Nucleolin binds to and regulates transcription of hepatitis B virus covalently closed circular DNA minichromosome.

Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.

PI3K-AKT activation resculpts integrin signaling to drive filamentous tau-induced proinflammatory astrogliosis.

Background Microtubule-binding protein tau is a misfolding-prone protein associated with tauopathies. As tau undergoes cell-to-cell transmission, extracellular tau aggregates convert astrocytes into a pro-inflammatory state via integrin activation, causing them to release unknown neurotoxic factors. Results Here, we combine transcriptomics with isotope labeling-based quantitative mass spectrometry analysis of mouse primary astrocyte secretome to establish PI3K-AKT as a critical differentiator between pathogenic and physiological integrin activation; simultaneous activation of PI3K-AKT and focal adhesion kinase (FAK) in tau fibril-treated astrocytes changes the output of integrin signaling, causing pro-inflammatory gene upregulation, trans-Golgi network restructuring, and altered secretory flow. Furthermore, NCAM1, as a proximal signaling component in tau-stimulated integrin and PI3K-AKT activation, facilitates the secretion of complement C3 as a main neurotoxic factor. Significantly, tau fibrils-associated astrogliosis and C3 secretion can be mitigated by FAK or PI3K inhibitors. Conclusions These findings reveal an unexpected function for PI3K-AKT in tauopathy-associated reactive astrogliosis, which may be a promising target for anti-inflammation-based Alzheimer's therapy.

PI3K-AKT activation resculpts integrin signaling to drive filamentous tau-induced proinflammatory astrogliosis.

BACKGROUND: Microtubule-binding protein tau is a misfolding-prone protein associated with tauopathies. As tau undergoes cell-to-cell transmission, extracellular tau aggregates convert astrocytes into a pro-inflammatory state via integrin activation, causing them to release unknown neurotoxic factors. RESULTS: Here, we combine transcriptomics with isotope labeling-based quantitative mass spectrometry analysis of mouse primary astrocyte secretome to establish PI3K-AKT as a critical differentiator between pathogenic and physiological integrin activation; simultaneous activation of PI3K-AKT and focal adhesion kinase (FAK) in tau fibril-treated astrocytes changes the output of integrin signaling, causing pro-inflammatory gene upregulation, trans-Golgi network restructuring, and altered secretory flow. Furthermore, NCAM1, as a proximal signaling component in tau-stimulated integrin and PI3K-AKT activation, facilitates the secretion of complement C3 as a main neurotoxic factor. Significantly, tau fibrils-associated astrogliosis and C3 secretion can be mitigated by FAK or PI3K inhibitors. CONCLUSIONS: These findings reveal an unexpected function for PI3K-AKT in tauopathy-associated reactive astrogliosis, which may be a promising target for anti-inflammation-based Alzheimer's therapy.

In vivo Polycystin-1 interactome using a novel Pkd1 knock-in mouse model.

PKD1 is the most commonly mutated gene causing autosomal dominant polycystic kidney disease (ADPKD). It encodes Polycystin-1 (PC1), a putative membrane protein that undergoes a set of incompletely characterized post-transcriptional cleavage steps and has been reported to localize in multiple subcellular locations, including the primary cilium and mitochondria. However, direct visualization of PC1 and detailed characterization of its binding partners remain challenging. We now report a new mouse model with HA epitopes and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. Using this model, we sought to visualize endogenous PC1-eGFP and performed affinity-purification mass spectrometry (AP-MS) and network analyses. We show that the modified Pkd1 allele is fully functional but the eGFP-tagged protein cannot be detected without signal amplification by secondary antibodies. Using nanobody-coupled beads and large quantities of tissue, AP-MS identified an in vivo PC1 interactome, which is enriched for mitochondrial proteins and components of metabolic pathways. These studies suggest this mouse model and interactome data will be useful to understand PC1 function, but that new methods and brighter tags will be required to track endogenous PC1.

VpdC is a ubiquitin-activated phospholipase effector that regulates Legionella vacuole expansion during infection.

Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that Legionella pneumophila controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature. Consequently, L. pneumophila overproducing VpdC were prevented from adequately expanding their surrounding membrane, trapping the replicating bacteria within spatially confined vacuoles and reducing their capability to proliferate intracellularly. Quantitative lipidomics confirmed a VpdC-dependent increase in several types of lysophospholipids during infection, and VpdC production in transiently transfected cells caused tubulation of organelle membranes as well as mitochondria fragmentation, processes that can be phenocopied by supplying cells with exogenous lysophospholipids. Together, these results demonstrate an important role for bacterial phospholipases in vacuolar expansion.

A clostripain-like protease plays a major role in generating the secretome of enterotoxigenic Bacteroides fragilis.

Bacteroides fragilis toxin (BFT) is a protein secreted by enterotoxigenic (ETBF) strains of B. fragilis. BFT is synthesized as a proprotein (proBFT) that is predicted to be a lipoprotein and that is cleaved into two discrete fragments by a clostripain-like protease called fragipain (Fpn). In this study, we obtained evidence that Fpn cleaves proBFT following its transport across the outer membrane. Remarkably, we also found that the disruption of the fpn gene led to a strong reduction in the level of >100 other proteins, many of which are predicted to be lipoproteins, in the culture medium of an ETBF strain. Experiments performed with purified Fpn provided direct evidence that the protease releases at least some of these proteins from the cell surface. The observation that wild-type cells outcompeted an fpn- strain in co-cultivation assays also supported the notion that Fpn plays an important role in cell physiology and is not simply dedicated to toxin biogenesis. Finally, we found that purified Fpn altered the adhesive properties of HT29 intestinal epithelial cells. Our results suggest that Fpn is a broad-spectrum protease that not only catalyzes the protein secretion on a wide scale but that also potentially cleaves host cell proteins during colonization.

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease.

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

Chlorcyclizine Inhibits Viral Fusion of Hepatitis C Virus Entry by Directly Targeting HCV Envelope Glycoprotein 1.

Chlorcyclizine (CCZ) is a potent hepatitis C virus (HCV) entry inhibitor, but its molecular mechanism is unknown. Here, we show that CCZ directly targets the fusion peptide of HCV E1 and interferes with the fusion process. Generation of CCZ resistance-associated substitutions of HCV in vitro revealed six missense mutations in the HCV E1 protein, five being in the putative fusion peptide. A viral fusion assay demonstrated that CCZ blocked HCV entry at the membrane fusion step and that the mutant viruses acquired resistance to CCZ's action in blocking membrane fusion. UV cross-linking of photoactivatable CCZ-diazirine-biotin in both HCV-infected cells and recombinant HCV E1/E2 protein demonstrated direct binding to HCV E1 glycoprotein. Mass spectrometry analysis revealed that CCZ cross-linked to an E1 sequence adjacent to the putative fusion peptide. Docking simulations demonstrate a putative binding model, wherein CCZ binds to a hydrophobic pocket of HCV E1 and forms extensive interactions with the fusion peptide.

Exosomes mediate sensory hair cell protection in the inner ear.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

Dual Roles for Yeast Sti1/Hop in Regulating the Hsp90 Chaperone Cycle.

The Hsp90 chaperone is regulated by many cochaperones that tune its activities, but how they act to coordinate various steps in the reaction cycle is unclear. The primary role of Saccharomyces cerevisiae Hsp70/Hsp90 cochaperone Sti1 (Hop in mammals) is to bridge Hsp70 and Hsp90 to facilitate client transfer. Sti1 is not essential, so Hsp90 can interact with Hsp70 in vivo without Sti1. Nevertheless, many Hsp90 mutations make Sti1 necessary. We noted that Sti1-dependent mutations cluster in regions proximal to N-terminal domains (SdN) or C-terminal domains (SdC), which are known to be important for interaction with Hsp70 or clients, respectively. To uncover mechanistic details of Sti1-Hsp90 cooperation, we identified intramolecular suppressors of the Hsp90 mutants and assessed their physical, functional, and genetic interactions with Hsp70, Sti1, and other cochaperones. Our findings suggest Hsp90 SdN and SdC mutants depend on the same interaction with Sti1, but for different reasons. Sti1 promoted an essential Hsp70 interaction in the SdN region and supported SdC-region function by establishing an Hsp90 conformation crucial for capturing clients and progressing through the reaction cycle. We find the Hsp70 interaction and relationship with Sti1/Hop is conserved in the human Hsp90 system. Our work consolidates and clarifies much structural, biochemical, and computational data to define in vivo roles of Sti1/Hop in coordinating Hsp70 binding and client transfer with progression of the Hsp90 reaction cycle.

Folding of a bacterial integral outer membrane protein is initiated in the periplasm.

The Bam complex promotes the insertion of ß-barrel proteins into the bacterial outer membrane, but it is unclear whether it threads ß-strands into the lipid bilayer in a stepwise fashion or catalyzes the insertion of pre-folded substrates. Here, to distinguish between these two possibilities, we analyze the biogenesis of UpaG, a trimeric autotransporter adhesin (TAA). TAAs consist of three identical subunits that together form a single ß-barrel domain and an extracellular coiled-coil ("passenger") domain. Using site-specific photocrosslinking to obtain spatial and temporal insights into UpaG assembly, we show that UpaG ß-barrel segments fold into a trimeric structure in the periplasm that persists until the termination of passenger-domain translocation. In addition to obtaining evidence that at least some ß-barrel proteins begin to fold before they interact with the Bam complex, we identify several discrete steps in the assembly of a poorly characterized class of virulence factors.

The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble proteins for degradation.

In eukaryotes, many proteins function in multi-subunit complexes that require proper assembly. To maintain complex stoichiometry, cells use the endoplasmic reticulum-associated degradation system to degrade unassembled membrane subunits, but how unassembled soluble proteins are eliminated is undefined. Here we show that degradation of unassembled soluble proteins (referred to as unassembled soluble protein degradation, USPD) requires the ubiquitin selective chaperone p97, its co-factor nuclear protein localization protein 4 (Npl4), and the proteasome. At the ubiquitin ligase level, the previously identified protein quality control ligase UBR1 (ubiquitin protein ligase E3 component n-recognin 1) and the related enzymes only process a subset of unassembled soluble proteins. We identify the homologous to the E6-AP carboxyl terminus (homologous to the E6-AP carboxyl terminus) domain-containing protein HUWE1 as a ubiquitin ligase for substrates bearing unshielded, hydrophobic segments. We used a stable isotope labeling with amino acids-based proteomic approach to identify endogenous HUWE1 substrates. Interestingly, many HUWE1 substrates form multi-protein complexes that function in the nucleus although HUWE1 itself is cytoplasmically localized. Inhibition of nuclear entry enhances HUWE1-mediated ubiquitination and degradation, suggesting that USPD occurs primarily in the cytoplasm. Altogether, these findings establish a new branch of the cytosolic protein quality control network, which removes surplus polypeptides to control protein homeostasis and nuclear complex assembly.

A Longitudinal Study of the Development of Emotional Deception Detection Within New Same-Sex Friendships.

Previous studies show that close friends improve at lie detection over time. However, is this improvement due to an increase in the ability to decode the feelings of close friends or a change in how close friends communicate their true and deceptive emotions? In a study of 45 pairs of friends, one friend from each pair (the "sender") was videotaped showing truthful and faked affect in response to pleasant and unpleasant movie clips. The other friend from each pair (the "judge") guessed the true emotions of both the friend and a stranger 1 month and 6 months into the friendship. Judges were better at guessing the true emotions of friends than strangers, and this advantage in judging friends increased among close friends over time. Surprisingly, improvement over time was due mostly to a change in the sender's communication, rather than an increase in judges' ability to decode their friends' feelings.

A proteomic approach to discover and compare interacting partners of papillomavirus E2 proteins from diverse phylogenetic groups.

Papillomaviruses are a very successful group of viruses that replicate persistently in localized regions of the stratified epithelium of their specific host. Infection results in pathologies ranging from asymptomatic infection, benign warts, to malignant carcinomas. Despite this diversity, papillomavirus genomes are small (7-8 kbp) and contain at most eight genes. To sustain the complex papillomaviral life cycle, each viral protein has multiple functions and interacts with and manipulates a plethora of cellular proteins. In this study, we use tandem affinity purification and MS to identify host factors that interact with 11 different papillomavirus E2 proteins from diverse phylogenetic groups. The E2 proteins function in viral transcription and replication and correspondingly interact with host proteins involved in transcription, chromatin remodeling and modification, replication, and RNA processing.

Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM) and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

Structure of the Plasmodium 6-cysteine s48/45 domain.

The s48/45 domain was first noted in Plasmodium proteins more than 15 y ago. Previously believed to be unique to Plasmodium, the s48/45 domain is present in other aconoidasidans. In Plasmodium, members of the s48/45 family of proteins are localized on the surface of the parasite in different stages, mostly by glycosylphosphatydylinositol-anchoring. Members such as P52 and P36 seem to play a role in invasion of hepatocytes, and Pfs230 and Pfs48/45 are involved in fertilization in the sexual stages and have been consistently studied as targets of transmission-blocking vaccines for years. In this report, we present the molecular structure for the s48/45 domain corresponding to the C-terminal domain of the blood-stage protein Pf12 from Plasmodium falciparum, obtained by NMR. Our results indicate that this domain is a ß-sandwich formed by two sheets with a mixture of parallel and antiparallel strands. Of the six conserved cysteines, two pairs link the ß-sheets by two disulfide bonds, and the third pair forms a bond outside the core. The structure of the s48/45 domain conforms well to the previously defined surface antigen 1 (SAG1)-related-sequence (SRS) fold observed in the SAG family of surface antigens found in Toxoplasma gondii. Despite extreme sequence divergence, remarkable spatial conservation of one of the disulfide bonds is observed, supporting the hypothesis that the domains have evolved from a common ancestor. Furthermore, a homologous domain is present in ephrins, raising the possibility that the precursor of the s48/45 and SRS domains emerged from an ancient transfer to Apicomplexa from metazoan hosts.

Protein phosphatase 2A-SUR-6/B55 regulates centriole duplication in C. elegans by controlling the levels of centriole assembly factors.

Centrioles play a crucial role in mitotic spindle assembly and duplicate precisely once per cell cycle. In worms, flies, and humans, centriole assembly is dependent upon a key regulatory kinase (ZYG-1/Sak/Plk4) and its downstream effectors SAS-5 and SAS-6. Here we report a role for protein phosphatase 2A (PP2A) in centriole duplication. We find that the PP2A catalytic subunit LET-92, the scaffolding subunit PAA-1, and the B55 regulatory subunit SUR-6 function together to positively regulate centriole assembly. In PP2A-SUR-6-depleted embryos, the levels of ZYG-1 and SAS-5 are reduced and the ZYG-1- and SAS-5-dependent recruitment of SAS-6 to the nascent centriole fails. We show that PP2A physically associates with SAS-5 in vivo and that inhibiting proteolysis can rescue SAS-5 levels and the centriole duplication defect of PP2A-depleted embryos. Together, our findings indicate that PP2A-SUR-6 promotes centriole assembly by protecting ZYG-1 and SAS-5 from degradation.

Structural basis of HIV-1 neutralization by affinity matured Fabs directed against the internal trimeric coiled-coil of gp41.

The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

The assessment of human erythroid output in NOD/SCID mice reconstituted with human hematopoietic stem cells.

The third-generation NOD/LtSz-scid/IL2Rγ(null) (NOD/SCID IL2Rγ(null)) mouse represents a significantly improved xenograft model allowing high levels of human leukocyte engraftment over extended follow up. One remaining limitation of this mouse model, however, is the low level of circulating human erythrocytes. We established a practical ex vivo erythroid culture system of xenograft marrow progenitors to enrich for human erythroid progeny. At various time points after transplant, erythroid cells were easily assayed after 17 days of ex vivo culture of xenograft marrow, with nearly all nucleated cells of human origin and approximately 60% human GPA or CD71 positive. We then transplanted cord blood CD34(+) cells marked with a lentiviral vector encoding green fluorescent protein (GFP). Three months later, ex vivo culture of xenograft marrow progenitors showed 41.3% of the cultured erythroid cells were positive for GFP and human CD71, and 56.2% were positive for GFP and human GPA, similar to that of circulating leukocytes at the same time point. Next, G-CSF mobilized peripheral blood CD34(+) cells from a sickle cell trait subject were infused in this mouse model to determine if the hemoglobin pattern could be modeled. CD34(+) cells from the sickle cell trait subject engrafted equally compared to CD34(+) cells from normal subjects, establishing the sickle cell trait phenotype. Lastly, a comparison of adult-derived peripheral blood CD34(+) cells and cord blood-derived CD34(+) cells xenografted mice was made, and long term follow-up demonstrated a recapitulation of the fetal to adult hemoglobin switch. This approach should prove a useful tool for testing strategies for genetic manipulation of erythroid progeny and the study of hemoglobin switching.

Casein kinase 1alpha governs antigen-receptor-induced NF-kappaB activation and human lymphoma cell survival.

The transcription factor NF-kappaB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-kappaB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-kappaB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1alpha (CK1alpha) as a bifunctional regulator of NF-kappaB. CK1alpha dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1alpha kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1alpha has thus a dual 'gating' function which first promotes and then terminates receptor-induced NF-kappaB. ABC DLBCL cells required CK1alpha for constitutive NF-kappaB activity, indicating that CK1alpha functions as a conditionally essential malignancy gene-a member of a new class of potential cancer therapeutic targets.