Production of extracellular membrane vesicles plays an important role in communication in bacterial populations and in bacteria-host interactions. Vesicles as carriers of various regulatory and signaling molecules may be potentially used as disease biomarkers and promising therapeutic agents, including vaccine preparations. The composition of membrane vesicles has been deciphered for a limited number of Gram-negative and Gram-positive bacteria. In this work, for the first time, extracellular membrane vesicles of a streptomycin-resistant strain Bacillus pumilus 3-19, a producer of extracellular guanyl-preferring ribonuclease binase, are isolated, visualized, and characterized by their genome and proteome composition. It has been established that there is no genetic material in the vesicles and the spectrum of the proteins differs depending on the phosphate content in the culture medium of the strain. Vesicles from a phosphate-deficient medium carry 49 unique proteins in comparison with 101 from a medium with the high phosphate content. The two types of vesicles had 140 mutual proteins. Flagellar proteins, RNase J, which is the main enzyme of RNA degradosomes, phosphatases, peptidases, iron transporters, signal peptides, were identified in vesicles. Antibiotic resistance proteins and amyloid-like proteins whose genes are present in B. pumilus 3-19 cells are absent. Phosphate deficiency-induced binase was found only in vesicles from a phosphate-deficient medium.

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) acts as a negative regulator of autophagy by interacting with Beclin 1 at Golgi membranes in mammalian cells. The molecular mechanism of this interaction is largely unknown. We recently showed that human GAPR-1 (hGAPR-1) has amyloidogenic properties resulting in the formation of protein condensates upon overexpression in Saccharomyces cerevisiae. Here we show that human Beclin 1 (hBeclin 1) has several predicted amyloidogenic regions and that overexpression of hBeclin 1-mCherry in yeast also results in the formation of fluorescent protein condensates. Surprisingly, co-expression of hGAPR-1-GFP and hBeclin 1-mCherry results in a strong reduction of hBeclin 1 condensates. Mutations of the known interaction site on the hGAPR-1 and hBeclin 1 surface abolished the effect on condensate formation during co-expression without affecting the condensate formation properties of the individual proteins. Similarly, a hBeclin 1-derived B18 peptide that is known to bind hGAPR-1 and to interfere with the interaction between hGAPR-1 and hBeclin 1, abolished the reduction of hBeclin 1 condensates by co-expression of hGAPR-1. These results indicate that the same type of protein-protein interactions interfere with condensate formation during co-expression of hGAPR-1 and hBeclin 1 as previously described for their interaction at Golgi membranes. The amyloidogenic properties of the B18 peptide were, however, important for the interaction with hGAPR-1, as mutant peptides with reduced amyloidogenic properties also showed reduced interaction with hGAPR-1 and reduced interference with hGAPR-1/hBeclin 1 condensate formation. We propose that amyloidogenic interactions take place between hGAPR-1 and hBeclin 1 prior to condensate formation.

Biofilms have a significant role in microbial persistence, antibiotic resistance, and chronic infections; consequently, there is a pressing need for development of novel \"anti-biofilm strategies.\" One of the fundamental mechanisms involved in biofilm formation is protein-protein interactions of \"amyloid-like proteins\" (ALPs) in the extracellular matrix. Such interactions could be potential targets for development of novel anti-biofilm strategies; therefore, assessing the structural features of these interactions could be of great scientific value. Characterization of structural features the of protein-protein interaction with conventional structure biology tools including X-ray diffraction and nuclear magnetic resonance is technically challenging, expensive, and time-consuming. In contrast, modeling such interactions is time-efficient and economical, and might provide deeper understanding of structural basis of interactions. Although it is often acknowledged that molecular modeling methods have varying accuracy, their careful implementation with supplementary verification methods can provide valuable insight and directions for future studies. With this reasoning, during the present study, the protein-protein interaction of TasA(28-261)-TapA(33-253) (which is a decisive process for biofilm formation by Bacillus subtilis) was modeled using in silico approaches, viz., molecular modeling, protein-protein docking, and molecular dynamics simulations. Results obtained here identified amino acid residues present within intrinsically disordered regions of both proteins to be critical for interaction. These results were further supported with principal component analyses (PCA) and free energy landscape (FEL) analyses. Results presented here represent novel finding, and we hypothesize that amino acid residues identified during the present study could be targeted for inhibition of biofilm formation by B. subtilis.

Amyloids cause incurable diseases in humans and animals and regulate vital processes in bacteria and eukaryotes. Amyloid fibrils have unique properties, such as amazing resistance to a variety of agents, mechanical strength, and elasticity, and it is not surprising that in the course of evolution eukaryotes have learned to employ amyloid structures to regulate various vital processes. Proteins exhibiting amyloid properties have been detected in lower eukaryotes and in diverse cell lines of arthropods and vertebrates. A growing number of studies of eukaryotic proteins that demonstrate certain amyloid-like properties require clear criteria to systematize modern knowledge about the functional amyloids. In this review, we propose to separate eukaryotic proteins, whose amyloid properties are clearly proven, and proteins, which show some amyloid characteristics in vivo or in vitro. In order to assert that a protein is a functional amyloid, it is necessary to prove that it has a cross-β structure in vivo. Here, we consider the advantages and disadvantages of various methods for the analysis of the amyloid properties of a protein. Analysis of the current data shows that amyloids play an important role in the regulation of vital processes in eukaryotes, and new functional amyloids should be searched primarily among structural, protective, and storage proteins. A systematic search for functional amyloids in eukaryotes is only beginning, and the use of novel proteomic methods opens up great prospects for identification of amyloids in any organs and tissues of various organisms.

The progression of many neurodegenerative diseases is assumed to be caused by misfolding of specific characteristic diseases related proteins, resulting in aggregation and fibril formation of these proteins. Protein misfolding associated age related diseases, although different in disease manifestations, share striking similarities. In all cases, one disease protein aggregates and loses its function or additionally shows a toxic gain of function. However, the clear link between these individual amyloid-like protein aggregates and cellular toxicity is often still uncertain. The similar features of protein misfolding and aggregation in this group of proteins, all involved in age related neurodegenerative diseases, results in high interest in characterization of their structural properties. We review here recent findings on structural properties of some age related disease proteins, in the context of their biological importance in disease.