57 research outputs found
Characterization of Crn7, a novel mammalian Golgi protein
Coronins constitute an evolutionarily conserved family of WD-repeat actin-binding proteins, which can be clearly classified into two distinct groups based on their structural features. All coronins possess a conserved basic N-terminal motif and three to ten WD repeats clustered in one or two core domains. Dictyostelium and mammalian coronins are important regulators of the actin cytoskeleton, while the fly Dpod1 and the yeast coronin proteins crosslink both actin and microtubules. Apart from that, several coronins have been shown to be involved in vesicular transport. C. elegans POD-1 and Drosophila coro regulate the actin cytoskeleton, but also govern vesicular trafficking as indicated by mutant phenotypes. In both organisms, defects in cytoskeleton and trafficking lead to severe developmental defects ranging from abnormal cell division to aberrant formation of morphogen gradients. Crn7 is a ubiquitous mammalian coronin family member. The protein is distributed between the cytosol and Golgi, where it is present at the outer side of the membrane. Golgi localization of Crn7 depends on tyrosine phosphorylation and the integrity of ER-to-Golgi transport. The protein intimately associates with the Golgi membrane and does not require coatomer for its localization. Crn7 is an essential protein, as its knockdown by RNAi leads to a dramatic time- and concentration-dependent decrease in cell viability. Crn7 RNAi cells display scattered Golgi morphology, as demonstrated by electron and light microscopy. Most importantly, the knockdown leads to the block of protein export from the Golgi complex, while the import into the organelle, both anterograde and retrograde, remains unaffected. Further, I established that Crn7 interacts with AP-1 adaptor protein complex participating in the Golgi export by linking cargoes to the clathrin coat. The Golgi complex is the central protein sorting organelle in eukaryotic cells. The Golgi architecture varies significantly between species and cell types, but the organelle executes principally the same function. Upon the cargo protein entry from the endoplasmic reticulum, resident Golgi enzymes modify the cargo in a way that proteins destined to take different transport routes can be biochemically distinguished between and selectively recruited to the corresponding export carriers. We suggest that Golgi-localized Crn7 can function by regulating the cargo export from the Golgi, and thus affect protein sorting and trafficking along the biosynthetic pathway. We anticipate that Crn7 is recruited to the Golgi membranes by cytosolic portions of non-YxxPhi cargoes and cargo proteins, and interacts with AP-1 to allow the Golgi export of such cargoes/receptors
Initiation of TCR Phosphorylation and Signal Transduction
Recent data with CD8+ T cells show that the initial phase of T cell receptor (TCR) binding to MHC–peptide (MHCp) is quickly followed by a second, stronger, binding phase representing the binding of CD8 to the MHCp. This second phase requires signaling by a Src-family kinase such as Lck. These data point out two aspects of the initial stage of TCR signaling that have not yet been clearly resolved. Firstly, how and by which Src-family kinase, is the initial phosphorylation of CD3ζ accomplished, given that the Lck associated with the co-receptors (CD4 or CD8) is not yet available. Secondly, what is the mechanism by which the co-receptor is brought close to the bound TCR before the co-receptor binds to MHCp
Coronin 7, the mammalian POD-1 homologue, localizes to the Golgi apparatus
AbstractCoronins constitute an evolutionary conserved family of WD-repeat actin-binding proteins. Their primary function is thought to be regulating the actin cytoskeleton. Apart from that, several coronins were indirectly shown to participate in vesicular transport, establishment of cell polarity and cytokinesis. Here, we report a novel mammalian protein, coronin 7 (crn7), which is significantly different from other mammalian coronins in its domain architecture. Crn7 possesses two stretches of WD repeats in contrast to the other coronins only having one. The protein is expressed throughout the mouse embryogenesis and is strongly upregulated in brain and developing structures of the immune system in the course of development. In adult animals, both crn7 mRNA and protein are abundantly present in most organs, with significantly higher amounts in brain, kidney, thymus and spleen and lower amounts in muscle. At the subcellular level, the bulk of the protein appears to be present in the cytosol and in large cytosolic complexes. However, a significant portion of the protein is detected on vesicle-like cytoplasmic structures as well as on the cis-Golgi. In the Golgi region, crn7 staining appears broader than that of the cis-Golgi markers Erd2p and β-COP, still, the trans-Golgi network appears predominantly crn7-negative. Importantly, the membrane-associated form of crn7 protein is phosphorylated on tyrosine residues, whereas the cytosolic form is not. Crn7 is the first coronin protein proven to localize to the Golgi membrane. We conclude that it plays a role in the organization of intracellular membrane compartments and vesicular trafficking rather than in remodeling the cytoskeleton
MMP-9/Gelatinase B Degrades Immune Complexes in Systemic Lupus Erythematosus
Systemic Lupus Erythematosus (SLE) is a common and devastating autoimmune disease, characterized by a dysregulated adaptive immune response against intracellular antigens, which involves both autoreactive T and B cells. In SLE, mainly intracellular autoantigens generate autoantibodies and these assemble into immune complexes and activate the classical pathway of the complement system enhancing inflammation. Matrix metalloproteinase-9 (MMP-9) levels have been investigated in the serum of SLE patients and in control subjects. On the basis of specific studies, it has been suggested to treat SLE patients with MMP inhibitors. However, some of these inhibitors induce SLE. Analysis of LPR−/−MMP-9−/− double knockout mice suggested that MMP-9 plays a protective role in autoantigen clearance in SLE, but the effects of MMP-9 on immune complexes remained elusive. Therefore, we studied the role of MMP-9 in the clearance of autoantigens, autoantibodies and immune complexes and demonstrated that the lack of MMP-9 increased the levels of immune complexes in plasma and local complement activation in spleen and kidney in the LPR−/− mouse model of SLE. In addition, we showed that MMP-9 dissolved immune complexes from plasma of lupus-prone LPR−/−/MMP-9−/− mice and from blood samples of SLE patients. Surprisingly, autoantigens incorporated into immune complexes, but not immunoglobulin heavy or light chains, were cleaved by MMP-9. We discovered Apolipoprotein-B 100 as a new substrate of MMP-9 by analyzing the degradation of immune complexes from human plasma samples. These data are relevant to understand lupus immunopathology and side-effects observed with the use of known drugs. Moreover, we caution against the use of MMP inhibitors for the treatment of SLE
Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide-MHC
Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide–MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4–MHCII interaction is generally weaker than CD8–MHCI.National Institutes of Health (U.S.). Pioneer Awar
CD8αα and ‐αβ isotypes are equally recruited to the immunological synapse through their ability to bind to MHC class I
Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide–MHC
Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide–MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4–MHCII interaction is generally weaker than CD8–MHCI.National Institutes of Health (U.S.). Pioneer Awar
Thymocyte development in the absence of matrix metalloproteinase-9/gelatinase B
Matrix metalloproteinases (MMP) play critical roles in a variety of immune reactions by facilitating cell migration, and affect cell communication by processing both cytokines and cell surface receptors. Based on published data indicating that MMP-9 is upregulated upon T cell activation and also in the thymus upon the induction of negative selection, we investigated the contribution of MMP-9 into mouse T cell development and differentiation in the thymus. Our data suggest that MMP-9 deficiency does not result in major abnormalities in the development of any conventionally selected or agonist selected subsets and does not interfere with thymocyte apoptosis and clearance, and that MMP-9 expression is not induced in immature T cells at any stage of their thymic development.status: publishe
TCR signal strength and T cell development
Thymocyte selection involves the positive and negative selection of the repertoire of T cell receptors (TCR), such that the organism does not suffer autoimmunity, and yet has the benefit of the ability to recognize any invading pathogen. The signal transduced through the TCR is translated into a number of different signaling cascades that result in transcription factor activity in the nucleus and changes to the cytoskeleton and motility. Negative selection involves inducing apoptosis in thymocytes that express strongly self-reactive TCRs, whereas positive selection must induce survival and differentiation programs in cells that are more weakly self-reactive. The TCR recognition event is analog by nature, but the outcome of signaling is not. There are a large number of molecules that regulate the strength of the TCR-derived signal at various points in the cascades. This review will discuss the various factors that can act to regulate the strength of the TCR signal during thymocyte development.status: publishe
Assay specificity and possible additional analyses.
<p>(A) ActCasp3 staining (left) and side scatter signal (right) in the DP gate with and without A2 tetramer stimulation for 24 h in the presence or absence of caspase inhibitor Z-VAD (OMe)-FMK. (B) Simultaneous stimulation of PD670-labeled and unlabeled cells in the same well with K<sup>b</sup>-Q7 tetramer for 24 h. ActCasp3 staining in the PD670 positive and negative DP gate with and without stimulation with tetramer for 24 h. (C) PD670 signal (X axis) in the DP gate with and without stimulation with A2, Q7, G4, and E1 tetramers for 24 h. (D) Vα2 (left) and CD69 (middle) staining in the DP gate with and without stimulation with A2, Q7, G4, and E1 tetramers for 24 h. Tetramer-PE signal (right) in ActCasp3<sup>+</sup> and ActCasp3<sup>−</sup> DP gate compared with that of non-treated B6 DP thymocytes. (E) Vα2 (left), CD69 (middle) and tetramer-PE (right) signals in the DP gate with and without stimulation with K<sup>b</sup>-A2 tetramer for 24 h in the presence or absence of caspase inhibitor Z-VAD (OMe)-FMK.</p
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