28 research outputs found
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Identification of antiviral roles for the exon-junction complex and nonsense-mediated decay in flaviviral infection.
West Nile virus (WNV) is an emerging mosquito-borne flavivirus, related to dengue virus and Zika virus. To gain insight into host pathways involved in WNV infection, we performed a systematic affinity-tag purification mass spectrometry (APMS) study to identify 259 WNV-interacting human proteins. RNA interference screening revealed 26 genes that both interact with WNV proteins and influence WNV infection. We found that WNV, dengue and Zika virus capsids interact with a conserved subset of proteins that impact infection. These include the exon-junction complex (EJC) recycling factor PYM1, which is antiviral against all three viruses. The EJC has roles in nonsense-mediated decay (NMD), and we found that both the EJC and NMD are antiviral and the EJC protein RBM8A directly binds WNV RNA. To counteract this, flavivirus infection inhibits NMD and the capsid-PYM1 interaction interferes with EJC protein function and localization. Depletion of PYM1 attenuates RBM8A binding to viral RNA, suggesting that WNV sequesters PYM1 to protect viral RNA from decay. Together, these data suggest a complex interplay between the virus and host in regulating NMD and the EJC
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Multiple Routes to Oncogenesis Are Promoted by the Human Papillomavirus-Host Protein Network.
We have mapped a global network of virus-host protein interactions by purification of the complete set of human papillomavirus (HPV) proteins in multiple cell lines followed by mass spectrometry analysis. Integration of this map with tumor genome atlases shows that the virus targets human proteins frequently mutated in HPV- but not HPV+ cancers, providing a unique opportunity to identify novel oncogenic events phenocopied by HPV infection. For example, we find that the NRF2 transcriptional pathway, which protects against oxidative stress, is activated by interaction of the NRF2 regulator KEAP1 with the viral protein E1. We also demonstrate that the L2 HPV protein physically interacts with the RNF20/40 histone ubiquitination complex and promotes tumor cell invasion in an RNF20/40-dependent manner. This combined proteomic and genetic approach provides a systematic means to study the cellular mechanisms hijacked by virally induced cancers.Significance: In this study, we created a protein-protein interaction network between HPV and human proteins. An integrative analysis of this network and 800 tumor mutation profiles identifies multiple oncogenesis pathways promoted by HPV interactions that phenocopy recurrent mutations in cancer, yielding an expanded definition of HPV oncogenic roles. Cancer Discov; 8(11); 1474-89. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1333
A crystalline, internally-coordinated chloroborane for asymmetric hydroboration
Asymmetric hydroboration is an important method in the preparation of enantiomerically-enriched compounds that are necessary in many areas of chemistry. Here is reported the preparation of a unique chiral chloroborane-internal ether complex and its applications in asymmetric hydroboration. This chloroborane is easily obtained in crystalline, solvent-free and enantiomerically pure form. The chemical shift of the Lewis-basic methoxy group is sensitive to the boron environment, making this reagent especially amenable to NMR studies in which diastereomer ratios can, in many cases, be determined before oxidation, and even kinetics are relatively easily carried out. It reacts readily with prochiral alkenes to give, after oxidation, alcohols in up to 82% ee. The dialkylchloroborane intermediates can be diastereomerically purified by recrystallization either directly, or as air-stable ethanolamine complexes, to give after oxidation alcohols in up to 99.8% ee. The chloro group is easily replaced by hydride in-situ, allowing for intramolecular asymmetric hydroborations and deuteroborations with a high degree of regiochemical control that intermolecular hydroborations cannot provide
A scaffold protein connects type IV pili with the Chp chemosensory system to mediate activation of virulence signaling in Pseudomonas aeruginosa
Type IV pili (TFP) function as mechanosensors to trigger acute virulence programs in Pseudomonas aeruginosa. On surface contact, TFP retraction activates the Chp chemosensory system phosphorelay to upregulate 3', 5'-cyclic monophosphate (cAMP) production and transcription of virulence-associated genes. To dissect the specific interactions mediating the mechanochemical relay, we used affinity purification/mass spectrometry, directed co-immunoprecipitations in P. aeruginosa, single cell analysis of contact-dependent transcriptional reporters, subcellular localization and bacterial two hybrid assays. We demonstrate that FimL, a Chp chemosensory system accessory protein of unknown function, directly links the integral component of the TFP structural complex FimV, a peptidoglycan binding protein, with one of the Chp system output response regulators PilG. FimL and PilG colocalize at cell poles in a FimV-dependent manner. While PilG phosphorylation is required for TFP function and mechanochemical signaling, it is not required for polar localization or binding to FimL. Phylogenetic analysis reveals other bacterial species simultaneously encode TFP, the Chp system, FimL, FimV and adenylate cyclase homologs, suggesting that surface sensing may be widespread among TFP-expressing bacteria. We propose that FimL acts as a scaffold enabling spatial colocalization of TFP and Chp system components to coordinate signaling leading to cAMP-dependent upregulation of virulence genes on surface contact
Chlamydia interfere with an interaction between the mannose-6-phosphate receptor and sorting nexins to counteract host restriction
Chlamydia trachomatis is an obligate intracellular pathogen that resides in a membrane-bound compartment, the inclusion. The bacteria secrete a unique class of proteins, Incs, which insert into the inclusion membrane and modulate the host-bacterium interface. We previously reported that IncE binds specifically to the Sorting Nexin 5 Phox domain (SNX5-PX) and disrupts retromer trafficking. Here, we present the crystal structure of the SNX5-PX:IncE complex, showing IncE bound to a unique and highly conserved hydrophobic groove on SNX5. Mutagenesis of the SNX5-PX:IncE binding surface disrupts a previously unsuspected interaction between SNX5 and the cation-independent mannose-6-phosphate receptor (CI-MPR). Addition of IncE peptide inhibits the interaction of CI-MPR with SNX5. Finally, C. trachomatis infection interferes with the SNX5:CI-MPR interaction, suggesting that IncE and CI-MPR are dependent on the same binding surface on SNX5. Our results provide new insights into retromer assembly and underscore the power of using pathogens to discover disease-related cell biology
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Chlamydia interfere with an interaction between the mannose-6-phosphate receptor and sorting nexins to counteract host restriction
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Characterization of an A3G-VifHIV-1-CRL5-CBFβ Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host-Pathogen Complexes.
Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFβ) to determine the structure of the (A3G-Vif-CRL5-CBFβ) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFβ complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes
PEAK3/C19orf35 pseudokinase, a new NFK3 kinase family member, inhibits CrkII through dimerization
Members of the New Kinase Family 3 (NKF3), PEAK1/SgK269 and Pragmin/SgK223 pseudokinases, have emerged as important regulators of cell motility and cancer progression. Here, we demonstrate that C19orf35 (PEAK3), a newly identified member of the NKF3 family, is a kinase-like protein evolutionarily conserved across mammals and birds and a regulator of cell motility. In contrast to its family members, which promote cell elongation when overexpressed in cells, PEAK3 overexpression does not have an elongating effect on cell shape but instead is associated with loss of actin filaments. Through an unbiased search for PEAK3 binding partners, we identified several regulators of cell motility, including the adaptor protein CrkII. We show that by binding to CrkII, PEAK3 prevents the formation of CrkII-dependent membrane ruffling. This function of PEAK3 is reliant upon its dimerization, which is mediated through a split helical dimerization domain conserved among all NKF3 family members. Disruption of the conserved DFG motif in the PEAK3 pseudokinase domain also interferes with its ability to dimerize and subsequently bind CrkII, suggesting that the conformation of the pseudokinase domain might play an important role in PEAK3 signaling. Hence, our data identify PEAK3 as an NKF3 family member with a unique role in cell motility driven by dimerization of its pseudokinase domain
Characterization of an A3G-VifHIV-1-CRL5-CBFβ Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host-Pathogen Complexes.
Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFβ) to determine the structure of the (A3G-Vif-CRL5-CBFβ) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFβ complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes