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1.
Anal Chem ; 94(51): 17751-17756, 2022 12 27.
Artículo en Inglés | MEDLINE | ID: mdl-36510358

RESUMEN

Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein cross-link has been identified within a given data set are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data.


Asunto(s)
Proteoma , Espectrometría de Masas , Proteoma/química , Reactivos de Enlaces Cruzados/química
2.
Mol Syst Biol ; 15(9): e8994, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31556486

RESUMEN

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.


Asunto(s)
Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteómica/métodos , Citosol/química , Humanos , Células K562 , Modelos Moleculares , Fragmentos de Péptidos/química , Conformación Proteica , Programas Informáticos
3.
Proc Natl Acad Sci U S A ; 112(7): E607-15, 2015 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-25646488

RESUMEN

Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation. Uniquely protein profiling on the same single cells before and after LPS stimulation identified a role for macrophage inhibitory factor (MIF) to potentiate the activation of LPS-induced cytokine production. Advanced clustering analysis identified functional subsets including quiescent, polyfunctional fully activated, partially activated populations with different cytokine profiles. This population architecture is conserved throughout the cell activation process and prevails as it is extended to other TLR ligands and to primary macrophages derived from a healthy donor. This work demonstrates that the phenotypically similar cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level. This technology enables full-spectrum dissection of immune functional states in response to pathogenic or environmental stimulation, and opens opportunities to quantify deep functional heterogeneity for more comprehensive and accurate immune monitoring.


Asunto(s)
Análisis de la Célula Individual , Virulencia , Humanos , Ligandos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Receptor Toll-Like 4/metabolismo , Células U937
4.
Proc Natl Acad Sci U S A ; 110(17): E1545-54, 2013 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-23569223

RESUMEN

TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1. This ATPase activator function controls the activities of other members of the Torsin family in distinct fashion, leading to an acceleration of the hydrolysis step by up to two orders of magnitude. The dystonia-causing mutant of TorsinA is defective in this activation mechanism, suggesting a loss-of-function mechanism for this congenital disorder.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Distonía Muscular Deformante/genética , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Cromatografía en Gel , Clonación Molecular , Distonía Muscular Deformante/metabolismo , Células HEK293 , Células HeLa , Humanos , Hidrólisis , Immunoblotting , Inmunoprecipitación , Chaperonas Moleculares/genética
5.
Nat Commun ; 13(1): 838, 2022 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-35149681

RESUMEN

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome.


Asunto(s)
Endopeptidasas/química , Endopeptidasas/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Dominio Catalítico , Microscopía por Crioelectrón , Citoplasma , Endopeptidasas/genética , Complejo de la Endopetidasa Proteasomal/genética , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo
6.
Biochim Biophys Acta Proteins Proteom ; 1869(3): 140583, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33321258

RESUMEN

In eukaryotic cells, the ubiquitin-proteasome system serves to remove proteins that are either dysfunctional or no longer needed. The 26S proteasome is a 2.5 MDa multisubunit complex comprising the 20S core particle, where degradation is executed, and one or two regulatory particles which prepare substrates for degradation. Whereas the 20S core particles of several species had been studied extensively by X-ray crystallography, the 26S holocomplex structure had remained elusive for a long time. Recent advances in single-particle cryo-electron microscopy have changed the situation and provided atomic resolution models of this intriguing molecular machine and its dynamics. Besides, cryo-electron tomography enables structural studies in situ, providing molecular resolution images of macromolecules inside pristinely preserved cellular environments. This has greatly contributed to our understanding of proteasome dynamics in the context of cells.


Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Complejo de la Endopetidasa Proteasomal/química , Conformación Proteica , Fracciones Subcelulares/metabolismo
7.
Cell Rep ; 24(5): 1301-1315.e5, 2018 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-30067984

RESUMEN

The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo-electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics. We demonstrate unique impacts of ATP binding by individual ATPases on the proteasome conformational distribution and report two conformational states of the proteasome suggestive of a rotary ATP hydrolysis mechanism. These structures, coupled with functional analyses, reveal key roles for the ATPases Rpt1 and Rpt6 in gating substrate entry into the core particle. This deepened knowledge of proteasome conformational dynamics reveals key elements of intersubunit communication within the proteasome and clarifies the regulation of substrate entry into the proteolytic chamber.


Asunto(s)
Simulación de Dinámica Molecular , Complejo de la Endopetidasa Proteasomal/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Sci Signal ; 8(381): ra59, 2015 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-26082435

RESUMEN

Macrophages not only produce multiple cytokines but also respond to multiple cytokines, which likely shapes the ultimate response of the population. To determine the role of paracrine signaling in shaping the profile of inflammatory cytokines secreted by macrophages in response to stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS), we combined multiplexed, microwell-based measurements of cytokine secretion by single cells with analysis of cytokine secretion by cell populations. Loss of paracrine signaling as a result of cell isolation reduced the secretion by macrophage-like U937 cells and human monocyte-derived macrophages (MDMs) of a subset of LPS-stimulated cytokines, including interleukin-6 (IL-6) and IL-10. Graphical Gaussian modeling (GGM) of the single-cell data defined a regulatory network of paracrine signals, which was validated experimentally in the population through antibody-mediated neutralization of individual cytokines. Tumor necrosis factor-α (TNF-α) was the most influential cytokine in the GGM network. Paracrine signaling by TNF-α secreted from a small subpopulation of "high-secreting" cells was necessary, but not sufficient, for the secretion of large amounts of IL-6 and IL-10 by the cell population. Decreased relative IL-10 secretion by isolated MDMs was linked to increased TNF-α secretion, suggesting that inhibition of the inflammatory response also depends on paracrine signaling. Our results reveal a previously uncharacterized role for cell-to-cell communication within a population in coordinating a rapid innate immune response despite underlying cell-to-cell heterogeneity.


Asunto(s)
Citocinas/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Comunicación Paracrina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/inmunología , Humanos , Macrófagos/citología , Comunicación Paracrina/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/agonistas , Células U937
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