PaCS Is a Novel Cytoplasmic Structure Containing Functional Proteasome and Inducible by Cytokines/Trophic Factors (original) (raw)
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2014
Cytoplasmic structures showing a selective concentration of both polyubiquitinated proteins and proteasome have been described in various epithelial, hematopoietic, mesenchymal and neural cells in vitro or in fetal tissues, as well as in chronically-infected, mutated preneoplastic and neoplastic tissues. These cytoplasmic structures differ from other ubiquitin-reactive cytoplasmic bodies, like sequestosomes, aggresome-like-induced structures in dendritic cells (DALIS)/non-dendritic cells (ALIS) and aggresomes in showing distinctive ultrastructural organization (particle-rich cytoplasmic structure or PaCS), a cytochemical pattern and a functional profile. Their formation can be induced in vitro in dendritic or natural killer cells by trophic factors and interleukin treatment. They originate in close connection with ribosomes, while, as a result of their growth, the cytoskeleton and other surrounding organelles are usually dislocated outside their core. Interestingly, these particulate cytoplasmic structures are often found to fill cytoplasmic blebs forming proteasome-and polyubiquitinated protein-discharging vesicles, called ectosomes, which are found to detach from the cell and freely float in the extracellular space. To clearly point out the importance of the polyubiquitinated proteins and proteasome containing cytoplasmic structures, their role in cell biology and pathology has been carefully analyzed.
International Journal of Molecular Sciences, 2018
In this article, we outline and discuss available information on the cellular site and mechanism of proteasome interaction with cytosolic polyubiquitinated proteins and heat-shock molecules. The particulate cytoplasmic structure (PaCS) formed by barrel-like particles, closely reproducing in vivo the high-resolution structure of 26S proteasome as isolated in vitro, has been detected in a variety of fetal and neoplastic cells, from living tissue or cultured cell lines. Specific trophic factors and interleukins were found to induce PaCS during in vitro differentiation of dendritic, natural killer (NK), or megakaryoblastic cells, apparently through activation of the MAPK-ERK pathway. Direct interaction of CagA bacterial oncoprotein with proteasome was shown inside the PaCSs of a Helicobacter pylori-infected gastric epithelium, a finding suggesting a role for PaCS in CagA-mediated gastric carcinogenesis. PaCS dissolution and autophagy were seen after withdrawal of inducing factors. PaCS-...
A protocol to visualize cytosolic aggresome-like bodies using confocal microscopy
STAR Protocols, 2021
A protocol to visualize cytosolic aggresomelike bodies using confocal microscopy Ubiquitin stress-induced NEDDylation leads to the formation of aggresome-like bodies (ALBs) in the perinuclear region of cells. Therefore, imaging analysis is essential for characterizing the biological phenotypes of ALBs. Here, we describe a protocol to monitor ALBs induced by ubiquitin stress using immunocytochemistry and to quantify cells containing ALBs. This optimized protocol details the use of readily available materials and reagents and can be applied to explore diverse molecules involved in stress-induced ALBs.
Molecular and Cellular Biology, 2004
Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.
Cell, 2013
Immunoproteasomes are alternative forms of proteasomes that have an enhanced ability to generate antigenic peptides. Recently, Seifert and colleagues reported surprising observations concerning the functions of immunoproteasomes and cellular responses to interferon-g: (1) that immunoproteasomes degrade ubiquitinated proteins faster than the constitutive proteasomes, (2) that polyubiquitin conjugates accumulate after interferon-g treatment but then are preferentially degraded by immunoproteasomes, and (3) that immunoproteasome deficiency causes the formation of inclusions and more severe experimental autoimmune encephalomyelitis (EAE). In contrast, we find that polyubiquitin conjugates do not transiently accumulate following IFNgtreatment and that immunoproteasomes do not prevent the formation of intracellular inclusions or protect against EAE. Furthermore, purified 26S constitutive and immunoproteasomes bind ubiquitin conjugates similarly and degrade them at similar rates. We conclude that, although immunoproteasomes can increase the generation of peptides appropriate for MHC class I presentation, they do not degrade ubiquitinated proteins more efficiently than constitutive particles.
Journal of Experimental Medicine, 2002
Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.
The Journal of cell biology, 1990
Cytolytic lymphocytes contain specialized lytic granules whose secretion during cell-mediated cytolysis results in target cell death. Using serial section EM of RNK-16, a natural killer cell line, we show that there are structurally distinct types of granules. Each type is composed of varying proportions of a dense core domain and a multivesicular cortical domain. The dense core domains contain secretory proteins thought to play a role in cytolysis, including cytolysin and chondroitin sulfate proteoglycan. In contrast, the multivesicular domains contain lysosomal proteins, including acid phosphatase, alpha-glucosidase, cathepsin D, and LGP-120. In addition to their protein content, the lytic granules have other properties in common with lysosomes. The multivesicular regions of the granules have an acidic pH, comparable to that of endosomes and lysosomes. The granules take up exogenous cationized ferritin with lysosome-like kinetics, and this uptake is blocked by weak bases and low t...
Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity
Proceedings of The National Academy of Sciences, 2000
Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.