Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in Jurkat cells

Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling

First report of maize redness disease in Hungary

Abstract During 2010, several maize production areas in Hungary were surveyed for the occurrence of maize redness (MR) disease symptoms associated with stolbur phytoplasma, as well as for the presence of the known vector of the disease, a planthopper Reptalus panzeri (Low). Incidence of maize plants with symptoms of reddening was low in all surveyed areas. Altogether, 25 symptomatic maize plants were collected at 9 localities and tested for phytoplasma presence. In addition, from one locality specimens of cixiids R. panzeri and Hyalesthes obsoletus Signoret were collected and PCR analyzed. Presence of stolbur phytoplasma in MR symptomatic maize plants and stolbur-infected R. panzeri was identified at the single locality Monorierdő in central Hungary. This finding represents the first report of MR presence in Hungary

First report of maize redness disease in Hungary

During 2010, several maize production areas in Hungary were surveyed for the occurrence of maize redness (MR) disease symptoms associated with stolbur phytoplasma, as well as for the presence of the known vector of the disease, a planthopper Reptalus panzeri (Low). Incidence of maize plants with symptoms of reddening was low in all surveyed areas. Altogether, 25 symptomatic maize plants were collected at 9 localities and tested for phytoplasma presence. In addition, from one locality specimens of cixiids R. panzeri and Hyalesthes obsoletus Signoret were collected and PCR analyzed. Presence of stolbur phytoplasma in MR symptomatic maize plants and stolbur-infected R. panzeri was identified at the single locality Monorierdo in central Hungary. This finding represents the first report of MR presence in Hungary

First report of maize redness disease in Hungary

During 2010, several maize production areas in Hungary were surveyed for the occurrence of maize redness (MR) disease symptoms associated with stolbur phytoplasma, as well as for the presence of the known vector of the disease, a planthopper Reptalus panzeri (Low). Incidence of maize plants with symptoms of reddening was low in all surveyed areas. Altogether, 25 symptomatic maize plants were collected at 9 localities and tested for phytoplasma presence. In addition, from one locality specimens of cixiids R. panzeri and Hyalesthes obsoletus Signoret were collected and PCR analyzed. Presence of stolbur phytoplasma in MR symptomatic maize plants and stolbur-infected R. panzeri was identified at the single locality Monorierdo in central Hungary. This finding represents the first report of MR presence in Hungary

Thymic Atrophy and Apoptosis of CD4+CD8+ Thymocytes in the Cuprizone Model of Multiple Sclerosis.

Previous studies on the degenerative animal model of multiple sclerosis suggested that the copper-chelator cuprizone might directly suppress T-cell functions. Peripheral T-cell function in the cuprizone model has already been explored; therefore, in the present study, we investigated, for the first time, how cuprizone feeding affects the thymus, the organ of T-cell maturation and selection. We found that even one week of cuprizone treatment induced significant thymic atrophy, affecting the cortex over the medulla. Fluorescent microscopy and flow-cytometric analyses of thymi from cuprizone- and vehicle-treated mice indicated that eradication of the cluster of the differentiation-4 (CD4)-CD8 double-positive T-cell subset was behind the substantial cell loss. This result was confirmed with CD3-CD4-CD8 triple-staining experiments. Ultrastructurally, we observed degraded as well as enlarged mitochondria, myelin-bodies, large lipid droplets, and large lysosomes in the thymi of cuprizone-treated mice. Some of these features were similar to those in physiological and steroid-induced accelerated aging. According to our results, apoptosis was mainly of mitochondrial origin mediated by both caspase-3- and apoptosis inducing factor-mediated mechanisms. Additionally, mitogen activated protein kinase activation and increased pro-apoptotic B cell lymphoma-2 family protein expression were the major underlying processes. Our results do not indicate a functional relationship between cuprizone-induced thymus involution and the absence of inflammatory responses or the selective demyelination observed in the cuprizone model. On the other hand, due to the reversible nature of cuprizone's deleterious effects, the cuprizone model could be valuable in studying thymus regeneration as well as remyelination processes

Wnt-4 Protects Thymic Epithelial Cells Against Dexamethasone-Induced Senescence

Glucocorticoids are widely used immunosuppressive drugs in treatment of autoimmune diseases and hematological malignancies. Glucocorticoids are particularly effective immune suppressants, because they induce rapid peripheral T cell and thymocyte apoptosis resulting in impaired T cell–dependent immune responses. Although glucocorticoids can induce apoptotic cell death directly in developing thymocytes, how exogenous glucocorticoids affect the thymic epithelial network that provides the microenvironment for T cell development is still largely unknown. In the present work, we show that primary thymic epithelial cells (TECs) express glucocorticoid receptors and that high-dosage dexamethasone induces degeneration of the thymic epithelium within 24 h of treatment. Changes in organ morphology are accompanied by a decrease in the TEC transcription factor FoxN1 and its regulator Wnt-4 parallel with upregulation of lamina-associated polypeptide 2α and peroxisome proliferator activator receptor γ, two characteristic molecular markers for adipose thymic involution. Overexpression of Wnt-4, however, can prevent upregulation of adipose differentiation-related aging markers, suggesting an important role of Wnt-4 in thymic senescence

HRES-1/Rab4 Promotes the Formation of LC3⁺ Autophagosomes and the Accumulation of Mitochondria during Autophagy

<div><p>HRES-1/Rab4 is a small GTPase that regulates endocytic recycling. It has been colocalized to mitochondria and the mechanistic target of rapamycin (mTOR), a suppressor of autophagy. Since the autophagosomal membrane component microtubule-associated protein light chain 3 (LC3) is derived from mitochondria, we investigated the impact of HRES-1/Rab4 on the formation of LC3⁺ autophagosomes, their colocalization with HRES-1/Rab4 and mitochondria, and the retention of mitochondria during autophagy induced by starvation and rapamycin. HRES-1/Rab4 exhibited minimal baseline colocalization with LC3, which was enhanced 22-fold upon starvation or 6-fold upon rapamycin treatment. Colocalization of HRES-1/Rab4 with mitochondria was increased >2-fold by starvation or rapamycin. HRES-1/Rab4 overexpression promoted the colocalization of mitochondria with LC3 upon starvation or rapamycin treatment. A dominant-negative mutant, HRES-1/Rab4^S27N had reduced colocalization with LC3 and mitochondria upon starvation but not rapamycin treatment. A constitutively active mutant, HRES-1/Rab4^Q72L showed diminished colocalization with LC3 but promoted the partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. Phosphorylation-resistant mutant HRES-1/Rab4^S204Q showed diminished colocalization with LC3 but increased partitioning to mitochondria. A newly discovered C-terminally truncated native isoform, HRES-1/Rab4^1–121, showed enhanced localization to LC3 and mitochondria without starvation or rapamycin treatment. HRES-1/Rab4^1–121 increased the formation of LC3⁺ autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab4^1–121, HRES-1/Rab4^Q72L and HRES-1/Rab4^S204Q promoted the accumulation of mitochondria during starvation. The specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated by its abrogation by dominant-negative HRES-1/Rab4^S27N mutation. The formation of interconnected mitochondrial tubular networks was markedly enhanced by HRES-1/Rab4^Q72L upon starvation, which may contribute to the retention of mitochondria during autophagy. The present study thus indicates that HRES-1/Rab4 regulates autophagy through promoting the formation of LC3⁺ autophagosomes and the preservation of mitochondria.</p></div

Confocal microscopy of HRES-1/Rab4, mitochondria, and LC3⁺ autophagosomes in HeLa cells under starvation (Star) and treatment with rapamycin (Rapa) and bafilomycin A1 (Baf).

<p>eGFP-tagged HRES-1/Rab4 isoforms were identified by green fluorescence. Mitochondria were stained with MTDR and visualized by blue fluorescence. LC3⁺ autophagosomes were visualized by red fluorescence of FP650-LC3. Individual and composite color channels are shown for each experimental condition. A, HeLa cells were transfected with FP650-LC3 alone. B, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4. C, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4^1–121. D, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4^S27N. E, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4^Q72L. F, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4^S204Q. The areas showing the formation of mitochondrial tubular networks are delineated by white dotted rectangles in panels A and E.</p

Detection of LC3 fused to FP650 (FP650-LC3) and HRES-1/Rab4 isoforms, including wild-type HRES-1/Rab4, C-terminally truncated HRES-1/Rab4^1–121, dominant-negative/GTP binding-deficient HRES-1/Rab4^S27N, constitutively active/GTPase-deficient HRES-1/Rab4^Q72L and phosphorylation-resistant form HRES-1/Rab4^S204Q, tagged with eGFP.

<p>A, Functional domains of proteins encoded by the HRES-1/Rab4 cDNA at 1q42 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Nagy1" target="_blank">[12]</a> (Genbank accession number: AY585832). Amino acid changes previously shown to affect Rab4 activity are typed in red. HRES-1/Rab4^S27N prevents GTP binding and acts as a dominant negative mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Lazzarino1" target="_blank">[20]</a>. HRES-1/Rab4^Q72L is constitutively active due to elimination of GTPase activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Cormont1" target="_blank">[21]</a>. HRES-1/Rab4^S204Q will not be phosphorylated by p34cdc2 kinase in mitotic cells and remains endosome-associated throughout the cell cycle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-vanderSluis1" target="_blank">[22]</a>. B, Amino acid sequence of HRES-1/Rab4^1–121, representing a 36-nucleotide out-of-frame deletion, is attributed to alternative splicing (GenBank submission number 1591873). This results in a frameshift with an amino acid sequence corresponding to the 96 N-terminal residues of HRES-1/Rab4 continuing into 25 C-terminal residues (typed in red characters), which are unrelated to the amino acid sequence of residues 97-218 in wild-type HRES-1/Rab4. C, Confocal microscopy of HeLa cells transfected with expression vectors producing FP650-LC3 (emitting red fluorescence) and HRES-1/Rab4 isoforms fused to eGFP (emitting green fluorescence) relative to control cells transfected with vectors expressing fluorescent proteins FP650 and eGFP alone. D, Western blot analysis of HeLa cells transduced with expression vectors producing eGFP-HRES-1/Rab4 and FP650-LC3 fusion proteins. HRES-1/Rab4 isoforms were detected with antibody SC312 directed to the C-terminus which is absent in HRES-1/Rab4^1–121. E, Western blot analysis of HRES-1/Rab4^1–121 expression in HeLa cells transfected with pAAV-HRES-1/Rab4^1–121-IRES-GFP vector (clone 8466), pAAV-hrGFP-HRES-1/Rab4^1–121 vector (clone 9214), and pAAV-HRES-1/Rab4-IRES-GFP vector (clone 8467). Cells were incubated without (control) or with 0.1% DMSO, bafilomycin A1 (200 nM), or leupeptin (10 μg/ml). HRES-1/Rab4^1–121 and HRES-1/Rab4^1–121-GFP fusion protein were detected with rabbit antibody G1432. HRES-1/Rab4 was detected with rabbit antibody 13407 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Nagy1" target="_blank">[12]</a>.</p

Confocal microscopy of HeLa cells transfected with expression vector producing LC3 tagged with FP650 (emitting red fluorescence) and different isoforms of HRES-1/Rab4, including wild-type (HRES-1/Rab4), C-terminally truncated form (HRES-1/Rab4^1–121), dominant-negative form (Rab4^S27N), constitutively active (HRES-1/Rab4^Q72L) and phosphorylation-resistant form (HRES-1/Rab4^S204Q) tagged with eGFP (emitting green fluorescence).

<p>Cells were kept in complete medium (Control), starved in serum-free medium without glutamine (Star), or treated with autophagy-modifying agents, bafilomycin A1 (Baf) and rapamycin (Rapa) for 4 hours. Starved and rapamycin treated cells were also treated with Baf. Images are representative of 6–40 cells analyzed during three independent experiments. Image inserts bracketed by broken lines correspond to LC3 vesicles magnified from the original image. Individual and composite color channels are shown for each experimental condition.</p