reducing its aggregation tendency. All of the mutants were at least as potent as rhKGF in stimulating proliferation of MCF-7 epithelial cells. Our results identified A51E as an equally potent, more stable, and less aggregation-prone analog of rhKGF which could be a promising alternative drug candidate for the commercially available rhKGF (Palifermin).

The aggregation-prone region (APR) is a hydrophobic polypeptide motif that promotes protein aggregation, most commonly in the unfolded or misfolded state. It has been described that chaperones can shield the APRs of proteins, thereby preventing aggregate formation during de novo protein synthesis and stress response. Glutamyl-tRNA reductase (GluTR) is a key enzyme in tetrapyrrole biosynthesis (TBS) which catalyzes the rate-limiting step of 5-aminolevulinic acid synthesis. The GluTR sequence contains two APRs located at the N-terminus, which are suggested to be associated with the dysregulation of protein homeostasis during folding and refolding processes or under stress conditions. It remains open if these APRs directly contribute to GluTR aggregation in vivo , and how their removal or the modification might impact the aggregation and stability. In this study, we altered and removed the GluTR-APRs to investigate their effects on the stability and enzymatic activity of GluTR. Deletion of the APRs has been shown to be highly disruptive to the structure of GluTR, and a substitution mutation of V→P in each APR has also lowered the GluTR stability and activity. In contrast, the mutation V→T resulted in a modest reduction (18–30%) in GluTR aggregation in vitro , which was associated with a 27% improvement in GluTR stability in vivo relative to the wild-type enzyme. These results indicate that a point mutation in APR can improve GluTR stability without significantly affecting enzyme activity, thus imposing a potential direction for bioengineering of GluTR to improve productivity of the TBS pathway in plants.

Alzheimer’s disease (AD) is a complex neurodegenerative disorder characterized by extracellular amyloid plaques, predominantly consisting of amyloid-β (Aβ) peptides. The oligomeric form of Aβ is acknowledged as the most neurotoxic, propelling the pathological progression of AD. Interestingly, besides Aβ, other proteins are co-localized within amyloid plaques. Peptide analogs corresponding to the “aggregation-prone” regions (APRs) of these proteins could exhibit high-affinity binding to Aβ and significant inhibitory potential against the Aβ oligomerization process. The peptide analogs of co-localized protease, Cathepsin B, may act as such potent inhibitors. In silico studies on the complexes of the oligomeric state of Aβ and Cathepsin B peptide analogs were performed utilizing molecular docking and molecular dynamics simulations, revealing that these analogs disrupt the β-sheet-rich core of Aβ oligomers, a critical structural feature of their stability. Of the four peptide analogs evaluated, two demonstrated considerable potential by effectively destabilizing oligomers while maintaining low self-aggregation propensity, i.e., a crucial consideration for therapeutic safety. These findings point out the potential of APR-derived peptide analogs from co-localized proteins as innovative agents against AD, paving the way for further exploration in peptide-based therapeutic development.

This chapter contains sections titled: Introduction Energy Landscapes, Protein Folding, and Aggregation Prediction of APRs in Proteins and Biotherapeutics Conclusions and Future Directions Acknowledgments References

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.