RELT family members activate p38 and induce apoptosis by a mechanism distinct from TNFR1.

Receptor Expressed in Lymphoid Tissues (RELT) is a human Tumor Necrosis Factor Receptor (TNFR) family member that has two identified homologous binding partners, RELL1 and RELL2. This study sought to further understand the pattern of RELT expression, the functional role of RELT family members, and the mechanism of RELT-induced apoptosis. RELT protein expression was detected in the spleen, lymph node, brain, breast and peripheral blood leukocytes (PBLs). A smaller than expected size of RELT was observed in PBLs, suggesting a proteolytically cleaved form of RELT. RELL1 and RELL2 overexpression activated the p38 MAPK pathway more substantially than RELT in HEK-293 cells, and this activation of p38 by RELT family members was blocked by dominant-negative mutant forms of OSR1 or TRAF2, implicating these molecules in RELT family member signaling. RELT was previously shown to induce apoptosis in human epithelial cells despite lacking the characteristic death domain (DD) found in other TNFRs. Seven deletion mutants of RELT that lacked differing portions of the intracellular domain were created to assess whether RELT possesses a novel DD. None of the deletion mutants induced apoptosis as efficiently as full-length RELT, a result that is consistent with a novel DD being located at the carboxyl-terminus. Interestingly, induction of apoptotic morphology by RELT overexpression was not prevented when signaling by FADD or Caspase-8 was blocked, indicating RELT induces apoptosis by a pathway distinct from other death-inducing TNFRs such as TNFR1. Collectively, this study provides more insights into RELT expression, RELT family member function, and the mechanism of RELT-induced death.

High-level pacidamycin resistance in Pseudomonas aeruginosa is mediated by an opp oligopeptide permease encoded by the opp-fabI operon.

Pacidamycins (or uridyl peptide antibiotics) possess selective in vivo activity against Pseudomonas aeruginosa. An important limitation for the therapeutic use of pacidamycins with P. aeruginosa is the high frequency (10(-6) to 10(-7)) at which resistant mutants emerge. To elucidate the mechanism(s) of this resistance, pacidamycin-resistant P. aeruginosa mutants were isolated. Two types of mutants were obtained. Type 1, or high-level resistance mutants with a pacidamycin MIC of 512 µg/ml, were more abundant, with a frequency of~2 × 10(-6), and did not show cross-resistance with other antibiotics. Type 2, low-level resistance mutants, were isolated with a frequency of ~10(-8) and had a pacidamycin MIC of 64 µg/ml (the MIC for the wild-type strain was 4 to 16 µg/ml). These mutants were cross-resistant to levofloxacin, tetracycline, and erythromycin and were shown to overexpress either the MexAB-OprM or MexCD-OprJ multidrug resistance efflux pumps. High-level resistant mutants were isolated by transposon mutagenesis and one insertion was localized to oppB, one of two periplasmic binding protein components of an oligopeptide transport system which is encoded by the opp-fabI operon. The Opp system is required for uptake of pacidamycin across the inner membrane, since various opp, but not fabI, mutants were resistant to high levels of pacidamycin. Both of the two putative Opp periplasmic binding proteins, OppA and OppB, were required for pacidamycin uptake. Although both impaired uptake into and efflux from the cell can cause pacidamycin resistance in P. aeruginosa, our data suggest that impaired uptake is the primary reason for the high-frequency and high-level pacidamycin resistance.

The RELT Family of Proteins: An Increasing Awareness of Their Importance for Cancer, the Immune System, and Development.

This review highlights Receptor Expressed in Lymphoid Tissues (RELT), a Tumor Necrosis Factor Superfamily member, and its two paralogs, RELL1 and RELL2. Collectively, these three proteins are referred to as RELTfms and have gained much interest in recent years due to their association with cancer and other human diseases. A thorough knowledge of their physiological functions, including the ligand for RELT, is lacking, yet emerging evidence implicates RELTfms in a variety of processes including cytokine signaling and pathways that either promote cell death or survival. T cells from mice lacking RELT exhibit increased responses against tumors and increased inflammatory cytokine production, and multiple lines of evidence indicate that RELT may promote an immunosuppressive environment for tumors. The relationship of individual RELTfms in different cancers is not universal however, as evidence indicates that individual RELTfms may be risk factors in certain cancers yet appear to be protective in other cancers. RELTfms are important for a variety of additional processes related to human health including microbial pathogenesis, inflammation, behavior, reproduction, and development. All three proteins have been strongly conserved in all vertebrates, and this review aims to provide a clearer understanding of the current knowledge regarding these interesting proteins.

Identification of PLSCR1 as a protein that interacts with RELT family members.

Receptor expressed in lymphoid tissues (RELT) proteins are recently described surface receptors belonging to the larger TNF receptor family. To improve our understanding of RELT-mediated signal transduction, we performed a screen for RELT-interacting proteins. Phospholipid Scramblase 1 (PLSCR1) was identified through a yeast two-hybrid genetic screen utilizing the intracellular portion of the RELT family member, RELL1, as bait. PLSCR1 was observed to physically interact with all known RELT family members as determined by co-immunoprecipitation experiments. The protein kinase, oxidative stress responsive 1 (OSR1) was previously shown to interact and phosphorylate all three RELT family members. In our study, no physical association was observed between OSR1 and PLSCR1 alone. However, in the presence of RELT, OSR1 was capable of co-immunoprecipitating PLSCR1, suggesting the formation of a protein complex between RELT, OSR1, and PLSCR1. In addition, OSR1 phosphorylated PLSCR1 in an in vitro kinase assay, but only in the presence of RELT, suggesting a functional multiprotein complex. RELT and PLSCR1 co-localized in intracellular regions of human embryonic kidney-293 cells, with RELT overexpression appearing to alter the localization of PLSCR1. These studies demonstrate that RELT family members physically interact with PLSCR1, and that these interactions may regulate the phosphorylation of PLSCR1 by OSR1.

RELT induces cellular death in HEK 293 epithelial cells.

RELT is a recently identified Tumor Necrosis Factor Receptor that possess two homologues in humans named RELL1 and RELL2. We investigated whether RELT and its homologues could induce cellular death when transiently transfected into HEK 293 epithelial cells. Transfection of RELT family members into HEK 293 epithelial cells induced cell death characterized by rounding and lifting of cells accompanied by DNA fragmentation, characteristics that are consistent with the activation of an apoptotic pathway. Overexpression of RELT in COS-7 cells resulted in cell rounding and lifting without DNA fragmentation, suggesting that the effects of RELT signaling may vary among different cell types. In summary, we report that overexpression of RELT or its homologues RELL1 and RELL2 in HEK 293 epithelial cells results in cell death with morphological characteristics consistent with the activation of an apoptotic pathway.

RELT stains prominently in B-cell lymphomas and binds the hematopoietic transcription factor MDFIC.

Receptor Expressed in Lymphoid Tissues (RELT) is a human tumor necrosis factor receptor superfamily member (TNFRSF) that is expressed most prominently in cells and tissues of the hematopoietic system. RELL1 and RELL2 are two homologs that physically interact with RELT and co-localize with RELT at the plasma membrane. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members. The transcription factor MyoD family inhibitor domain-containing (MDFIC) was identified in a yeast two-hybrid genetic screen using RELL1 as bait. MDFIC co-localizes with RELT family members at the plasma membrane; this co-localization was most prominently observed with RELL1 and RELL2. In vitro co-immunoprecipitation (Co-IP) was utilized to demonstrate that MDFIC physically interacts with RELT, RELL1, and RELL2. Co-IP using deletion mutants of MDFIC and RELT identified regions important for physical association between MDFIC and RELT family members and a computational analysis revealed that RELT family members are highly disordered proteins. Immunohistochemistry of normal human lymph nodes revealed RELT staining that was most prominent in macrophages. Interestingly, the level of RELT staining significantly increased progressively in low and high-grade B-cell lymphomas versus normal lymph nodes. RELT co-staining with CD20 was observed in B-cell lymphomas, indicating that RELT is expressed in malignant B cells. Collectively, these results further our understanding of RELT-associated signaling pathways, the protein structure of RELT family members, and provide preliminary evidence indicating an association of RELT with B-cell lymphomas.

Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program.

Identification of RELT homologues that associate with RELT and are phosphorylated by OSR1.

RELL1 and RELL2 are two newly identified RELT homologues that bind to the TNF receptor family member RELT. The expression of RELL1 at the mRNA level is ubiquitous, whereas expression of RELL2 mRNA is more restricted to particular tissues. RELT, RELL1, and RELL2 co-localized with one another at the plasma membrane. The three proteins interacted with one another as demonstrated by in vitro co-immunoprecipitation experiments. We propose that RELL1 and RELL2 be considered RELT family members based on their similar amino acid sequences and on their ability to physically interact with one another. OSR1 was identified through a yeast two-hybrid screen utilizing the intracellular portion of RELL1 as bait, and OSR1 was shown to interact with the three RELT family members by in vitro co-immunoprecipitation experiments. Additionally, OSR1 phosphorylated the RELT family members in an in vitro kinase assay. These results report two novel homologues of RELT that interact with RELT and are phosphorylated by the OSR1 kinase.

The bcsA gene influences multiple aspects of development in Myxococcus xanthus.

M. xanthus strains containing a mutation in the bcsA gene are able to bypass the B and C signaling requirements for development. The bcsA mutant was examined with regards to several aspects of development to better ascertain the function of the bcsA gene. The bcsA mutant developed on nutrient levels sufficient to support vegetative growth in wild-type cells, supporting previous evidence that the bcsA gene inhibits development. The earliest effect of the bcsA mutation on the development program was when cells were beginning to aggregate together to form fruiting bodies. Spores produced by bcsA mutants were hypersusceptible to sodium dodecyl sulfate, suggesting that the bcsA gene is important for optimal spore production. Transcription of the bcsA gene was induced significantly during development at a time when cells were beginning to aggregate together. Collectively, these results indicate that the bcsA gene inhibits development and is also transcriptionally upregulated during development.

Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development.

The BsgA protease is required for starvation-induced development in Myxococcus xanthus. Bypass suppressors of a bsgA mutant were isolated to identify genes that may encode additional components of BsgA protease-dependent regulation of development. Strain M951 was isolated following Tn5 mutagenesis of a bsgA mutant and was capable of forming fruiting bodies and viable spores in the absence of the BsgA protease. The Tn5Omega951 insertion was localized to a gene, bcsA, that encodes a protein that has significant amino acid similarity to a group of recently described flavin-containing monooxygenases involved in styrene catabolism. Mutations in bcsA bypassed the developmental requirements for both extracellular B and C signaling but did not bypass the requirement for A signaling. Bypass of the B-signaling requirement by the bcsA mutation was accompanied by restored expression of a subset of developmentally induced lacZ fusions to the BsgA protease-deficient strain. bcsA mutant cells developed considerably faster than wild-type cells at low cell density and altered transcriptional levels of a developmentally induced, cell-density-regulated gene (Omega4427), suggesting that the bcsA gene product may normally act to inhibit development in a cell-density-regulated fashion. Bypass of the requirements for both B and C signaling by bcsA mutations suggests a possible link between these two genetically, biochemically, and temporally distinct signaling requirements.

Beta-ketoacyl acyl carrier protein reductase (FabG) activity of the fatty acid biosynthetic pathway is a determining factor of 3-oxo-homoserine lactone acyl chain lengths.

The two acyl-homoserine lactones (AHLs) N-(butyryl)-L-homoserine lactone and N-[3-oxododecanoyl]-L-homoserine lactone (3-oxo-C(12)-HSL) are required for quorum sensing in Pseudomonas aeruginosa. These AHLs derive their invariant lactone rings from S-adenosylmethionine and their variable acyl chains from the cellular acyl-acyl carrier protein (ACP) pool. This reaction is catalysed by specific AHL synthases, which exhibit acyl chain specificity. Culture supernatants of P. aeruginosa contain multiple 3-oxo-AHLs that differ in their acyl chain lengths but their physiological role, if any, remains unknown. An in vitro fatty acid-3-oxo-AHL synthesis system was established utilizing purified P. aeruginosa Fab proteins, ACP and the LasI 3-oxo-AHL synthase. In the presence of excess protein, substrates and cofactors, this system produced almost exclusively 3-oxo-C(12)-HSL. When the beta-ketoacyl-ACP reductase (FabG) catalysed step was made rate-limiting by switching from the preferred NADPH cofactor to NADH, increased levels of short chain 3-oxo-AHLs were produced, presumably because shorter-chain ketoacyl-ACPs accumulated and thus became LasI substrates. Consistent with these in vitro observations, a fabG(Ts) mutant produced increased amounts of 3-oxo-AHLs in vivo. Thus, in vitro and in vivo evidence indicated that modulation of FabG activity of the fatty acid biosynthetic pathway may determine the acyl chain lengths of these 3-oxo-AHLs and that the LasI 3-oxo-AHL synthase is sufficient for their synthesis.