Phage Display Screening for Alba Superfamily Proteins from the Human Malaria Parasite, Plasmodium falciparum Reveals a High Level of Association with Protein Modification Pathways and Hints at New Drug Targets.

PURPOSE: A 2016 study estimated that over 3 billion people are currently at risk of contracting malaria. Although a wide variety of medications are available to treat malaria, the parasites have started to exhibit resistance to many commonly used therapeutics necessitating a push for new investigations to identify novel drug targets. METHODS: In this study, nucleic acid-binding Alba superfamily proteins of the human malaria parasite, Plasmodium falciparum were investigated to identify interacting protein motifs. A high-throughput molecular screening technique, phage display, coupled with next-generation sequencing was applied to assess large data sets. RESULTS: Four P. falciparum Alba proteins were used for screening which appear to have distinct roles in parasite biology based on the results of this work. The majority of the peptide motifs identified from phage display were involved in post-translational modification pathways, thus suggesting that parasite-specific gene regulatory mechanisms are involved which could serve as drug targets for novel therapeutics. CONCLUSION: This study found 18 peptide motifs which potentially have strong interactions with one or more of the Alba superfamily proteins from P. falciparum. Considering the large fraction of post-translational modification-related peptide motifs identified from this work, one or more of the protein modification pathways could serve as a good target for malaria treatment.

A near-infrared fluorescent probe based on a hemicyanine dye with an oxazolidine switch for mitochondrial pH detection.

A near-infrared fluorescent probe (AH+) has been prepared by incorporating an oxazolidine switch into a near-infrared hemicyanine dye. The probe shows fast and sensitive responses to pH from an oxazolidine switch to the hemicyanine dye upon pH decreases from 10.0 to 5.0. The probe shows good photostability, low cytotoxicity, and reversible fluorescence responses to pH changes with a pKa value of 7.6. It has been successfully used to determine pH changes in mitochondria.

Ratiometric fluorescent probes based on through-bond energy transfer of cyanine donors to near-infrared hemicyanine acceptors for mitochondrial pH detection and monitoring of mitophagy.

Two ratiometric near-infrared fluorescent probes have been developed to selectively detect mitochondrial pH changes based on highly efficient through-bond energy transfer (TBET) from cyanine donors to near-infrared hemicyanine acceptors. The probes consist of identical cyanine donors connected to different hemicyanine acceptors with a spirolactam ring structure linked via a biphenyl linkage. At neutral or basic pH, the probes display only fluorescence of the cyanine donors when they are excited at 520 nm. However, acidic pH conditions trigger spirolactam ring opening, leading to increased π-conjugation of the hemicyanine acceptors, resulting in new near-infrared fluorescence peaks at 740 nm and 780 nm for probes A and B, respectively. This results in ratiometric fluorescence responses of the probes to pH changes indicated by decreases of the donor fluorescence and increases of the acceptor fluorescence under donor excitation at 520 nm due to a highly efficient TBET from the donors to the acceptors. The probes only show cyanine donor fluorescence in alkaline-pH mitochondria. However, the probes show moderate fluorescence decreases of the cyanine donor and considerable fluorescence increases of hemicyanine acceptors during the mitophagy process induced by nutrient starvation or under drug treatment. The probes display rapid, selective, and sensitive responses to pH changes over metal ions, good membrane penetration, good photostability, large pseudo-Stokes shifts, low cytotoxicity, mitochondria-targeting, and mitophagy-tracking capabilities.

Fluorescent probes with high pKa values based on traditional, near-infrared rhodamine, and hemicyanine fluorophores for sensitive detection of lysosomal pH variations.

Sterically hindered fluorescent probes (A-C) have been developed by introducing 2-aminophenylboronic acid pinacol ester to a traditional, A, a near-infrared rhodamine dye, B, and a near-infrared hemicyanine dye, C, forming closed spirolactam ring structures. Probe A was non-fluorescent under basic pH conditions whereas probes B and C were moderately fluorescent with fluorescence quantum yields of 9% and 5% in pH 7.4 PBS buffer containing 1% ethanol, respectively. With all probes increasing acidity leads to significant increases in fluorescence at 580 nm, 644 and 744 nm for probes A, B and C with fluorescence quantum yields of 26%, 21% and 10% in pH 4.5 PBS buffer containing 1% ethanol, respectively. Probes A, B and C were calculated to have pKa values of 5.81, 5.45 and 6.97. The difference in fluorescence under basic conditions is ascribed to easier opening of the closed spirolactam ring configurations due to significant steric hindrance between the 2-aminophenylboronic acid pinacol ester residue and an adjacent H atom in the xanthene derivative moiety in probe B or C. The probes show fast, reversible, selective and sensitive fluorescence responses to pH changes, and are capable of sensing lysosomal pH variations in living cells.

Archaeal RNA polymerase and transcription regulation.

To elucidate the mechanism of transcription by cellular RNA polymerases (RNAPs), high-resolution X-ray crystal structures together with structure-guided biochemical, biophysical, and genetics studies are essential. The recently solved X-ray crystal structures of archaeal RNAP allow a structural comparison of the transcription machinery among all three domains of life. The archaea were once thought of closely related to bacteria, but they are now considered to be more closely related to the eukaryote at the molecular level than bacteria. According to these structures, the archaeal transcription apparatus, which includes RNAP and general transcription factors (GTFs), is similar to the eukaryotic transcription machinery. Yet, the transcription regulators, activators and repressors, encoded by archaeal genomes are closely related to bacterial factors. Therefore, archaeal transcription appears to possess an intriguing hybrid of eukaryotic-type transcription apparatus and bacterial-like regulatory mechanisms. Elucidating the transcription mechanism in archaea, which possesses a combination of bacterial and eukaryotic transcription mechanisms that are commonly regarded as separate and mutually exclusive, can provide data that will bring basic transcription mechanisms across all life forms.

Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature-sensitive.

All archaeal genomes encode RNA polymerase (RNAP) subunits E and F that share a common ancestry with the eukaryotic RNAP subunits A43 and A14 (Pol I), Rpb7 and Rpb4 (Pol II), and C25 and C17 (Pol III). By gene replacement, we have isolated archaeal mutants of Thermococcus kodakarensis with the subunit F-encoding gene (rpoF) deleted, but we were unable to isolate mutants lacking the subunit E-encoding gene (rpoE). Wild-type T. kodakarensis grows at temperatures ranging from 60 degrees C to 100 degrees C, optimally at 85 degrees C, and the DeltarpoF cells grew at the same rate as wild type at 70 degrees C, but much slower and to lower cell densities at 85 degrees C. The abundance of a chaperonin subunit, CpkB, was much reduced in the DeltarpoF strain growing at 85 degrees C and increased expression of cpkB, rpoF or rpoE integrated at a remote site in the genome, using a nutritionally regulated promoter, improved the growth of DeltarpoF cells. RNAP preparations purified from DeltarpoF cells lacked subunit F and also subunit E and a transcription factor TFE that co-purifies with RNAP from wild-type cells, but in vitro, this mutant RNAP exhibited no discernible differences from wild-type RNAP in promoter-dependent transcription, abortive transcript synthesis, transcript elongation or termination.