Multiple Protein Domains Mediate Interaction between Bcl10 and MALT1*

Bcl10 and MALT1 are essential mediators of NF-κB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological importance. Previous studies have indicated that a 13-amino acid region downstream of the Bcl10 caspase recruitment domain (CARD) is responsible for interacting with the immunoglobulin-like domains of MALT1. We now provide evidence that the death domain of MALT1 and the CARD of Bcl10 also contribute to Bcl10-MALT1 interactions. Although a direct interaction between the MALT1 death domain and Bcl10 cannot be detected via immunoprecipitation, FRET data strongly suggest that the death domain of MALT1 contributes significantly to the association between Bcl10 and MALT1 in T cells in vivo. Furthermore, analysis of point mutants of conserved residues of Bcl10 shows that the Bcl10 CARD is essential for interaction with the MALT1 N terminus. Mutations that disrupt proper folding of the Bcl10 CARD strongly impair Bcl10-MALT1 interactions. Molecular modeling and functional analyses of Bcl10 point mutants suggest that residues Asp80 and Glu84 of helix 5 of the Bcl10 CARD directly contact MALT1. Together, these data demonstrate that the association between Bcl10 and MALT1 involves a complex interaction between multiple protein domains. Moreover, the Bcl10-MALT1 interaction is the second reported example of interactions between a CARD and a non-CARD protein region, which suggests that many signaling cascades may utilize CARD interactions with non-CARD domains

POLKADOTS Are Foci of Functional Interactions in T-Cell Receptor–mediated Signaling to NF-κB

Stimulation of the T-cell receptor (TCR) results in the activation of several transcription factors, including NF-κB, that are crucial for T-cell proliferation and gain of effector functions. On TCR engagement, several proteins within the TCR-directed NF-κB signaling pathway undergo dynamic spatial redistribution, but the significance of these redistribution events is largely unknown. We have previously described TCR-induced cytoplasmic structures called POLKADOTS (punctate and oligomeric killing or activating domains transducing signals) that are enriched in the NF-κB signaling intermediate, Bcl10. We now show that these structures are formed only under conditions that promote efficient NF-κB activation. Furthermore, POLKADOTS formation is dependent on functional domains of specific NF-κB signal transducers. Through use of a photoactivatable GFP, we demonstrate that POLKADOTS contain both a highly stable and a rapidly equilibrating protein component. FRET analyses show that POLKADOTS are sites of enriched interactions between Bcl10 and partner signaling proteins. These observations strongly suggest that POLKADOTS are focal sites of dynamic information exchange between cytosolic intermediates in the process of TCR activation of NF-κB

Alveolar Macrophages Infected with Ames or Sterne Strain of <i>Bacillus anthracis</i> Elicit Differential Molecular Expression Patterns

<div><p>Alveolar macrophages (AMs) phagocytose <i>Bacillus anthracis</i> following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of <i>B. anthracis</i>, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of <i>B. anthracis</i>, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the <i>B. anthracis.</i> Meta-analyses revealed genes differentially expressed in response to <i>B. anthracis</i> infection were also induced upon infections with multiple pathogens such as <i>Francisella Novicida</i> or <i>Staphylococcus aureus</i>. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of <i>B. anthracis</i>. The reported differences may account for the marked difference in pathogenicity between these two strains.</p></div

Overlap between the genes identified from current study and studies referenced.

<p>Explanation of the column headings in table are as follows: Pathogens: name of the pathogen used in the referenced microarray studies; # of overlapping genes: the number of genes that are overlapping between the current study and the referenced studies; Cells used in the study: the type of cells used in the referenced microarray studies; Selected Overlapping Genes: selected gene symbols that are overlapping between the two studies; P-value: the P-value of the overlap; PMID or GEO ID: the reference of the published microarray studies.</p

Microarray analyses identified 528 human orthologs which were differentially expressed between Ames and Sterne infected AMs.

<p>AMs obtained from five rhesus macaque donors were infected with Ames or Sterne spores at an MOI of 10 for indicated time points. Total mRNAs were purified and hybridized to rhesus macaque cDNA microarrays. “Ames vs Sterne” bar depicts the ratio of gene expressions (in logarithmic scale) between Ames-infected AMs to Sterne-infected counterparts. “Ames” and “Sterne” bar depict fold change of a gene expression (in logarithmic scale) by normalizing Ames or Sterne treated AMs with the 0 h time point. The level of fold changes are colored coded, where red stands for high values (>1) and blue for low fold changes (<1). To highlight statistically significant changes for “Ames vs. Sterne” for the ease of visualization, we decreased the contrast by three folds for the portion of the heat map, where differential expression is not significant.</p

Gene ontology (GO) analysis.

<p>Statistically significant overrepresentation of selected functional classes and protein families based upon gene ontology (GO) analysis.</p

AMs infected with Ames or Sterne spores show differential pro-inflammatory cytokine/chemokine protein expression patterns.

<p>AMs were infected with either Ames or Sterne spores at an MOI of 10. Supernatants were collected at indicated time points and cytokine/chemokines levels were quantified. The experiment was performed at least three times and data for three rhesus macaque donors are shown. Scatter plots are presented as mean ± Standard Deviation. Experiments for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087201#pone-0087201-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087201#pone.0087201.s001" target="_blank">Figure S1</a> were performed concurrently and shared the same controls. P-value is calculated using a paired Student’s t-test. *represents p-value <0.05 and **represents p-value <0.01.</p

A Schematic diagram of observed differential modulation of host responses to Ames and Sterne spores of <i>B. anthracis</i>.

<p>The diagram is based on the observed differences in the cytokine/chemokine and host responses in AMs infected with either Ames or Sterne spores. TFs: Transcription factors.</p

Time-dependent kinetic difference in the induction of COX-2 and PGE₂ expression (A) AMs were infected with either Ames or Sterne spores for 90 min, 4 h or 18 h.

<p>Cells were lysed and the mRNA was purified and quantified by real time PCR. Fold expression was calculated by normalizing to time 0. (B) AMs were infected with Ames or Sterne spores at an MOI of 10. The amount of PGE₂ was quantified by ELISA. Data shown in (A) and (B) are representative of n = 3 experiments. Scatter plots are presented as mean ± Standard deviation. P-value is calculated using a paired two tailed Student’s t-test. *represent p-value <0.05.</p