Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor.

The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms

The Discovery of a Reciprocal Relationship between Tyrosine-Kinase Signaling and Cullin Neddylation

<div><p>While neddylation is known to activate cullin (CUL)-RING ubiquitin ligases (CRLs), its role in regulating T cell signaling is poorly understood. Using the investigational NEDD8 activating enzyme (NAE) inhibitor, MLN4924, we found that neddylation negatively regulates T cell receptor (TCR) signaling, as its inhibition increases IL-2 production, T cell proliferation and Treg development <i>in vitro</i>. We also discovered that loss of CUL neddylation occurs upon TCR signaling, and CRLs negatively regulate IL-2 production. Additionally, we found that tyrosine kinase signaling leads to CUL deneddylation in multiple cell types. These studies indicate that CUL neddylation is a global regulatory mechanism for tyrosine kinase signaling.</p></div

Activated CRLs containing CUL1 and especially CUL2 and CUL3 regulate IL-2 production.

<p>(A) MA5.8ζ IL-2 secretion at 12 h normalized to the number of live cells measured by annexin V and propidium iodide staining and (B) transcription at 12 h with 4 µg/mL α-CD3 and the indicated concentrations of OPT (n = 3). (C–G) IL-2 secretion by MA5.8ζ cells, cells expressing control shRNA, and cells expressing an shRNA for (C) CUL1, (D) CUL2, (E) CUL3, (F) CUL4a/b and (G) CUL5 after 48 h of stimulation with indicated concentrations of α-CD3. Data are averaged from two independent experiments, where each experiment examined three independently created lines for each shRNA. (H) IL-2 secretion at 48 h by MA5.8ζ cells and cells expressing either control shRNA, CUL2 shRNA or CUL3 shRNA with 1 µg/mL α-CD3 and 200 nM MLN4924. Data are averaged as in (C–G). Values represent mean ± s.e.m.; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Pervanadate stimulation reduces CRL neddylation.

<p>(A) Representative immunoblot and (B) quantification of NEDD8 relative density from primary mouse CD4⁺ T cells stimulated with 100 µM pervanadate or incubated either with 3 µM MLN4924 for 2 h as a dennedylated control or 2 mM OPT for 30 min as a neddylated control (n = 3). (C) Representative immunoblot and (D) quantification of NEDD8 relative density from primary mouse CD43⁻ B cells stimulated as in (B) (n = 3). (E) Representative immunoblot and quantification (F) of NEDD8 relative density from MEFs stimulated as in (B) (n = 3). (G) Representative immunoblot and (H) quantification of NEDD8 relative density from BT549 cells stimulated as in (B) (n = 3). (I) Representative immunoblot and (J) quantification of NEDD8 relative density from HT29 colon cancer cells stimulated as in (B) (n = 3). Values represent mean ± s.e.m.; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p