Hepatitis B virus (HBV) infects hepatocytes that are in the G0/G1 phase with intact nuclear membrane and organized chromosome architecture. In the nucleus of the infected cells, HBV covalently closed circular (ccc) DNA, an episomal minichromosome, serves as the template for all viral transcripts and the reservoir of persistent infection. Nuclear positioning of cccDNA can be assessed by the spatial distance between viral DNA and host chromosomal DNA through Circular Chromosome Conformation Capture (4C) combined with high-throughput sequencing (4C-seq). The 4C-seq analysis relies on proximity ligation and is commonly used for mapping genomic DNA regions that communicate within a host chromosome. The method has been tailored for studying nuclear localization of HBV episomal cccDNA in relation to the host chromosomes. In this study, we present a step-by-step protocol for 4C-seq analysis of HBV infection, including sample collection and fixation, 4C DNA library preparation, sequence library preparation, and data analysis. Although limited by proximity ligation of DNA fragments, 4C-seq analysis provides useful information of HBV localization in 3D genome, and aids the understanding of viral transcription in light of host chromatin conformation.

BACKGROUND: During the bloom season, the colonial cyanobacterium Microcystis forms complex aggregates which include a diverse microbiome within an exopolymer matrix. Early research postulated a simple mutualism existing with bacteria benefitting from the rich source of fixed carbon and Microcystis receiving recycled nutrients. Researchers have since hypothesized that Microcystis aggregates represent a community of synergistic and interacting species, an interactome, each with unique metabolic capabilities that are critical to the growth, maintenance, and demise of Microcystis blooms. Research has also shown that aggregate-associated bacteria are taxonomically different from free-living bacteria in the surrounding water. Moreover, research has identified little overlap in functional potential between Microcystis and members of its microbiome, further supporting the interactome concept. However, we still lack verification of general interaction and know little about the taxa and metabolic pathways supporting nutrient and metabolite cycling within Microcystis aggregates.
RESULTS: During a 7-month study of bacterial communities comparing free-living and aggregate-associated bacteria in Lake Taihu, China, we found that aerobic anoxygenic phototrophic (AAP) bacteria were significantly more abundant within Microcystis aggregates than in free-living samples, suggesting a possible functional role for AAP bacteria in overall aggregate community function. We then analyzed gene composition in 102 high-quality metagenome-assembled genomes (MAGs) of bloom-microbiome bacteria from 10 lakes spanning four continents, compared with 12 complete Microcystis genomes which revealed that microbiome bacteria and Microcystis possessed complementary biochemical pathways that could serve in C, N, S, and P cycling. Mapping published transcripts from Microcystis blooms onto a comprehensive AAP and non-AAP bacteria MAG database (226 MAGs) indicated that observed high levels of expression of genes involved in nutrient cycling pathways were in AAP bacteria.
CONCLUSIONS: Our results provide strong corroboration of the hypothesized Microcystis interactome and the first evidence that AAP bacteria may play an important role in nutrient cycling within Microcystis aggregate microbiomes. Video Abstract.

The ECM (extracellular matrix) is a major component of the vascular microenvironment that modulates vascular homeostasis. ECM proteins include collagens, elastin, noncollagen glycoproteins, and proteoglycans/glycosaminoglycans. ECM proteins form complex matrix structures, such as the basal lamina and collagen and elastin fibers, through direct interactions or lysyl oxidase-mediated cross-linking. Moreover, ECM proteins directly interact with cell surface receptors or extracellular secreted molecules, exerting matricellular and matricrine modulation, respectively. In addition, extracellular proteases degrade or cleave matrix proteins, thereby contributing to ECM turnover. These interactions constitute the ECM interactome network, which is essential for maintaining vascular homeostasis and preventing pathological vascular remodeling. The current review mainly focuses on endogenous matrix proteins in blood vessels and discusses the interaction of these matrix proteins with other ECM proteins, cell surface receptors, cytokines, complement and coagulation factors, and their potential roles in maintaining vascular homeostasis and preventing pathological remodeling.

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.

The cell-to-cell communication primarily occurs through cell-surface and secreted proteins, which form a sophisticated network that coordinates systemic immune function. Uncovering these protein-protein interactions (PPIs) is indispensable for understanding the molecular mechanism and elucidating immune system aberrances under diseases. Traditional biological studies typically focus on a limited number of PPI pairs due to the relative low throughput of commonly used techniques. Encouragingly, classical methods have advanced, and many new systems tailored for large-scale protein-protein screening have been developed and successfully utilized. These high-throughput PPI investigation techniques have already made considerable achievements in mapping the immune cell interactome, enriching PPI databases and analysis tools, and discovering therapeutic targets for cancer and other diseases, which will definitely bring unprecedented insight into this field.

OBJECTIVE: Our study is applying a community-based approach to examine the influence of exercise on gut microbiota (GM) and discover GM structures linked with NAFLD improvements during exercise. The majority of microbiome research has focused on finding specific species that may contribute to the development of human diseases. However, we believe that complex diseases, such as NAFLD, would be more efficiently treated using consortia of species, given that bacterial functionality is based not only on its own genetic information but also on the interaction with other microorganisms. Our results revealed that exercise significantly changes the GM interaction and that structural alterations can be linked with improvements in intrahepatic lipid content and metabolic functions. We believe that the identification of these characteristics in the GM enhances the development of exercise treatment for NAFLD and will attract general interest in this field.

Proteome-wide characterization of protein-protein interactions (PPIs) is crucial to understand the functional roles of protein machinery within cells systematically. With the accumulation of PPI data in different plants, the interaction details of binary PPIs, such as the three-dimensional (3D) structural contexts of interaction sites/interfaces, are urgently demanded. To meet this requirement, we have developed a comprehensive and easy-to-use database called PlaPPISite ( http://zzdlab.com/plappisite/index.php ) to present interaction details for 13 plant interactomes. Here, we provide a clear guide on how to search and view protein interaction details through the PlaPPISite database. Firstly, the running environment of our database is introduced. Secondly, the input file format is briefly introduced. Moreover, we discussed which information related to interaction sites can be achieved through several examples. In addition, some notes about PlaPPISite are also provided. More importantly, we would like to emphasize the importance of interaction site information in plant systems biology through this user guide of PlaPPISite. In particular, the easily accessible 3D structures of PPIs in the coming post-AlphaFold2 era will definitely boost the application of plant interactome to decipher the molecular mechanisms of many fundamental biological issues.

Proteins play an essential role in the vital biological processes governing cellular functions. Most proteins function as members of macromolecular machines, with the network of interacting proteins revealing the molecular mechanisms driving the formation of these complexes. Profiling the physiology-driven remodeling of these interactions within different contexts constitutes a crucial component to achieving a comprehensive systems-level understanding of interactome dynamics. Here, we apply co-fractionation mass spectrometry and computational modeling to quantify and profile the interactions of ∼2000 proteins in the bacterium Escherichia coli cultured under 10 distinct culture conditions. The resulting quantitative co-elution patterns revealed large-scale condition-dependent interaction remodeling among protein complexes involved in diverse biochemical pathways in response to the unique environmental challenges. The network-level analysis highlighted interactome-wide biophysical properties and structural patterns governing interaction remodeling. Our results provide evidence of the local and global plasticity of the E. coli interactome along with a rigorous generalizable framework to define protein interaction specificity. We provide an accompanying interactive web application to facilitate the exploration of these rewired networks.

There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.

Chikungunya virus (CHIKV) is a re-emerging mosquito-transmitted RNA virus causing joint and muscle pain. To better understand how CHIKV rewires the host cell and usurps host cell functions, we generated a systematic CHIKV-human protein-protein interaction map and revealed several novel connections that will inform further mechanistic studies. One of these novel interactions, between the viral protein E1 and STIP1 homology and U-box containing protein 1 (STUB1), was found to mediate ubiquitination of E1 and degrade E1 through the proteasome. Capsid associated with G3BP1, G3BP2 and AAA+ ​ATPase valosin-containing protein (VCP). Furthermore, VCP inhibitors blocked CHIKV infection, suggesting VCP could serve as a therapeutic target. Further work is required to fully understand the functional consequences of these interactions. Given that CHIKV proteins are conserved across alphaviruses, many virus-host protein-protein interactions identified in this study might also exist in other alphaviruses. Construction of interactome of CHIKV provides the basis for further studying the function of alphavirus biology.