HTT-OMNI: A Web-based Platform for Huntingtin Interaction Exploration and Multi-omics Data Integration.

Huntington's disease (HD) is a progressive neurological disorder that is caused by polyglutamine expansion of the huntingtin (HTT) protein. With the hope to uncover key modifiers of disease, a focus of the field of HD research has been on characterizing HTT-interacting proteins (HIPs) and the effect of the HTT polyglutamine expansion on the cellular omics landscape. However, while hundreds of studies have uncovered over 3000 potential HIPs to date, a means to interrogate these complementary interaction and omics datasets does not exist. The lack of a unified platform for exploring this breadth of potential HIPs and associated omics data represents a substantial barrier toward understanding the impact of HTT polyQ expansion and identifying interactions proximal to HD pathogenesis. Here, we describe the development of a web-based platform called HTT-OMNI (HTT OMics and Network Integration). This application facilitates the visualization and exploration of ∼3400 potential HTT interactors (from the HINT database) and their associated polyQ-dependent omics measurements, such as transcriptome and proteome abundances. Additionally, HTT-OMNI allows for the integration of user-generated datasets with existing HIPs and omic measurements. We first demonstrate the utility of HTT-OMNI for filtering existing HTT PPIs based on a variety of experimental metadata parameters, highlighting its capacity to select for HIPs detected in specific model organisms and tissues. Next, we leverage our application to visualize the relationships between HTT PPIs, genetic disease modifiers, and their multiomic landscape. Finally, we generate and analyze a previously unreported dataset of HTT PPIs, aimed at defining tissue-specific HTT interactions and the polyQ-dependent modulation of their relative stabilities in the cortex and striatum of HD mouse models.

The DNA Sensor cGAS is Decorated by Acetylation and Phosphorylation Modifications in the Context of Immune Signaling.

The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. Post-translational modification (PTM) has started to be recognized as a critical component of cGAS regulation, yet the extent of these modifications remains unclear. Here, we report the identification and functional analysis of cGAS phosphorylations and acetylations in several cell types under basal and immune-stimulated conditions. cGAS was enriched by immunoaffinity purification from human primary fibroblasts prior to and after infection with herpes simplex virus type 1 (HSV-1), as well as from immune-stimulated STING-HEK293T cells. Six phosphorylations and eight acetylations were detected, of which eight PTMs were not previously documented. PTMs were validated by parallel reaction monitoring (PRM) mass spectrometry in fibroblasts, HEK293T cells, and THP-1 macrophage-like cells. Primary sequence and structural analysis of cGAS highlighted a subset of PTM sites with elevated surface accessibility and high evolutionary sequence conservation. To assess the functional relevance of each PTM, we generated a series of single-point cGAS mutations. Stable cell lines were constructed to express cGAS with amino acid substitutions that prevented phosphorylation (Ser-to-Ala) and acetylation (Lys-to-Arg) or that mimicked the modification state (Ser-to-Asp and Lys-to-Gln). cGAS-dependent apoptotic and immune signaling activities were then assessed for each mutation. Our results show that acetyl-mimic mutations at Lys384 and Lys414 inhibit the ability of cGAS to induce apoptosis. In contrast, the Lys198 acetyl-mimic mutation increased cGAS-dependent interferon signaling when compared with the unmodified charge-mimic. Moreover, targeted PRM quantification showed that Lys198 acetylation is decreased upon infections with two herpesviruses-HSV-1 and human cytomegalovirus (HCMV), highlighting this residue as a regulatory point during virus infection.

Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles.

Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV's three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3's ion channel function is further evidenced by its ability to mediate ionic currents when expressed in Xenopus laevis oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals.

Diverse mechanisms evolved by DNA viruses to inhibit early host defenses.

In mammalian cells, early defenses against infection by pathogens are mounted through a complex network of signaling pathways shepherded by immune-modulatory pattern-recognition receptors. As obligate parasites, the survival of viruses is dependent on the evolutionary acquisition of mechanisms that tactfully dismantle and subvert the cellular intrinsic and innate immune responses. Here, we review the diverse mechanisms by which viruses that accommodate DNA genomes are able to circumvent activation of cellular immunity. We start by discussing viral manipulation of host defense protein levels by either transcriptional regulation or protein degradation. We next review viral strategies used to repurpose or inhibit these cellular immune factors by molecular hijacking or by regulating their post-translational modification status. Additionally, we explore the infection-induced temporal modulation of apoptosis to facilitate viral replication and spread. Lastly, the co-evolution of viruses with their hosts is highlighted by the acquisition of elegant mechanisms for suppressing host defenses via viral mimicry of host factors. In closing, we present a perspective on how characterizing these viral evasion tactics both broadens the understanding of virus-host interactions and reveals essential functions of the immune system at the molecular level. This knowledge is critical in understanding the sources of viral pathogenesis, as well as for the design of antiviral therapeutics and autoimmunity treatments.

Recent Advances in ZnO Nanomaterial-Mediated Biological Applications and Action Mechanisms.

In recent years, with the deepening research, metal zinc oxide (ZnO) nanomaterials have become a popular research object in the biological field, particularly in biomedicine and food safety, which is attributed to their unique physicochemical properties such as high surface area and volume ratio, luminescence effect, surface characteristics and biological activities. Herein, this review provides a detailed overview of the ZnO nanomaterial-mediated biological applications that involve anti-bacterial, anti-tumor, anti-inflammation, skin care, biological imaging and food packaging applications. Importantly, the corresponding action mechanisms of ZnO nanomaterials are pointed. Additionally, the structure and structure-dependent physicochemical properties, the common synthesis methods and the biosafety of ZnO nanoparticles are revealed in brief. Finally, the significance and future challenges of ZnO nanomaterial applications are concluded.

Intercellular communication within the virus microenvironment affects the susceptibility of cells to secondary viral infections.

Communication between infected cells and cells in the surrounding tissue is a determinant of viral spread. However, it remains unclear how cells in close or distant proximity to an infected cell respond to primary or secondary infections. We establish a cell-based system to characterize a virus microenvironment, distinguishing infected, neighboring, and distal cells. Cell sorting, microscopy, proteomics, and cell cycle assays allow resolving cellular features and functional consequences of proximity to infection. We show that human cytomegalovirus (HCMV) infection primes neighboring cells for both subsequent HCMV infections and secondary infections with herpes simplex virus 1 and influenza A. Neighboring cells exhibit mitotic arrest, dampened innate immunity, and altered extracellular matrix. Conversely, distal cells are poised to slow viral spread due to enhanced antiviral responses. These findings demonstrate how infection reshapes the microenvironment through intercellular signaling to facilitate spread and how spatial proximity to an infection guides cell fate.

Post-translational modification control of viral DNA sensors and innate immune signaling.

The vertebrate innate immune system confers host cells with mechanisms to protect against both evolutionarily ancient pathogens and newly emerging pathogenic strains. Innate immunity relies on the host cell's ability to distinguish between self and pathogen-derived molecules. To achieve this, the innate immune system uses germline encoded receptors called pattern recognition receptors (PRRs), which recognize various molecular signatures, including nucleic acids, proteins, lipids, glycans and glycolipids. Among these molecules, the recognition of pathogenic, mislocalized, or damaged DNA by cellular protein receptors, commonly called DNA sensors, represents a major surveillance pathway for initiating immune signaling. The ability of cells to temporally regulate DNA sensor activation and subsequent signal termination is critical for effective immune signaling. These same mechanisms are also co-opted by pathogens to promote their replication. Therefore, there is significant interest in understanding DNA sensor regulatory networks during microbial infections and autoimmune disease. One emerging aspect of DNA sensor regulation is through post-translational modifications (PTMs), including phosphorylation, acetylation, ubiquitination, ADP-ribosylation, SUMOylation, methylation, deamidation, glutamylation. In this chapter, we discuss how PTMs have been shown to positively or negatively impact DNA sensor functions via diverse mechanisms, including direct regulation of enzymatic activity, protein-protein and protein-DNA interactions, protein translocations and protein turnover. In addition, we highlight the ability of virus-induced PTMs to promote immune evasion. We also discuss the recent evidence linking PTMs on DNA sensors with human diseases and more broadly, highlight promising directions for future research on PTM-mediated regulation of DNA sensor-dependent immune signaling.

Systematic profiling of protein complex dynamics reveals DNA-PK phosphorylation of IFI16 en route to herpesvirus immunity.

Dynamically shifting protein-protein interactions (PPIs) regulate cellular responses to viruses and the resulting immune signaling. Here, we use thermal proximity coaggregation (TPCA) mass spectrometry to characterize the on-off behavior of PPIs during infection with herpes simplex virus 1 (HSV-1), a virus with an ancient history of coevolution with hosts. Advancing the TPCA analysis to infer associations de novo, we build a time-resolved portrait of thousands of host-host, virus-host, and virus-virus PPIs. We demonstrate that, early in infection, the DNA sensor IFI16 recruits the active DNA damage response kinase, DNA-dependent protein kinase (DNA-PK), to incoming viral DNA at the nuclear periphery. We establish IFI16 T149 as a substrate of DNA-PK upon viral infection or DNA damage. This phosphorylation promotes IFI16-driven cytokine responses. Together, we characterize the global dynamics of PPIs during HSV-1 infection, uncovering the co-regulation of IFI16 and DNA-PK functions as a missing link in immunity to herpesvirus infection.

Workflows and considerations for investigating protein interactions of viral DNA sensors.

DNA sensors are a core component of innate immunity in mammalian cells. In response to pathogen infection, these specialized proteins sense pathogenic DNA from bacteria or viruses and initiate immune signaling cascades. These defense mechanisms rely on the rapid formation and temporal regulation of protein-protein interactions. Similarly, protein interactions underlie virus immune evasion mechanisms, as proteins from diverse viruses associate with and inhibit DNA sensors. Here, we describe experimental protocols for identifying protein interactions of DNA sensors, and discuss considerations for optimal isolation of protein complexes when targeting either endogenous or tagged proteins. Additionally, as viral infections and immune responses are known to induce prominent changes in cellular protein abundances, we provide a workflow for investigating these protein associations in the context of proteome alterations.

Interactome and Proteome Dynamics Uncover Immune Modulatory Associations of the Pathogen Sensing Factor cGAS.

Viral DNA sensing is an essential component of the mammalian innate immune response. Upon binding viral DNA, the cyclic-GMP-AMP synthase (cGAS) catalyzes the production of cyclic dinucleotides to induce type I interferons. However, little is known about how cGAS is homeostatically maintained or regulated upon infection. Here, we define cytoplasmic cGAS interactions with cellular and viral proteins upon herpes simplex virus type 1 (HSV-1) infection in primary human fibroblasts. We compare several HSV-1 strains (wild-type, d109, d106) that induce cytokine responses and apoptosis and place cGAS interactions in the context of temporal proteome alterations using isobaric-labeling mass spectrometry. Follow-up analyses establish a functional interaction between cGAS and 2'-5'-oligoadenylate synthase-like protein OASL. The OAS-like domain interacts with the cGAS Mab21 domain, while the OASL ubiquitin-like domain further inhibits cGAS-mediated interferon response. Our findings explain how cGAS may be inactively maintained in cellular homeostasis, with OASL functioning as a negative feedback loop for cytokine induction.