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71.
Leslie S. Wolfe Bradford J. Stanley Chang Liu William K. Eliason Yong Xiong 《Journal of virology》2010,84(14):7135-7139
The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitination of APOBEC3G and subsequently its proteasomal degradation. Canonical ECS-type ubiquitin ligases consist of a cullin scaffold protein to which adaptor and substrate receptor proteins bind at the N terminus. HIV Vif serves as a substrate receptor protein—its N terminus recruits APOBEC3G, while multiple C-terminal regions assemble with the E3 ligase (9, 13, 24). The E3 ligase interacting regions include a zinc finger (residues 100 to 140), a BC box (residues 141 to 154), and a cullin box (residues 155 to 176) (Fig. (Fig.11).Open in a separate windowFIG. 1.(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).Vif binds the cullin adaptor proteins EloB and EloC through the BC-box region (24). The BC box is a loop-helix motif with the consensus sequence (T/S)LxxxCxxx(V/L/I) (7), and it also exists in cellular proteins that interact with EloBC. While Vif does not fit this consensus perfectly, it still binds EloBC with high affinity, and this interaction is lost upon mutation or deletion of consensus BC-box residues (10, 24, 25). This interaction has been described previously for the cellular proteins VHL (15), SOCS2 (3), SOCS3 (1), SOCS4 (4), and recently HIV Vif (14).Both the Vif zinc finger and cullin box interact with the E3 ligase scaffold protein cullin 5 (Cul5) (11, 12, 20, 21). It has been established that the zinc finger is required for Vif to bind Cul5. Mutation of critical histidine or cysteine residues in this region or the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) abolishes the Vif-Cul5 interaction (8, 11, 25). The sequence of the Vif cullin box is not as conserved as those of cellular SOCS-box proteins, which have a defined structure and determine the specificities of their respective cullins (6). The role of the Vif cullin box is not clear, but it has been suggested to promote dimerization of Vif, involving the conserved PPLP region (22, 23), and has recently been implicated in APOBEC3G binding (5, 17). While its importance in Cul5 binding has been demonstrated in coimmunoprecipitation experiments (14), experimental data also exist showing that the Vif zinc finger alone still immunoprecipitates Cul5 (11, 21).To dissect the assembly of the Vif-E3 ubiquitin ligase, we quantified the binding interactions between various C-terminal Vif constructs, EloBC, and Cul5 by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). We additionally probed the effects of the cullin box on Vif dimerization. 相似文献
72.
To explore the residue interactions in the glycophorin A dimerization motif, an alanine scan double mutant analysis at the helix-helix interface was carried out. These data reveal a combination of additive and coupled effects. The majority of the double mutants are found to be equally or slightly more stable than would be predicted by the sum of the energetic cost of the single-point mutants. The proximity of the mutated sites is not related to the presence of coupling between those sites. Previous studies reveal that a single face of the glycophorin A monomer contains a specific glycine-containing motif (GxxxG) that is thought to be a driving force for the association of transmembrane helices. Double mutant cycles suggest that the relationship of the GxxxG motif to the remainder of the helix-helix interface is complex. Sequences containing mutations that abolish the GxxxG motif retain an ability to dimerize, while a sequence containing a GxxxG motif appears unable to form dimers. The energetic effects of weakly coupled and additive double mutants can be explained by changes in van der Waals interactions at the dimer interface. These results emphasize the fact that the sequence context of the dimer interface modulates the strength of the glycophorin A GxxxG-mediated transmembrane dimerization reaction. 相似文献
73.
Megan J. Kelly-Slatten Catherine E. Stewart Malak M. Tfaily Julie D. Jastrow Abigail Sasso Marie-Anne de Graaff 《Global Change Biology Bioenergy》2023,15(5):613-629
Recent studies have indicated that the C4 perennial bioenergy crops switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii) accumulate significant amounts of soil carbon (C) owing to their extensive root systems. Soil C accumulation is likely driven by inter- and intraspecific variability in plant traits, but the mechanisms that underpin this variability remain unresolved. In this study we evaluated how inter- and intraspecific variation in root traits of cultivars from switchgrass (Cave-in-Rock, Kanlow, Southlow) and big bluestem (Bonanza, Southlow, Suther) affected the associations of soil C accumulation across soil fractions using stable isotope techniques. Our experimental field site was established in June 2008 at Fermilab in Batavia, IL. In 2018, soil cores were collected (30 cm depth) from all cultivars. We measured root biomass, root diameter, specific root length, bulk soil C, C associated with coarse particulate organic matter (CPOM) and fine particulate organic matter plus silt- and clay-sized fractions, and characterized organic matter chemical class composition in soil using high-resolution Fourier-transform ion cyclotron resonance mass spectrometry. C4 species were established on soils that supported C3 grassland for 36 years before planting, which allowed us to use differences in the natural abundance of stable C isotopes to quantify C4 plant-derived C. We found that big bluestem had 36.9% higher C4 plant-derived C compared to switchgrass in the CPOM fraction in the 0–10 cm depth, while switchgrass had 60.7% higher C4 plant-derived C compared to big bluestem in the clay fraction in the 10–20 cm depth. Our findings suggest that the large root system in big bluestem helps increase POM-C formation quickly, while switchgrass root structure and chemistry build a mineral-bound clay C pool through time. Thus, both species and cultivar selection can help improve bioenergy management to maximize soil carbon gains and lower CO2 emissions. 相似文献
74.
Clutterbuck AL Smith JR Allaway D Harris P Liddell S Mobasheri A 《Journal of Proteomics》2011,74(5):704-715
This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1β increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis. 相似文献
75.
76.
Yan‐Hui Zhao Amparo Lzaro Zong‐Xin Ren Wei Zhou Hai‐Dong Li Zhi‐Bin Tao Kun Xu Zhi‐Kun Wu Lorne M. Wolfe De‐Zhu Li Hong Wang 《Oikos》2019,128(4):551-562
Pollination networks are usually constructed and assessed by direct field observations which commonly assume that all flower visitors are true pollinators. However, this assumption is often invalid and the use of data based on mere visitors to flowers may lead to a misunderstanding of intrinsic pollination networks. Here, using a large dataset by both sampling floral visitors and analyzing their pollen loads, we constructed 32 networks pairs (visitation versus pollen transport) across one flowering season at four elevation sites in the Himalaya–Hengduan Mountains region. Pollen analysis was conducted to determine which flower visitors acted as potential pollinators (pollen vectors) or as cheaters (those not carrying pollen of the visited plants). We tested whether there were topological differences between visitation and pollen transport networks and whether different taxonomic groups of insect visitors differed in their ability to carry pollen of the visited plants. Our results indicated that there was a significantly higher degree of specialization at both the network and species levels in the pollen transport networks in contrast to the visitation networks. Modularity was lower but nestedness was higher in the visitation networks compared to the pollen transport networks. All the cheaters were identified as peripheral species and most of them contributed positively to the nested structure. This may explain in part the differences in modularity and nestedness between the two network types. Bees carried the highest proportion of pollen of the visited plants. This was followed by Coleoptera, other Hymenoptera and Diptera. Lepidoptera carried the lowest proportion of pollen of the visited plants. Our study shows that the construction of pollen transport networks could provide a more in‐depth understanding of plant–pollinator interactions. Moreover, it suggests that detecting and removing cheater interactions when studying the topology of other mutualistic networks might be also important. 相似文献
77.
The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules 总被引:10,自引:0,他引:10
Miller MK Bang ML Witt CC Labeit D Trombitas C Watanabe K Granzier H McElhinny AS Gregorio CC Labeit S 《Journal of molecular biology》2003,333(5):951-964
CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression. 相似文献
78.
79.
Nicole A. Kruh-Garcia Lisa M. Wolfe Lelia H. Chaisson William O. Worodria Payam Nahid Jeff S. Schorey J. Lucian Davis Karen M. Dobos 《PloS one》2014,9(7)
The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states. 相似文献
80.