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Individuals at distinct stages of Alzheimer’s disease (AD) show abnormal electroencephalographic activity, which has been linked to network hyperexcitability and cognitive decline. However, whether pro-excitatory changes at the synaptic level are observed in brain areas affected early in AD, and if they are emergent in MCI, is not clearly known. Equally important, it is not known whether global synaptic E/I imbalances correlate with the severity of cognitive impairment in the continuum of AD. Measuring the amplitude of ion currents of human excitatory and inhibitory synaptic receptors microtransplanted from the hippocampus and temporal cortex of cognitively normal, mildly cognitively impaired and AD individuals into surrogate cells, we found regional differences in pro-excitatory shifts of the excitatory to inhibitory (E/I) current ratio that correlates positively with toxic proteins and degree of pathology, and impinges negatively on cognitive performance scores. Using these data with electrophysiologically anchored analysis of the synapto-proteome in the same individuals, we identified a group of proteins sustaining synaptic function and those related to synaptic toxicity. We also found an uncoupling between the function and expression of proteins for GABAergic signaling in the temporal cortex underlying larger E/I and worse cognitive performance. Further analysis of transcriptomic and in situ hybridization datasets from an independent cohort across the continuum of AD confirm regional differences in pro-excitatory shifts of the E/I balance that correlate negatively with the most recent calibrated composite scores for memory, executive function, language and visuospatial abilities, as well as overall cognitive performance. These findings indicate that early shifts of E/I balance may contribute to loss of cognitive capabilities in the continuum of AD clinical syndrome.

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on the impact of receptor binding domain changes, mutations in the C-terminal of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replicationin vitroand less diseasein vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Together, the data show that N679K is a loss-of-function mutation reducing overall spike levels during omicron infection, which may have important implications for disease severity, immunity, and vaccine efficacy.One Sentence SummarySpike substitution N679K attenuates SARS-CoV-2 Omicron variants by decreasing spike protein and has potential implications for immunity and vaccine efficacy.

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.

The purpose of this study was to conduct initial characterization of membrane vesicles isolated from human placenta by agitation of villous tissue (apical and basal) as well as vesicles obtained following dual perfusion of placental lobule. The morphology, physical and biological properties of the isolated vesicles were determined by electron microscopy, dynamic light scattering, and immunoblotting as well as nanoflow liquid chromatography-mass spectrometry proteomics analysis. CD-1 male mice were used to test the biocompatibility of the vesicles in vivo and assess the biodistribution of fluorescently labeled apical and perfusion vesicles. The vesicles obtained following placental perfusion and the apical vesicles had Z-average diameters of 199±23 nm and 246±24 nm, respectively, and demonstrated nanocarrier stability, low toxicity, and low immunogenicity. On the other hand, administration of basal vesicles resulted in animal demise with LD50 of 0.85 μgprotein/g. Both fluorescently labeled apical and perfusion vesicles were detected in the lungs, liver, kidneys, and spleen of CD-1 mice within 24 h of administration. However, there were differences in organ distribution of these vesicles over 24 hours time period. These data suggest that placental apical and perfusion vesicles have a potential for further development as biological vehicles for drug delivery.

Cancer cells utilize secretory pathways for paracrine signaling and extracellular matrix remodeling to facilitate directional cell migration, invasion, and metastasis. The Golgi apparatus is a central secretory signaling hub that is often deregulated in cancer. Here we described technologies that utilize microscopic, biochemical, and proteomic approaches to analyze Golgi secretory functions in genetically heterogeneous cancer cell lines.

Substantial increases in the conjugation of the main human SUMO paralogs, SUMO1, SUMO2, and SUMO3, are observed upon exposure to different cellular stressors, and such increases are considered important to facilitate cell survival to stress. Despite their critical cellular role, little is known about how the levels of the SUMO modifiers are regulated in the cell, particularly as it relates to the changes observed upon stress. Here we characterize the contribution of alternative splicing towards regulating the expression of the main human SUMO paralogs under normalcy and three different stress conditions, heat-shock, cold-shock, and Influenza A Virus infection. Our data reveal that the normally spliced transcript variants are the predominant mature mRNAs produced from the SUMO genes and that the transcript coding for SUMO2 is by far the most abundant of all. We also provide evidence that alternatively spliced transcripts coding for protein isoforms of the prototypical SUMO proteins, which we refer to as the SUMO alphas, are also produced, and that their abundance and nuclear export are affected by stress in a stress- and cell-specific manner. Additionally, we provide evidence that the SUMO alphas are actively synthesized in the cell as their coding mRNAs are found associated with translating ribosomes. Finally, we provide evidence that the SUMO alphas are functionally different from their prototypical counterparts, with SUMO1α and SUMO2α being non-conjugatable to protein targets, SUMO3α being conjugatable but targeting a seemingly different subset of proteins from those targeted by SUMO3, and all three SUMO alphas displaying different cellular distributions from those of the prototypical SUMOs. Thus, alternative splicing appears to be an important contributor to the regulation of the expression of the SUMO proteins and the cellular functions of the SUMOylation system.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site—the FCS, loop length, and glycosylation—are required for efficient SARS-CoV-2 replication and pathogenesis.

MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors) are synaptic cell surface molecules that regulate the formation of trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs), which promote synaptic development. Mutations in MDGAs are implicated in various neuropsychiatric diseases. MDGAs bind NLGNs in cis on the postsynaptic membrane and physically block NLGNs from binding to NRXNs. In crystal structures, the six immunoglobulin (Ig) and single fibronectin III domains of MDGA1 reveal a striking compact, triangular shape, both alone and in complex with NLGNs. Whether this unusual domain arrangement is required for biological function or other arrangements occur with different functional outcomes is unknown. Here, we show that WT MDGA1 can adopt both compact and extended 3D conformations that bind NLGN2. Designer mutants targeting strategic molecular elbows in MDGA1 alter the distribution of 3D conformations while leaving the binding affinity between soluble ectodomains of MDGA1 and NLGN2 intact. In contrast, in a cellular context, these mutants result in unique combinations of functional consequences, including altered binding to NLGN2, decreased capacity to conceal NLGN2 from NRXN1β, and/or suppressed NLGN2-mediated inhibitory presynaptic differentiation, despite the mutations being located far from the MDGA1-NLGN2 interaction site. Thus, the 3D conformation of the entire MDGA1 ectodomain appears critical for its function, and its NLGN-binding site on Ig1-Ig2 is not independent of the rest of the molecule. As a result, global 3D conformational changes to the MDGA1 ectodomain via strategic elbows may form a molecular mechanism to regulate MDGA1 action within the synaptic cleft.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform–specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform–specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders.

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a “kissing” interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor’s inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.

Complete LC-MS-based protein primary sequence characterization requires measurement of intact protein profiles under denaturing and/or reducing conditions. To address issues of protein overcharging of unstructured proteins under acidic, denaturing conditions and sample heterogeneity (macro- and micro-scales) which often confound denaturing intact mass analysis of a wide variety of protein samples, we propose the use of broadband isolation of entire charge state distributions of intact proteins followed by ion-ion proton transfer charge reduction, which we have termed "full scan PTCR" (fsPTCR). Using rapid denaturing size exclusion chromatography coupled to fsPTCR-Orbitrap MS and time-resolved deconvolution data analysis, we demonstrate a strategy for method optimization, leading to significant analytical advantages over conventional MS1. Denaturing analysis of the flexible bacterial translation initiation factor 2 (91 kDa) using fsPTCR reduced overcharging and showed an 11-fold gain in S/N compared to conventional MS1. Analysis by fsPTCR-MS of the microheterogeneous glycoprotein fetuin revealed twice as many proteoforms as MS1 (112 vs 56). In a macroheterogeneous mixture of proteins ranging from 14 to 148 kDa, fsPTCR provided more than 10-fold increased sensitivity and quantitative accuracy for diluted bovine serum albumin (66 kDa). Finally, our analysis shows that collisional gas pressure is a key parameter which can be utilized during fsPTCR to retain or remove larger proteins from acquired spectra.

Mitochondrial DNA (mtDNA) resides in a high ROS environment and suffers more mutations than its nuclear counterpart. Increasing evidence suggests that mtDNA mutations are not the results of direct oxidative damage, rather are caused, at least in part, by DNA replication errors. To understand how the mtDNA replicase, Pol γ, can give rise to elevated mutations, we studied the effect of oxidation of Pol γ on replication errors. Pol γ is a high fidelity polymerase with polymerase (pol) and proofreading exonuclease (exo) activities. We show that Pol γ exo domain is far more sensitive to oxidation than pol; under oxidative conditions, exonuclease activity therefore declines more rapidly than polymerase. The oxidized Pol γ becomes editing-deficient, displaying a 20-fold elevated mutations than the unoxidized enzyme. Mass spectrometry analysis reveals that Pol γ exo domain is a hotspot for oxidation. The oxidized exo residues increase the net negative charge around the active site that should reduce the affinity to mismatched primer/template DNA. Our results suggest that the oxidative stress induced high mutation frequency on mtDNA can be indirectly caused by oxidation of the mitochondrial replicase.

Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change., The way in which metastatic tumour cells control endocytic vesicular trafficking networks to establish a front-rear polarity axis that facilitates motility remains unclear. Here, the authors show that the EMT activator ZEB1 influences vesicular trafficking dynamics to execute cell polarity change.

Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput in vitro and in vivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression.

Upregulation of hydrogen sulfide (H2S) biosynthesis, at least in part related to the upregulation of cystathionine β-synthetase (CBS) in cancer cells, serves as a tumor-promoting factor and has emerged as a possible molecular target for antitumor drug development. To facilitate future clinical translation, we have synthesized a variety of novel CBS-targeting, esterase-cleavable prodrugs based on the structure of the prototypical CBS inhibitor aminooxyacetic acid (AOAA). The pharmacological properties of these compounds were evaluated in cell-free assays with recombinant human CBS protein, the human colon cancer cell line HCT116, and in vivo using various tumor-bearing mice models. The prodrug YD0251 (the isopropyl ester derivative of AOAA) was selected for detailed characterization. YD0251 exhibits improved antiproliferative efficacy in cell culture models when compared to AOAA. It is up to 18 times more potent than AOAA at suppressing HCT116 tumor growth in vivo and is effective when administered to tumor-bearing mice either via subcutaneous injection or oral gavage. Patient-derived xenografts (PDTXs) with higher levels of CBS protein grew significantly larger than tumors with lower levels, and YD0251 treatment inhibited the growth of PDTXs with elevated CBS, whereas it had no significant effect on PDTXs with low CBS protein levels. The toxicity of YD0251 was assessed in mice subjected to subchronic administration of supratherapeutic doses the inhibitor, no significant alteration in circulating markers of organ injury or histopathological alterations were noted, up to 60 mg/kg/day × 5 days. In preparation to a future theranostic concept (to match CBS inhibitor therapy to high-CBS expressors), we identified a potential plasma marker of CBS-expressing tumors. Colon cancer cells produced significant levels of lanthionine, a rare metabolic intermediate of CBS-mediated H2S biosynthesis, forced expression of CBS into non-transformed epithelial cells increased lanthionine biogenesis in vitro and in vivo (measured in the urine of tumor-bearing mice). These current results may be useful to facilitate the translation of a CBS inhibition-based antitumor concept into the clinical space.

Exposure of rodents to &lt, 20 cGy Space Radiation (SR) impairs performance in several hippocampus-dependent cognitive tasks, including spatial memory. However, there is considerable inter-individual susceptibility to develop SR-induced spatial memory impairment. In this study, a robust label-free mass spectrometry (MS)-based unbiased proteomic profiling approach was used to characterize the composition of the hippocampal proteome in adult male Wistar rats exposed to 15 cGy of 1 GeV/n 48Ti and their sham counterparts. Unique protein signatures were identified in the hippocampal proteome of: (1) sham rats, (2) Ti-exposed rats, (3) Ti-exposed rats that had sham-like spatial memory performance, and (4) Ti-exposed rats that impaired spatial memory performance. Approximately 14% (159) of the proteins detected in hippocampal proteome of sham rats were not detected in the Ti-exposed rats. We explored the possibility that the loss of the Sham-only proteins may arise as a result of SR-induced changes in protein homeostasis. SR-exposure was associated with a switch towards increased pro-ubiquitination proteins from that seen in Sham. These data suggest that the role of the ubiquitin-proteome system as a determinant of SR-induced neurocognitive deficits needs to be more thoroughly investigated.

Proton pump inhibitors (PPIs) are widely prescribed drugs for the treatment of gastroesophageal reflux disease (GERD). Several meta-analysis studies have reported associations between prolonged use of PPIs and major adverse cardiovascular events. However, interaction of PPIs with biological molecules involved in cardiovascular health is incompletely characterized. Dimethylarginine dimethylaminohydrolase (DDAH) is a cardiovascular enzyme expressed in cardiomyocytes, and other somatic cell types in one of two isotypes (DDAH1 and DDAH2) to metabolize asymmetric dimethylarginine (ADMA); a cardiovascular risk factor and competitive inhibitor of nitric oxide synthases (NOSs).We performed high throughput drug screening of over 130,000 small molecules to discover human DDAH1 inhibitors and found that PPIs directly inhibit DDAH1. We expressed and purified the enzyme for structural and mass spectrometry proteomics studies to understand how a prototype PPI, esomeprazole, interacts with DDAH1. We also performed molecular docking studies to model the interaction of DDAH1 with esomeprazole. X-ray crystallography was used to determine the structure of DDAH1 alone and bound to esomeprazole at resolutions ranging from 1.6 to 2.9 Å.Analysis of the enzyme active site shows that esomeprazole interacts with the active site cysteine (Cys273) of DDAH1. The structural studies were corroborated by mass spectrometry which indicated that cysteine was targeted by esomeprazole to inactivate DDAH1.The inhibition of this important cardiovascular enzyme by a PPI may help explain the reported association of PPI use and increased cardiovascular risk in patients and the general population.Our study calls for pharmacovigilance studies to monitor adverse cardiovascular events in chronic PPI users.

Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kDa (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45 (G45)–myosin IIA–containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a–dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8(+) T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55-G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.

A secretory process activated by p53 loss prevents cancer cell anoikis and drives immunosuppression in the tumor microenvironment., Cancer cells exhibit hyperactive secretory states that maintain cancer cell viability and remodel the tumor microenvironment. However, the oncogenic signals that heighten secretion remain unclear. Here, we show that p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi. p53 loss up-regulates the expression of a Golgi scaffolding protein, progestin and adipoQ receptor 11 (PAQR11), which recruits an adenosine diphosphate ribosylation factor 1–containing protein complex that loads cargos into secretory vesicles. PAQR11-dependent secretion of a protease, PLAU, prevents anoikis and initiates autocrine activation of a PLAU receptor/signal transducer and activator of transcription-3-dependent pathway that up-regulates PAQR11 expression, thereby completing a feedforward loop that amplifies prometastatic effector protein secretion. Pharmacologic inhibition of PLAU receptor impairs the growth and metastasis of p53-deficient cancers. Blockade of PAQR11-dependent secretion inhibits immunosuppressive processes in the tumor microenvironment. Thus, Golgi reprogramming by p53 loss is a key driver of hypersecretion in cancer.

Oral-facial-digital syndromes (OFD) are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. To date, mutations in 12 ciliary-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline mutations inINTS13, a subunit of the Integrator complex. This 14-component complex associates with RNAPII and can cleave nascent RNA to modulate gene expression. We determined that INTS13 utilizes a discrete domain within its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germlineINTS13mutations. Depletion ofINTS13disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown inXenopusembryos lead to motile cilia anomalies. Altogether, we show that mutations inINTS13cause an autosomal recessive ciliopathy, which reveals key interactions within Integrator components.

Efficient release of promoter-proximally paused Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here, we report that Integrator subunit 8 (IntS8) is critical for transcription repression and is required for association with Protein Phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the Pol II C-terminal domain and Spt5 and prevents the transition to productive elongation. Blocking PP2A association with Integrator thus stimulates pause release and gene activation. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and suggest a model for the control of productive elongation involving active competition between transcriptional kinases and phosphatases.

Heightened secretion of pro-tumorigenic effector proteins is a feature of malignant cells. Yet the molecular underpinnings and therapeutic implications of this feature remain unclear. Here we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell-biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence pro-metastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

BACKGROUND: Protein interactions between voltage-gated sodium (Nav) channels and accessory proteins play an essential role in neuronal firing and plasticity. However, a surprisingly limited number of kinases have been identified as regulators of these molecular complexes. We hypothesized that numerous as-of-yet unidentified kinases indirectly regulate the Nav channel via modulation of the intracellular fibroblast growth factor 14 (FGF14), an accessory protein with numerous unexplored phosphomotifs and required for channel function in neurons. METHODS: Here we present results from an in-cell high-throughput screening (HTS) against the FGF14:Nav1.6 complex using >3,000 diverse compounds targeting an extensive range of signaling pathways. Regulation by top kinase targets was then explored using in vitro phosphorylation, biophysics, mass-spectrometry and patch-clamp electrophysiology. RESULTS: Compounds targeting Janus kinase 2 (JAK2) were over-represented among HTS hits. Phosphomotif scans supported by mass spectrometry revealed FGF14(Y158), a site previously shown to mediate both FGF14 homodimerization and interactions with Nav1.6, as a JAK2 phosphorylation site. Following inhibition of JAK2, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation, but not in the presence of the FGF14(Y158A) mutant. Patch-clamp electrophysiology revealed that through Y158, JAK2 controls FGF14-dependent modulation of Nav1.6 channels. In hippocampal CA1 pyramidal neurons, the JAK2 inhibitor Fedratinib reduced firing by a mechanism that is dependent upon expression of FGF14. CONCLUSIONS: These studies point toward a novel mechanism by which levels of JAK2 in neurons could directly influence firing and plasticity by controlling the FGF14 dimerization equilibrium, and thereby the availability of monomeric species for interaction with Nav1.6.

Genome-wide association studies have identified thousands of noncoding variants associated with human traits and diseases. However, the functional interpretation of these variants is a major challenge. Here, we constructed a multi-tissue atlas of human 3'UTR alternative polyadenylation (APA) quantitative trait loci (3'aQTLs), containing approximately 0.4 million common genetic variants associated with the APA of target genes, identified in 46 tissues isolated from 467 individuals (Genotype-Tissue Expression Project). Mechanistically, 3'aQTLs can alter poly(A) motifs, RNA secondary structure and RNA-binding protein-binding sites, leading to thousands of APA changes. Our CRISPR-based experiments indicate that such 3'aQTLs can alter APA regulation. Furthermore, we demonstrate that mapping 3'aQTLs can identify APA regulators, such as La-related protein 4. Finally, 3'aQTLs are colocalized with approximately 16.1% of trait-associated variants and are largely distinct from other QTLs, such as expression QTLs. Together, our findings show that 3'aQTLs contribute substantially to the molecular mechanisms underlying human complex traits and diseases.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified several relevant cell-cell junction functions. Immunohistology study confirmed that both infected WT and infected ANXA2-KO mice were subjected to adherens junctional protein (VE-cadherin) damages. However, key blood-brain barrier (BBB) components, tight junctional proteins ZO-1 and occludin, were disorganized in the brains from R. australis-infected ANXA2-KO mice, but not those of infected WT mice. Similar ANXA2-KO dependent CMHs and fragments of ZO-1 and occludin were also observed in Ebola virus-infected ANXA2-KO mice, but not found in infected WT mice. Overall, our study revealed a novel role of ANXA2 in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated tight junctions and its role in stabilizing the BBB in these deadly infections.Author SummaryTraditionally, spontaneous intracerebral microhemorrhages (CMHs) were defined as small foci of intracerebral hemorrhages. Such atraumatic CMHs are due to the rupture of a weak blood vessel wall. Infections complicating cerebrovascular accidents have been extensively investigated. However, the role of CMHs complicating infections, in particularly acute systemic infections, has been poorly explored. Population-based retrospective cohort studies suggest there are potentially more undiscovered cases of CMHs accompanying acute systemic infections. Given both the lack of an animal model and cellular/molecular pathophysiology of CMHs following acute systemic infections, there is an urgent need to increase our comprehensive understanding of acute infection-induced CMHs. Overall, our study revealed a novel role of annexin a2 (ANXA2) in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated endothelial tight junctions and its role in stabilizing the blood-brain barrier in these deadly infections.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

The Diels–Alder reaction is one of the most common methods to chemically synthesize a six-membered carbocycle. While it has long been speculated that the cyclohexene moiety found in many secondary metabolites is also introduced via similar chemistry, the enzyme SpnF involved in the biosynthesis of the insecticide spinosyn A in Saccharopolyspora spinosa is the first enzyme for which catalysis of an intramolecular [ 4 + 2 ] -cycloaddition has been experimentally verified as its only known function. Since its discovery, a number of additional standalone [ 4 + 2 ] -cyclases have been reported as potential Diels–Alderases; however, whether their catalytic cycles involve a concerted or stepwise cyclization mechanism has not been addressed experimentally. Here, we report direct experimental interrogation of the reaction coordinate for the [ 4 + 2 ] -carbocyclase SpnF via the measurement of α -secondary deuterium kinetic isotope effects (KIEs) at all sites of s p 2 → s p 3 rehybridization for both the nonenzymatic and enzyme-catalyzed cyclization of the SpnF substrate. The measured KIEs for the nonenzymatic reaction are consistent with previous computational results implicating an intermediary state between formation of the first and second carbon–carbon bonds. The KIEs measured for the enzymatic reaction suggest a similar mechanism of cyclization within the enzyme active site; however, there is evidence that conformational restriction of the substrate may play a role in catalysis.

Bacterial Type I restriction-modification (R-M) systems present a major barrier to foreign DNA entering the bacterial cell. The temperate phage P1 packages several proteins into the virion that protect the phage DNA from host restriction. Isogenic P1 deletion mutants were used to reconstitute the previously described restriction phenotypes associated with darA and darB. While P1ΔdarA and P1ΔdarB produced the expected phenotypes, deletions of adjacent genes hdf and ddrA also produced darA-like phenotypes and deletion of ulx produced a darB-like phenotype, implicating several new proteins of previously unknown function in the P1 dar antirestriction system. Interestingly, disruption of ddrB decreased P1's sensitivity to EcoB and EcoK restriction. Proteomic analysis of purified virions suggests that packaging of antirestriction components into P1 virions follows a distinct pathway that begins with the incorporation of DarA and Hdf and concludes with DarB and Ulx. Electron microscopy analysis showed that hdf and darA mutants also produce abnormally high proportions of virions with aberrant small heads, which suggests Hdf and DarA play a role in capsid morphogenesis. The P1 antirestriction system is more complex than previously realized and is comprised of multiple proteins including DdrA, DdrB, Hdf, and Ulx in addition to DarA and DarB.

Substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs), including many refined petroleum products, present a major challenge in regulatory submissions under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and US High Production Volume regulatory regimes. The inherent complexity of these substances, as well as variability in composition obfuscates detailed chemical characterization of each individual substance and their grouping for human and environmental health evaluation through read-across. In this study, we applied ion mobility mass spectrometry in conjunction with cheminformatics-based data integration and visualization to derive substance-specific signatures based on the distribution and abundance of various heteroatom classes. We used petroleum substances from four petroleum substance manufacturing streams and evaluated their chemical composition similarity based on high-dimensional substance-specific quantitative parameters including m/z distribution, drift time, carbon number range, and associated double bond equivalents and hydrogen-to-carbon ratios. Data integration and visualization revealed group-specific similarities for petroleum substances. Observed differences within a product group were indicative of batch- or manufacturer-dependent variation. We demonstrate how high-resolution analytical chemistry approaches can be used effectively to support categorization of UVCBs based on their heteroatom composition and how such data can be used in regulatory decision-making.

Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified several relevant cell-cell junction functions. Immunohistology study confirmed that both infected WT and infected ANXA2-KO mice were subjected to adherens junctional protein (VE-cadherin) damages. However, key blood-brain barrier (BBB) components, tight junctional proteins ZO-1 and occludin, were disorganized in the brains from R. australis-infected ANXA2-KO mice, but not those of infected WT mice. Similar ANXA2-KO dependent CMHs and fragments of ZO-1 and occludin were also observed in Ebola virus-infected ANXA2-KO mice, but not found in infected WT mice. Overall, our study revealed a novel role of ANXA2 in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated tight junctions and its role in stabilizing the BBB in these deadly infections., Author summary Traditionally, spontaneous intracerebral microhemorrhages (CMHs) were defined as small foci of intracerebral hemorrhages. Such atraumatic CMHs are due to the rupture of a weak blood vessel wall. Infections complicating cerebrovascular accidents have been extensively investigated. However, the role of CMHs complicating infections, in particularly acute systemic infections, has been poorly explored. Population-based retrospective cohort studies suggest there are potentially more undiscovered cases of CMHs accompanying acute systemic infections. Given both the lack of an animal model and cellular/molecular pathophysiology of CMHs following acute systemic infections, there is an urgent need to increase our comprehensive understanding of acute infection-induced CMHs. Overall, our study revealed a novel role of annexin a2 (ANXA2) in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated endothelial tight junctions and its role in stabilizing the blood-brain barrier in these deadly infections.

Enteric viruses exploit bacterial components including lipopolysaccharides (LPS) and peptidoglycan (PG) to facilitate infection in humans. With origins in the bat enteric system, we wondered if severe acute respiratory syndrome-coronavirus (SARS-CoV) or Middle East respiratory syndrome-CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity finding no change following LPS treatment. However, PG from B. subtilis reduced infection >10,000-fold while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity and surfactin treatment of the virus inoculum ablated infection in vivo. Finally, similar cyclic lipopeptides had no effect on CoV infectivity and the inhibitory effect of surfactin extended broadly to enveloped viruses including influenza, Ebola, Zika, Nipah, Chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad virucidal activity and suggest bacteria byproducts may negatively modulate virus infection.ImportanceIn this manuscript, we considered a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.