Crystal structures of FolM alternative dihydrofolate reductase 1 from Brucella suis and Brucella canis

Members of the bacterial genus Brucella cause brucellosis, a zoonotic disease that affects both livestock and wildlife. Brucella are category B infectious agents that can be aerosolized for biological warfare. As part of the structural genomics studies at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), FolM alternative dihydrofolate reductases 1 from Brucella suis and Brucella canis were produced and their structures are reported. The enzymes share ∼95% sequence identity but have less than 33% sequence identity to other homologues with known structure. The structures are prototypical NADPH-dependent short-chain reductases that share their highest tertiary-structural similarity with protozoan pteridine reductases, which are being investigated for rational therapeutic development.

R354X Mutation in PRPF31 Affects Its Capacity in Inflammation Response and Splicing

Pre-mRNA processing factor 31(PRPF31) is a key component of RNA splicing and also a disease-causing gene of Retinitis Pigmentosa (RP). Previously, we found that the nonsense mutation R354X in PRPF31induces RP in a Chinese RP family. In order to investigate the underlining molecular mechanisms of RP pathogenesis induced by this mutation, we generated cell lines stably expressing R354X mutant, wild type (WT) of PRPF31 and corresponding empty vector using HEK293T cells ,the resulting cell lines were used for Long non-coding RNA sequencing(LncRNA -sequencing). The results of LncRNA sequencing showed that, comparing to WT, R354X mutation changed the expression and splicing of coding and non-coding transcripts. Interestingly, in HEK293T and APRE-19 cells, inflammation-associated genes such as IFI6, OAS3and STAT3, enhanced their expression in response to the overexpression of WT PRPF31; however, in R354X mutation cells, those genes’ expression remained basal levels. Moreover, increased H2AFX expression and attenuated growth capacity were found in cells expressing R354X PRPF31. In contrast with WT, R354X mutant showed varied splicing model for dihydrofolate reductase (DHFR) in HEK293T , reduced CPSF1 and SORBS1 mRNAs binding in ARPE-19 ,and its binding with CTNNBL1 was also interfered in ARPE-19 cells. On the other hand, the R354X mutant also increased the level of transcripts read-through. Taken together, R354X mutation in PRPF31 affected cell survival, changed gene’s expression and splicing. Those findings indicate that inflammation and oxidation might contribute the pathogenesis of RP induced by the R354X mutation.

Albumin–Methotrexate Prodrug Analogues That Undergo Intracellular Reactivation Following Entrance into Cancerous Glioma Cells

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.

Sesquiterpene Lactones with Dual Inhibitory Activity against the Trypanosoma brucei Pteridine Reductase 1 and Dihydrofolate Reductase

The parasite Trypanosoma brucei (T. brucei) is responsible for human African trypanosomiasis (HAT) and the cattle disease “Nagana” which to this day cause severe medical and socio-economic issues for the affected areas in Africa. So far, most of the available treatment options are accompanied by harmful side effects and are constantly challenged by newly emerging drug resistances. Since trypanosomatids are auxotrophic for folate, their pteridine metabolism provides a promising target for an innovative chemotherapeutic treatment. They are equipped with a unique corresponding enzyme system consisting of the bifunctional dihydrofolate reductase-thymidylate synthase (TbDHFR-TS) and the pteridine reductase 1 (TbPTR1). Previously, gene knockout experiments with PTR1 null mutants have underlined the importance of these enzymes for parasite survival. In a search for new chemical entities with a dual inhibitory activity against the TbPTR1 and TbDHFR, a multi-step in silico procedure was employed to pre-select promising candidates against the targeted enzymes from a natural product database. Among others, the sesquiterpene lactones (STLs) cynaropicrin and cnicin were identified as in silico hits. Consequently, an in-house database of 118 STLs was submitted to an in silico screening yielding 29 further virtual hits. Ten STLs were subsequently tested against the target enzymes in vitro in a spectrophotometric inhibition assay. Five compounds displayed an inhibition over 50% against TbPTR1 as well as three compounds against TbDHFR. Cynaropicrin turned out to be the most interesting hit since it inhibited both TbPTR1 and TbDHFR, reaching IC50 values of 12.4 µM and 7.1 µM, respectively.

Establishment of a Dihydrofolate Reductase Gene Knock-In Zebrafish Strain to Aid Preliminary Analysis of Congenital Heart Disease Mechanisms

Background: The dihydrofolate reductase (DHFR) gene is imperative in development, therefore it is essential to explore its effects on heart development. Thus, here a dhfr zebrafish knock-in (KI) strain was constructed.Methods: CRISPR/Cas9 technology was used to establish the dhfr KI zebrafish strain. This strain was hybridized with TgG fluorescent strain zebrafish to observe the phenotypes of heart shape, size, and circularization direction. Wild-type (WT) and KI zebrafish were then dissected and histologically stained to observe pathological changes. Western blot analysis was used to verify the increased expressions of zebrafish genes after KI. Hybridization experiments were used to confirm the presence of abnormal gonadal dysplasia.Results: The zebrafish dhfr KI strain was successfully constructed through CRISPR/Cas9 technology. At 6 days post fertilization (dpf), microscopic examinations of KI (homozygous) specimens revealed pericardial effusions, heart compressions, and curled tails. Compared with WT, the Hematoxylin and Eosin (H&E) tissue sections of KI-homozygous zebrafish showed defects such as reduced atria and ventricles. Western blot analysis indicated that the expression of the DHFR protein increased in both heterozygotes and homozygotes of dhfr KI zebrafish. Hybridization experiments revealed that dhfr KI may affect gonadal function.Conclusion: The DHFR gene plays an important regulatory role in the process of heart development, and copy number variations (CNVs) of this gene may constitute a new pathogenic mechanism of congenital heart disease (CHD).