The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection.

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where ‘n’ can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5′ of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel α-helical ‘tweezer’-like structure.

The human IgG repertoire contains endogenous antibodies against beta amyloid (Aβ) that may be relevant to the pathogenesis and treatment of Alzheimer's disease. There have been widely disparate estimates of the levels of these antibodies in human plasma. We identify factors that have contributed to these disparities and describe improved methods for measuring anti-Aβ antibodies in blood. These methods include isolating immunoglobulin by thiophilic chromatography and using chaotropic salts to dislodge weakly bound antibodies without significantly reducing the binding of specific anti-Aβ antibodies. Using these methods, we show that human blood contains polyvalent IgG antibodies that bind to Aβ with relatively low avidity and specificity, as well as IgG antibodies that bind to linear and conformational epitopes on amyloid monomers and aggregates with moderate to high avidity.

Ewing sarcoma (ES) is characterized by a reciprocal translocation t(11;22) that results in a fusion of the EWSR1 and FLI1 genes (EWS-FLI). The objective of this study is to identify protein binding partners of the purportedly undruggable chimeric transcription factor EWS-FLI1 to serve as alternative pharmacological targets for potential ES-selective drug therapies. The literature identifies the orphan nuclear receptor DAX1 (encoded by NR0B1) as a binding partner of EWS-FLI1 the expression of which is restricted outside ES, thereby providing a target for a potential ES selective drug. Yeast two-hybrid (Y2H) screening using EWS-FLI1 as the bait was employed to identify other potential targets. The importance of the binding partners identified by the Y2H screen was initially assessed by a series of proliferation assays involving the ES cell lines EW8, ES7 and ES6 as well as rhabdomyosarcoma cell line Rh30 (Non-Ewing control). Prior to measuring proliferation, cells were transfected a siRNA designed to target the putative binding partner of EWS-FLI1. These cells were then grown alongside cells transfected with a control siRNA over a period of 96 hr. The change in cell confluence in the wells was measured by a live-cell real-time measurement (Incucyte). Gene knockdown was confirmed by RT-PCR and western blot analysis. The proliferation assays showed that the knockdown of DAX1 produces a notable reduction in cell proliferation compared to siRNA control proliferation in all three of the Ewing cell lines (10% to 25%). No significant DAX1 siRNA based inhibition of cell proliferation was detected in the Rh30 non-Ewing control cell line. Three of the proteins identified by the Y2H screen showed significant reductions in ES cell proliferation. Methyl-CpG-Binding Domain Protein 1 (MBD1) knockdown caused the proliferation of the ES cell lines to level off at 60% to 80% within 2 to 4 days. Proliferation of MBD1 siRNA and control siRNA transfected Rh30 cells was similar for the full 4 days of the assay. Mannosidase-α-class 2B-member 2 (MAN2B2) knockdown caused the proliferation of the ES and Rh30 cell lines to level off at 25% to 65% within 1 to 2 days. Mixed-Lineage Leukemia Protein 3 (MLL3) knockdown caused the proliferation of the EW8 and ES7 cell lines to level off at 52% and 70% respectively. MLL3 knockdown did not inhibit the proliferation of Rh30 cells when compared to siRNA control cells. In contrast, ES6 cell proliferation was also not inhibited by MLL3 knockdown when compared to their siRNA control cells. The knockdowns of DAX1, MLL3 and MBD1 suggest that these EWS-FLI1 binding partners would likely provide potential targets for ES drug development. The effect of knocking down Y2H-identified EWS-FLI interactors on colony formation, migration and tumorigenicity is ongoing. Citation Format: Matthew L. Rotondi, Peter J. Houghton. Identification and characterization of EWS-FLI1 binding partners in Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2560. doi:10.1158/1538-7445.AM2017-2560