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91.
In addition to its high affinity for antibody Fc domains, staphylococcal Protein A has been shown to bind certain Fab domains. We investigated this in order to develop a small, recombinant Protein A-binding alternative to immunoglobulin G (IgG) from nanobodies, single-domain antibodies derived from a camelid variant IgG’s variable region. We engineered a nanobody with affinity solely for Protein A as well as a dimerized version of higher affinity for typical multidomain Protein A constructs. Because this recombinant nanobody can be immobilized using a cleavable crosslinker, it has proven to be suitable for the isolation and mild elution of protein complexes in native conditions. 相似文献
92.
Altering nuclear pore complex function impacts longevity and mitochondrial function in S. cerevisiae
Christopher L. Lord Benjamin L. Timney Michael P. Rout Susan R. Wente 《The Journal of cell biology》2015,208(6):729-744
The eukaryotic nuclear permeability barrier and selective nucleocytoplasmic transport are maintained by nuclear pore complexes (NPCs), large structures composed of ∼30 proteins (nucleoporins [Nups]). NPC structure and function are disrupted in aged nondividing metazoan cells, although it is unclear whether these changes are a cause or consequence of aging. Using the replicative life span (RLS) of Saccharomyces cerevisiae as a model, we find that specific Nups and transport events regulate longevity independent of changes in NPC permeability. Mutants lacking the GLFG domain of Nup116 displayed decreased RLSs, whereas longevity was increased in nup100-null mutants. We show that Nup116 mediates nuclear import of the karyopherin Kap121, and each protein is required for mitochondrial function. Both Kap121-dependent transport and Nup116 levels decrease in replicatively aged yeast. Overexpression of GSP1, the small GTPase that powers karyopherin-mediated transport, rescued mitochondrial and RLS defects in nup116 mutants and increased longevity in wild-type cells. Together, these studies reveal that specific NPC nuclear transport events directly influence aging. 相似文献
93.
Seung Joong Kim Javier Fernandez-Martinez Parthasarathy Sampathkumar Anne Martel Tsutomu Matsui Hiro Tsuruta Thomas M. Weiss Yi Shi Ane Markina-Inarrairaegui Jeffery B. Bonanno J. Michael Sauder Stephen K. Burley Brian T. Chait Steven C. Almo Michael P. Rout Andrej Sali 《Molecular & cellular proteomics : MCP》2014,13(11):2911-2926
94.
Yi Shi Javier Fernandez-Martinez Elina Tjioe Riccardo Pellarin Seung Joong Kim Rosemary Williams Dina Schneidman-Duhovny Andrej Sali Michael P. Rout Brian T. Chait 《Molecular & cellular proteomics : MCP》2014,13(11):2927-2943
Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (∼600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope.Macromolecular complexes are the building blocks that drive virtually all cellular and biological processes. In each eukaryotic cell, there exist many hundreds of these protein complexes (1–3), the majority of which are still poorly understood in terms of their structures, dynamics, and functions. The classical structure determination approaches of nuclear magnetic resonance, X-ray crystallography, and electron microscopy (EM)1 remain challenged in attempts to determine the high-resolution structures of large, dynamic, and flexible complexes in a living cell (4). Thus, additional robust and rapid methods are needed, ideally working in concert with these classical approaches, to allow the greatest structural and functional detail in characterizations of macromolecular assemblies.Integrative modeling approaches help address this need, providing powerful tools for determining the structures of endogenous protein complexes (5, 6) by relying on the collection of an extensive experimental dataset, preferably coming from diverse sources (both classical and new) and different levels of resolution. These data are translated into spatial restraints that are used to calculate an ensemble of structures by satisfying the restraints, which in turn can be analyzed and assessed to determine precision and estimate accuracy (5, 7). A major advantage of this approach is that it readily integrates structural data from different methods and a wide range of resolutions, spanning from a few angstroms to dozens of nanometers. This strategy has been successfully applied to a number of protein complexes (8–16). However, it has proven difficult and time-consuming to generate a sufficient number of accurate spatial restraints to enable high-resolution structural characterization; thus, the determination of spatial restraints currently presents a major bottleneck for widespread application of this integrative approach. An important step forward is therefore the development of technologies for collecting high-resolution and information-rich spatial restraints in a rapid and efficient manner, ideally from endogenous complexes isolated directly from living cells.Chemical cross-linking with mass spectrometric readout (CX-MS) (17, 18) has recently emerged as an enabling approach for obtaining residue-specific restraints on the structures of proteins and protein complexes (19–25). In a CX-MS experiment, the purified protein complex is chemically conjugated by a functional group-specific cross-linker, and this is followed by proteolytic digestion and analysis of the resulting peptide mixture by mass spectrometry (MS). However, because of the complexity of the peptide mixtures and low abundance of most of the informative cross-linked species, comprehensive detection of these cross-linked peptides has proven challenging. This challenge increases substantially in studies of endogenous complexes of modest to low abundance, which encompass the great majority of assemblies in any cell (26, 27). In addition, because most cross-linkers used for CX-MS target primary amines, comprehensive detection of cross-links is further limited by the occurrence of lysine, which constitutes only ∼6% of protein sequences, although these lysine residues are generally present on protein surfaces. The use of cross-linkers with different chemistries and reactive groups, especially toward abundant residues, would increase the cross-linking coverage and could be of great help for downstream structural analysis (28).The nuclear pore complex (NPC) is one of the largest protein assemblies in the cell and is the sole mediator of macromolecular transport between the nucleus and the cytoplasm. The NPC is formed by multiple copies of ∼30 different proteins termed nucleoporins (Nups) that are assembled into discrete subcomplexes (8, 29). These building blocks are arranged into eight symmetrical units called spokes that are radially connected to form several concentric rings. The outer rings of the NPC are mainly formed by the Nup84 complex (a conserved complex, termed the Nup107–Nup160 complex in vertebrates). In budding yeast, the Nup84 complex is an essential, Y-shaped assembly of ∼600 kDa that is formed by seven nucleoporins (Nup133, Nup120, Nup145c, Nup85, Nup84, Seh1, and Sec13 in Saccharomyces cerevisiae) (30). The Nup84 complex has been shown to have a common evolutionary origin with vesicle coating complexes (VCCs), such as COPII, COPI, and clathrin (31, 32), but the evolutionary relationships between these VCCs have not been fully delineated. The Nup84 complex has been extensively characterized; several of its components have been analyzed via X-ray crystallography (33, 34), its overall shape has been defined by means of negative-stain electron microscopy (14, 30, 35, 36), and recently efforts were made to define the protein contacts in the Nup84 complex via CX-MS in humans (35) and a thermophilic fungus (37). Finally, we recently used an integrative modeling approach combining domain mapping, negative-stain electron microscopy (38), and publicly available crystal structures to generate a medium-resolution map of the native Nup84 complex (14). However, despite all these efforts, the fine features of the complex, and in particular the intricate domain orientations and contacts within the complex''s hub, remain poorly described.To address these issues, we present here an optimized CX-MS strategy for robust and in-depth structural characterization of endogenous protein complexes. To test the strategy, we generated a comprehensive high-quality CX-MS dataset on the endogenous Nup84 complex using two complementary cross-linkers, disuccinimidyl suberate (DSS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Using the resulting cross-linking restraints together with other sources of information (including electron microscopy, X-ray crystallography, and comparative modeling), we computed a detailed structure of the endogenous Nup84 complex. In addition to providing the overall architecture of the yeast Nup84 complex, the resulting structure reveals the previously unknown architecture of its pentameric structural hub. Our results demonstrate that the present approach provides a robust framework for the standardized generation and use of CX-MS spatial restraints toward the structural characterization of endogenous protein complexes. 相似文献
95.
Simon P. Rout Jessica Radford Andrew P. Laws Francis Sweeney Ahmed Elmekawy Lisa J. Gillie Paul N. Humphreys 《PloS one》2014,9(9)
The anoxic, alkaline hydrolysis of cellulosic materials generates a range of cellulose degradation products (CDP) including α and β forms of isosaccharinic acid (ISA) and is expected to occur in radioactive waste disposal sites receiving intermediate level radioactive wastes. The generation of ISA''s is of particular relevance to the disposal of these wastes since they are able to form complexes with radioelements such as Pu enhancing their migration. This study demonstrates that microbial communities present in near-surface anoxic sediments are able to degrade CDP including both forms of ISA via iron reduction, sulphate reduction and methanogenesis, without any prior exposure to these substrates. No significant difference (n = 6, p = 0.118) in α and β ISA degradation rates were seen under either iron reducing, sulphate reducing or methanogenic conditions, giving an overall mean degradation rate of 4.7×10−2 hr−1 (SE±2.9×10−3). These results suggest that a radioactive waste disposal site is likely to be colonised by organisms able to degrade CDP and associated ISA''s during the construction and operational phase of the facility. 相似文献
96.
Purpose
Effective diagnosis of tuberculosis (TB) relies on accurate interpretation of radiological patterns found in a chest radiograph (CXR). Lack of skilled radiologists and other resources, especially in developing countries, hinders its efficient diagnosis. Computer-aided diagnosis (CAD) methods provide second opinion to the radiologists for their findings and thereby assist in better diagnosis of cancer and other diseases including TB. However, existing CAD methods for TB are based on the extraction of textural features from manually or semi-automatically segmented CXRs. These methods are prone to errors and cannot be implemented in X-ray machines for automated classification.Methods
Gabor, Gist, histogram of oriented gradients (HOG), and pyramid histogram of oriented gradients (PHOG) features extracted from the whole image can be implemented into existing X-ray machines to discriminate between TB and non-TB CXRs in an automated manner. Localized features were extracted for the above methods using various parameters, such as frequency range, blocks and region of interest. The performance of these features was evaluated against textural features. Two digital CXR image datasets (8-bit DA and 14-bit DB) were used for evaluating the performance of these features.Results
Gist (accuracy 94.2% for DA, 86.0% for DB) and PHOG (accuracy 92.3% for DA, 92.0% for DB) features provided better results for both the datasets. These features were implemented to develop a MATLAB toolbox, TB-Xpredict, which is freely available for academic use at http://sourceforge.net/projects/tbxpredict/. This toolbox provides both automated training and prediction modules and does not require expertise in image processing for operation.Conclusion
Since the features used in TB-Xpredict do not require segmentation, the toolbox can easily be implemented in X-ray machines. This toolbox can effectively be used for the mass screening of TB in high-burden areas with improved efficiency. 相似文献97.
Katherine E. Mues Michael Deming David G. Kleinbaum Philip J. Budge Mitch Klein Juan S. Leon Aishya Prakash Jonathan Rout LeAnne M. Fox 《PLoS neglected tropical diseases》2014,8(9)
Background
Lymphedema management programs have been shown to decrease episodes of adenolymphangitis (ADLA), but the impact on lymphedema progression and of program compliance have not been thoroughly explored. Our objectives were to determine the rate of ADLA episodes and lymphedema progression over time for patients enrolled in a community-based lymphedema management program. We explored the association between program compliance and ADLA episodes as well as lymphedema progression.Methodology/Principal Findings
A lymphedema management program was implemented in Odisha State, India from 2007–2010 by the non-governmental organization, Church''s Auxiliary for Social Action, in consultation with the Centers for Disease Control and Prevention. A cohort of patients was followed over 24 months. The crude 30-day rate of ADLA episodes decreased from 0.35 episodes per person-month at baseline to 0.23 at 24 months. Over the study period, the percentage of patients who progressed to more severe lymphedema decreased (P-value = 0.0004), while those whose lymphedema regressed increased over time (P-value<0.0001). Overall compliance to lymphedema management, lagged one time point, appeared to have little to no association with the frequency of ADLA episodes among those without entry lesions (RR = 0.87 (0.69, 1.10)) and was associated with an increased rate (RR = 1.44 (1.11, 1.86)) among those with entry lesions. Lagging compliance two time points, it was associated with a decrease in the rate of ADLA episodes among those with entry lesions (RR = 0.77 (95% CI: 0.59, 0.99)) and was somewhat associated among those without entry lesions (RR = 0.83 (95% CI: 0.64, 1.06)). Compliance to soap was associated with a decreased rate of ADLA episodes among those without inter-digital entry lesions.Conclusions/Significance
These results indicate that a community-based lymphedema management program is beneficial for lymphedema patients for both ADLA episodes and lymphedema. It is one of the first studies to demonstrate an association between program compliance and rate of ADLA episodes. 相似文献98.
99.
Cilia and flagella are membrane-sheathed, microtubule-based protrusions that decorate the surface of many eukaryotic cells. At their base, they form a selective barrier that concentrates certain proteins within the cilia but excludes others. Kee et al. (2012) now propose that nuclear pore complex proteins form a fundamental part of this diffusion barrier. 相似文献
100.
Ye L Liu X Rout SN Li Z Yan Y Lu L Kamala T Nanda NK Song W Samal SK Zhu X 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(4):2572-2585
The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-beta(2)-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-gamma-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-gamma. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain. 相似文献