In Silico Predictions of Ecological Plasticity Mediated by Protein Family Expansions in Early-Diverging Fungi

Early-diverging fungi (EDF) are ubiquitous and versatile. Their diversity is reflected in their genome sizes and complexity. For instance, multiple protein families have been reported to expand or disappear either in particular genomes or even whole lineages. The most commonly mentioned are CAZymes (carbohydrate-active enzymes), peptidases and transporters that serve multiple biological roles connected to, e.g., metabolism and nutrients intake. In order to study the link between ecology and its genomic underpinnings in a more comprehensive manner, we carried out a systematic in silico survey of protein family expansions and losses among EDF with diverse lifestyles. We found that 86 protein families are represented differently according to EDF ecological features (assessed by median count differences). Among these there are 19 families of proteases, 43 CAZymes and 24 transporters. Some of these protein families have been recognized before as serine and metallopeptidases, cellulases and other nutrition-related enzymes. Other clearly pronounced differences refer to cell wall remodelling and glycosylation. We hypothesize that these protein families altogether define the preliminary fungal adaptasome. However, our findings need experimental validation. Many of the protein families have never been characterized in fungi and are discussed in the light of fungal ecology for the first time.

In silico Molecular Docking of Anthraquinone Identified from Boerhavia diffusa Linn against Bax and Bcl-2 Gene

In today's medical environment, natural products have made a substantial contribution to the therapeutic approach in the treatment of diseases ranging from the simple to the complex. The old or traditional approach of standardization in medicinal plant research is a time-consuming, costly, and to some extent antiquated process. As a result, a computational technique that includes an in silico molecular docking simulation study has become an important tool for drug development, standardisation, and screening of phytochemicals. To investigate the cardioprotective research and the interaction of the strong chemical against Bax and Bcl-2 cardiomyocyte gene, docking was conducted using multiple Protein Data Bank files (3EOO, 3D2U, 2I42, and 3D2Y). The Anthraquinone has shown more potent interaction with apoptotic regulators Bcl-2 and Bax genes by showing good binding energy. The study also evident that Anthraquinone (UBA) was an ideal drug agent with better drug likeliness. Further, the compound can be used as therapeutic molecule for myocardial infarction. However, the results are preliminary and experimental evaluation will be carried out in near future.

DiMeLo-seq: Directed Methylation with Long-read sequencing v2

Directed Methylation and Long-read sequencing (DiMeLo-seq) is a powerful method to map protein-DNA interactions at a single-molecule level across the genome (including repetitive regions). It can be multiplexed to analyze multiple base modifications at once (e.g. endogenous CpG methylation and directed pA-Hia5 adenine methylation). Additionally, PCR amplification is not necessary for this protocol, which means that sequencing readout is proportional to protein-DNA interaction frequency. Finally, DiMeLo-seq can be used to map multiple protein interactions across a long single molecule.

A New Method for Recognizing Protein Complexes Based on Protein Interaction Networks and GO Terms

Motivation: A protein complex is the combination of proteins which interact with each other. Protein–protein interaction (PPI) networks are composed of multiple protein complexes. It is very difficult to recognize protein complexes from PPI data due to the noise of PPI.Results: We proposed a new method, called Topology and Semantic Similarity Network (TSSN), based on topological structure characteristics and biological characteristics to construct the PPI. Experiments show that the TSSN can filter the noise of PPI data. We proposed a new algorithm, called Neighbor Nodes of Proteins (NNP), for recognizing protein complexes by considering their topology information. Experiments show that the algorithm can identify more protein complexes and more accurately. The recognition of protein complexes is vital in research on evolution analysis.Availability and implementation: https://github.com/bioinformatical-code/NNP.

Sequestosome 1 Is Part of the Interaction Network of VAPB

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8). We performed RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC) to identify proteins that interact with or are in close proximity to P56S-VAPB. The mutation abrogates the interaction of VAPB with many known binding partners. Here, we identify Sequestosome 1 (SQSTM1), a well-known autophagic adapter protein, as a major interaction/proximity partner of P56S-VAPB. Remarkably, not only the mutant protein, but also wild-type VAPB interacts with SQSTM1, as shown by proximity ligation assays and co-immunoprecipiation experiments.

Modeling Alternate Conformations with Alphafold2 via Modification of the Multiple Sequence Alignment

The unprecedented performance of Deepmind's Alphafold2 in predicting protein structure in CASP XIV and the creation of a database of structures for multiple proteomes is reshaping structural biology. Moreover, the availability of Alphafold2's architecture and code has stimulated a number of questions on how to harness the capabilities of this remarkable tool. A question of central importance is whether Alphafold2's architecture is amenable to predict the intrinsic conformational heterogeneity of proteins. A general approach presented here builds on a simple manipulation of the multiple sequence alignment, via in silico mutagenesis, and subsequent modeling by Alphafold2. The approach is based in the concept that the multiple sequence alignment encodes for the structural heterogeneity, thus its rational manipulation will enable Alphafold2 to sample alternate conformations and potentially structural alterations due to point mutations. This modeling pipeline is benchmarked against canonical examples of protein conformational flexibility and applied to interrogate the conformational landscape of membrane proteins. This work broadens the applicability of Alphafold2 by generating multiple protein conformations to be tested biologically, biochemically, biophysically, and for use in structure-based drug design.

Synergistic Effect of Proteinase Activity by Purification and Identification of Toxic Protease From Nemopilema nomurai

Scyphozoan Nemopilema nomurai envenomation is an unresolved threat to human health in Asian waters. Nemopilema nomurai venom metalloproteinases show important toxicities in skin damage and inflammation, but there is still no purified protein for further studies. In this study, high proteinase activity fractions in tentacle autolysis were isolated by ammonium sulfate precipitation, DEAE Sepharose Fast Flow, and Superdex 75 chromatography successively. Purification was guided by azocasein hydrolysis activity and SDS-PAGE. The final products were analyzed by LC-MS/MS. Four elution peaks purified by Superdex 75 chromatography had multiple protein bands but did not show proteinase activity. These fractions would recover proteinase activity after mixing again. Regulation mechanisms were speculated as binding metalloproteinase regulator or disaggregating metalloproteinase inhibitor by LC-MS/MS analysis. For the first time, a synergistic effect in N. nomurai proteinase activity was found in the purification process.

BIND&MODIFY: Long-range single-molecule mapping of chromatin modification in eukaryotes v1

Here we describe a powerful method, BIND&MODIFY, for probing histone modifications and transcription factors at single molecular level. Our approach used the recombinant fused protein A-M.EcoGII, which tethers the methyltransferase M.EcoGII to the protein binding sites and locally labels the neighboring DNA regions via artificial methylations. This method could reveal ingle-molecule heterogenous histone modification status and CpG methylation at the same time, and could enable quantify the correlation between the distal elements. Further applications based on this method's concept could be applied to probe multiple protein binding events on the same single molecular DNA. The method proposed herein may soon become an essential tool for third-generation sequencing in the future.

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.