Inhibition of a malaria host-pathogen interaction by a computationally designed inhibitor.

Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. A unique characteristic of P. vivax is that it depends on the Duffy antigen/receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.

Rifampin resistance mutations in the rpoB gene of Enterococcus faecalis impact host macrophage cytokine production.

Antibiotic-resistant bacteria in the genus Enterococcus are a major cause of nosocomial infections and are an emergent public health concern. Similar to a number of bacterial species, resistance to the antibiotic rifampicin (RifR) in enterococci is associated with mutations in the gene encoding the ß subunit of RNA polymerase (rpoB). In Mycobacterium tuberculosis, RifRrpoB mutations alter mycobacterial surface lipid expression and are associated with an altered IL-1 cytokine response in macrophages upon infection. However, it is not clear if RifR mutations modulate host cytokine responses by other bacteria. To address this question, we utilized Enterococcus faecalis (E. faecalis). Here, we treated human monocyte-derived macrophages with heat-inactivated wild type or RifRrpoB mutants of E. faecalis and found that RifR mutations reduced IL-1ß cytokine production. However, RifR mutations elicited other potent pro- and anti-inflammatory responses, indicating that they can impact other immune pathways beyond IL-1R1 signaling. Our findings suggest that immunomodulation by mutations in rpoB may be conserved across diverse bacterial species and that subversion of IL-1R1 pathway is shared by RifR bacteria.

The structure of Acinetobacter-secreted protease CpaA complexed with its chaperone CpaB reveals a novel mode of a T2SS chaperone-substrate interaction.

Members of the Acinetobacter baumannii-calcoaceticus complex are nosocomial pathogens frequently causing multidrug-resistant infections that are increasing at alarming rates. A. baumannii has become the Gram-negative bacterium with the highest rate of multidrug resistance. As such, it is categorized by the World Health Organization as a critical priority for the research and development of new antimicrobial therapies. The zinc-dependent metalloendopeptidase CpaA is a predominant substrate of the type II secretion system (T2SS). CpaA is also a virulence factor of medically relevant Acinetobacter strains that specifically degrade the human glycoprotein coagulation factor XII and not its deglycosylated form, but the mechanism for this specificity is unknown. CpaB is a membrane-anchored T2SS chaperone that interacts with CpaA and is required for its stability and secretion. Here, we report the crystal structure of the CpaAB complex at 2.6-Å resolution, revealing four glycan-binding domains in CpaA that were not predicted from its primary sequence and may explain CpaA's glycoprotein-targeting activity. The structure of the complex identified a novel mode for chaperone-protease interactions in which the protease surrounds the chaperone. The CpaAB organization was akin to zymogen inactivation, with CpaB serving as a prodomain that inhibits catalytically active CpaA. CpaB contains a C-terminal tail that appears to block access to the CpaA catalytic site, and functional experiments with truncated variants indicated that this tail is dispensable for CpaA expression and secretion. Our results provide new insight into the mechanism of CpaA secretion and may inform the future development of therapeutic strategies for managing Acinetobacter infections.

Crystal structure of D-serine dehydratase from Escherichia coli.

D-Serine dehydratase from Escherichia coli is a member of the ß-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the ß-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.

Epigallocatechin-gallate suppresses tumorigenesis by directly targeting Pin1.

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). The human peptidyl prolyl cis/trans isomerase (Pin1) plays a critical role in oncogenic signaling. Herein, we report the X-ray crystal structure of the Pin1/EGCG complex resolved at 1.9 Å resolution. Notably, the structure revealed the presence of EGCG in both the WW and PPIase domains of Pin1. The direct binding of EGCG with Pin1 was confirmed and the interaction inhibited Pin1 PPIase activity. In addition, proliferation of cells expressing Pin1 was inhibited and tumor growth in a xenograft mouse model was suppressed. The binding of EGCG with Arg17 in the WW domain prevented the binding of c-Jun, a well-known Pin1 substrate. EGCG treatment corresponded with a decreased abundance of cyclin D1 and diminution of 12-O-tetradecanoylphorbol-l3-acetate-induced AP-1 or NF-κB promoter activity in cells expressing Pin1. Overall, these results showed that EGCG directly suppresses the tumor-promoting effect of Pin1.

T-LAK cell-originated protein kinase (TOPK) phosphorylation of Prx1 at Ser-32 prevents UVB-induced apoptosis in RPMI7951 melanoma cells through the regulation of Prx1 peroxidase activity.

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H(2)O(2)-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H(2)O(2) levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H(2)O(2) accumulation.