Identification of Histone Lysine Acetoacetylation as a Dynamic Post-Translational Modification Regulated by HBO1.

Ketone bodies have long been known as a group of lipid-derived alternative energy sources during glucose shortages. Nevertheless, the molecular mechanisms underlying their non-metabolic functions remain largely elusive. This study identified acetoacetate as the precursor for lysine acetoacetylation (Kacac), a previously uncharacterized and evolutionarily conserved histone post-translational modification. This protein modification is comprehensively validated using chemical and biochemical approaches, including HPLC co-elution and MS/MS analysis using synthetic peptides, Western blot, and isotopic labeling. Histone Kacac can be dynamically regulated by acetoacetate concentration, possibly via acetoacetyl-CoA. Biochemical studies show that HBO1, traditionally known as an acetyltransferase, can also serve as an acetoacetyltransferase. In addition, 33 Kacac sites are identified on mammalian histones, depicting the landscape of histone Kacac marks across species and organs. In summary, this study thus discovers a physiologically relevant and enzymatically regulated histone mark that sheds light on the non-metabolic functions of ketone bodies.

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.

Identification of 113 new histone marks by CHiMA, a tailored database search strategy.

Shotgun proteomics has been widely used to identify histone marks. Conventional database search methods rely on the "target-decoy" strategy to calculate the false discovery rate (FDR) and distinguish true peptide-spectrum matches (PSMs) from false ones. This strategy has a caveat of inaccurate FDR caused by the small data size of histone marks. To address this challenge, we developed a tailored database search strategy, named "Comprehensive Histone Mark Analysis (CHiMA)." Instead of target-decoy-based FDR, this method uses "50% matched fragment ions" as the key criterion to identify high-confidence PSMs. CHiMA identified twice as many histone modification sites as the conventional method in benchmark datasets. Reanalysis of our previous proteomics data using CHiMA led to the identification of 113 new histone marks for four types of lysine acylations, almost doubling the number of previously reported marks. This tool not only offers a valuable approach for identifying histone modifications but also greatly expands the repertoire of histone marks.

A TRUSTED targeted mass spectrometry assay for pan-herpesvirus protein detection.

The presence and abundance of viral proteins within host cells are part of the essential signatures of the cellular stages of viral infections. However, methods that can comprehensively detect and quantify these proteins are still limited, particularly for viruses with large protein coding capacity. Here, we design and experimentally validate a mass spectrometry-based Targeted herpesviRUS proTEin Detection (TRUSTED) assay for monitoring human viruses representing the three Herpesviridae subfamilies-herpes simplex virus type 1, human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus. We demonstrate assay applicability for (1) capturing the temporal cascades of viral replication, (2) detecting proteins throughout a range of virus concentrations and in in vivo models of infection, (3) assessing the effects of clinical therapeutic agents and sirtuin-modulating compounds, (4) studies using different laboratory and clinical viral strains, and (5) discovering a role for carbamoyl phosphate synthetase 1 in supporting HCMV replication.

CHD4 is recruited by GATA4 and NKX2-5 to repress noncardiac gene programs in the developing heart.

The nucleosome remodeling and deacetylase (NuRD) complex is one of the central chromatin remodeling complexes that mediates gene repression. NuRD is essential for numerous developmental events, including heart development. Clinical and genetic studies have provided direct evidence for the role of chromodomain helicase DNA-binding protein 4 (CHD4), the catalytic component of NuRD, in congenital heart disease (CHD), including atrial and ventricular septal defects. Furthermore, it has been demonstrated that CHD4 is essential for mammalian cardiomyocyte formation and function. A key unresolved question is how CHD4/NuRD is localized to specific cardiac target genes, as neither CHD4 nor NuRD can directly bind DNA. Here, we coupled a bioinformatics-based approach with mass spectrometry analyses to demonstrate that CHD4 interacts with the core cardiac transcription factors GATA4, NKX2-5, and TBX5 during embryonic heart development. Using transcriptomics and genome-wide occupancy data, we characterized the genomic landscape of GATA4, NKX2-5, and TBX5 repression and defined the direct cardiac gene targets of the GATA4-CHD4, NKX2-5-CHD4, and TBX5-CHD4 complexes. These data were used to identify putative cis-regulatory elements controlled by these complexes. We genetically interrogated two of these silencers in vivo: Acta1 and Myh11 We show that deletion of these silencers leads to inappropriate skeletal and smooth muscle gene misexpression, respectively, in the embryonic heart. These results delineate how CHD4/NuRD is localized to specific cardiac loci and explicates how mutations in the broadly expressed CHD4 protein lead to cardiac-specific disease states.

Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation.

Membrane proteins are involved in various cellular processes. However, purification of membrane proteins has long been a challenging task, as membrane protein stability in detergent is the bottleneck for purification and subsequent analyses. Therefore, the optimization of detergent conditions is critical for the preparation of membrane proteins. Here, we utilize analytical ultracentrifugation (AUC) to examine the effects of different detergents (OG, Triton X-100, DDM), detergent concentrations, and detergent supplementation on the behavior of membrane protein TmrA. Our results suggest that DDM is more suitable for the purification of TmrA compared with OG and TritonX-100; a high concentration of DDM yields a more homogeneous protein aggregation state; supplementing TmrA purified with a low DDM concentration with DDM maintains the protein homogeneity and aggregation state, and may serve as a practical and cost-effective strategy for membrane protein purification.

Cardiac proteomics reveals sex chromosome-dependent differences between males and females that arise prior to gonad formation.

Sex disparities in cardiac homeostasis and heart disease are well documented, with differences attributed to actions of sex hormones. However, studies have indicated sex chromosomes act outside of the gonads to function without mediation by gonadal hormones. Here, we performed transcriptional and proteomics profiling to define differences between male and female mouse hearts. We demonstrate, contrary to current dogma, cardiac sex disparities are controlled not only by sex hormones but also through a sex-chromosome mechanism. Using Turner syndrome (XO) and Klinefelter (XXY) models, we find the sex-chromosome pathway is established by X-linked gene dosage. We demonstrate cardiac sex disparities occur at the earliest stages of heart formation, a period before gonad formation. Using these datasets, we identify and define a role for alpha-1B-glycoprotein (A1BG), showing loss of A1BG leads to cardiac defects in females, but not males. These studies provide resources for studying sex-biased cardiac disease states.

The antiviral sirtuin 3 bridges protein acetylation to mitochondrial integrity and metabolism during human cytomegalovirus infection.

Regulation of mitochondrial structure and function is a central component of infection with viruses, including human cytomegalovirus (HCMV), as a virus means to modulate cellular metabolism and immune responses. Here, we link the activity of the mitochondrial deacetylase SIRT3 and global mitochondrial acetylation status to host antiviral responses via regulation of both mitochondrial structural integrity and metabolism during HCMV infection. We establish that SIRT3 deacetylase activity is necessary for suppressing virus production, and that SIRT3 maintains mitochondrial pH and membrane potential during infection. By defining the temporal dynamics of SIRT3-substrate interactions during infection, and overlaying acetylome and proteome information, we find altered SIRT3 associations with the mitochondrial fusion factor OPA1 and acetyl-CoA acyltransferase 2 (ACAA2), concomitant with changes in their acetylation levels. Using mutagenesis, microscopy, and virology assays, we determine OPA1 regulates mitochondrial morphology of infected cells and inhibits HCMV production. OPA1 acetylation status modulates these functions, and we establish K834 as a site regulated by SIRT3. Control of SIRT3 protein levels or enzymatic activity is sufficient for regulating mitochondrial filamentous structure. Lastly, we establish a virus restriction function for ACAA2, an enzyme involved in fatty acid beta-oxidation. Altogether, we highlight SIRT3 activity as a regulatory hub for mitochondrial acetylation and morphology during HCMV infection and point to global acetylation as a reflection of mitochondrial health.

Temporal dynamics of protein complex formation and dissociation during human cytomegalovirus infection.

The co-evolution and co-existence of viral pathogens with their hosts for millions of years is reflected in dynamic virus-host protein-protein interactions (PPIs) that are intrinsic to the spread of infections. Here, we investigate the system-wide dynamics of protein complexes throughout infection with the herpesvirus, human cytomegalovirus (HCMV). Integrating thermal shift assays and mass spectrometry quantification with virology and microscopy, we monitor the temporal formation and dissociation of hundreds of functional protein complexes and the dynamics of host-host, virus-host, and virus-virus PPIs. We establish pro-viral roles for cellular protein complexes and translocating proteins. We show the HCMV receptor integrin beta 1 dissociates from extracellular matrix proteins, becoming internalized with CD63, which is necessary for virus production. Moreover, this approach facilitates characterization of essential viral proteins, such as pUL52. This study of temporal protein complex dynamics provides insights into mechanisms of HCMV infection and a resource for biological and therapeutic studies.

Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

Triple-negative breast cancer (TNBC) patients have the worst prognosis and distant metastasis-free survival among all major subtypes of breast cancer. The poor clinical outlook is further exacerbated by a lack of effective targeted therapies for TNBC. Here we show that ectopic expression and therapeutic delivery of the secreted protein Tubulointerstitial nephritis antigen-like 1 (Tinagl1) suppresses TNBC progression and metastasis through direct binding to integrin α5ß1, αvß1, and epidermal growth factor receptor (EGFR), and subsequent simultaneous inhibition of focal adhesion kinase (FAK) and EGFR signaling pathways. Moreover, Tinagl1 protein level is associated with good prognosis and reversely correlates with FAK and EGFR activation status in TNBC. Our results suggest Tinagl1 as a candidate therapeutic agent for TNBC by dual inhibition of integrin/FAK and EGFR signaling pathways.

Proteomics and integrative omic approaches for understanding host-pathogen interactions and infectious diseases.

Organisms are constantly exposed to microbial pathogens in their environments. When a pathogen meets its host, a series of intricate intracellular interactions shape the outcome of the infection. The understanding of these host-pathogen interactions is crucial for the development of treatments and preventive measures against infectious diseases. Over the past decade, proteomic approaches have become prime contributors to the discovery and understanding of host-pathogen interactions that represent anti- and pro-pathogenic cellular responses. Here, we review these proteomic methods and their application to studying viral and bacterial intracellular pathogens. We examine approaches for defining spatial and temporal host-pathogen protein interactions upon infection of a host cell. Further expanding the understanding of proteome organization during an infection, we discuss methods that characterize the regulation of host and pathogen proteomes through alterations in protein abundance, localization, and post-translational modifications. Finally, we highlight bioinformatic tools available for analyzing such proteomic datasets, as well as novel strategies for integrating proteomics with other omic tools, such as genomics, transcriptomics, and metabolomics, to obtain a systems-level understanding of infectious diseases.

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.

Structure and mechanism of a type III secretion protease, NleC.

NleC is one of the virulence factors that is injected into infected host cells by enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) via a needle-like protein complex called the type III secretion system (T3SS). The cytosolic NleC specifically cleaves the p65 subunit of NF-κB in the p65-p50 heterodimeric complex just after the Cys38 site in its N-terminal domain. The degradation of the remainder of the p65 C-terminal domain by the proteasome disrupts the NF-κB signalling pathway, thus dampening the host inflammatory response. Here, the crystal structure of NleC is reported at 1.55 Šresolution. In conjunction with biochemical analyses, the structure reveals that NleC is a member of the zincin zinc protease family and that the configuration of the NleC active site resembles that of the metzincin clan of metallopeptidases but without the canonical Met turn of astacin. The extended zinc-binding motif of NleC (HEXXHXXTXXXD) includes three metal ligands. The fifth zinc ligand, a conserved tyrosine (a bound water molecule is the fourth ligand), lies 45 residues downstream of the zincin motif. Furthermore, the electrostatic potential complementarity between NleC and p65 also contributes to the cleavage activity of the protease. These results not only provide important insights into the mechanism of how NleC recognizes its substrates, but also shed light on the design of new antibiotics for the food-borne diseases arising from EPEC and EHEC.

Molecular basis for the selective and ABA-independent inhibition of PP2CA by PYL13.

PYR1/PYL/RCAR family proteins (PYLs) are well-characterized abscisic acid (ABA) receptors. Among the 14 PYL members in Arabidopsis thaliana, PYL13 is ABA irresponsive and its function has remained elusive. Here, we show that PYL13 selectively inhibits the phosphatase activity of PP2CA independent of ABA. The crystal structure of PYL13-PP2CA complex, which was determined at 2.4 Å resolution, elucidates the molecular basis for the specific recognition between PP2CA and PYL13. In addition to the canonical interactions between PYLs and PP2Cs, an extra interface is identified involving an element in the vicinity of a previously uncharacterized CCCH zinc-finger (ZF) motif in PP2CA. Sequence blast identified another 56 ZF-containing PP2Cs, all of which are from plants. The structure also reveals the molecular determinants for the ABA irresponsiveness of PYL13. Finally, biochemical analysis suggests that PYL13 may hetero-oligomerize with PYL10. These two PYLs antagonize each other in their respective ABA-independent inhibitions of PP2Cs. The biochemical and structural studies provide important insights into the function of PYL13 in the stress response of plant and set up a foundation for future biotechnological applications of PYL13.