Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson’s disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on 15N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain β-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.

SUMMARY Enteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by “chemical chaperones.”

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.

Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their antiadenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential antiadenoviral compounds. The assay is unique in that it is based on a replication-competent adenovirus type 11p green fluorescent protein (GFP)-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression in vitro by >80%, but only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid, turned out to be a potent inhibitor of adenovirus replication.

Background:Wilms' tumour 1 (WT1) gene was discovered as a tumour suppressor gene. Later findings have suggested that WT1 also can be oncogenic. This complexity is partly explained by the fact that WT1 has a number of target genes.Method:WT1 and its target gene human telomerase reverse transcriptase (hTERT) were analysed in clear cell renal cell carcinoma (ccRCC). In vitro experiments were performed to examine the functional link between WT1 and hTERT by overexpression of WT1 isoforms in the ccRCC cell line, TK-10.Results:WT1 demonstrated lower RNA expression in ccRCC compared with renal cortical tissue, whereas hTERT was increased, showing a negative correlation between WT1 and hTERT (P=0.005). These findings were experimentally confirmed in vitro. The WT1 generated effect on hTERT promoter activity seemed complex, as several negative regulators of hTERT transcription, such as SMAD3, JUN (AP-1) and ETS1, were activated by WT1 overexpression. Downregulation of potential positive hTERT regulators, such as cMyc, AP-2α, AP-2γ, IRF1, NFX1 and GM-CSF, were also observed. Chromatin immunoprecipitation analysis verified WT1 binding to the hTERT, cMyc and SMAD3 promoters.Conclusion:The collected data strongly indicate multiple pathways for hTERT regulation by WT1 in ccRCC.