Potent ClpP agonists with anticancer properties bind with improved structural complementarity and alter the mitochondrial N-terminome.

The mitochondrial ClpP protease is responsible for mitochondrial protein quality control through specific degradation of proteins involved in several metabolic processes. ClpP overexpression is also required in many cancer cells to eliminate reactive oxygen species (ROS)-damaged proteins and to sustain oncogenesis. Targeting ClpP to dysregulate its function using small-molecule agonists is a recent strategy in cancer therapy. Here, we synthesized imipridone-derived compounds and related chemicals, which we characterized using biochemical, biophysical, and cellular studies. Using X-ray crystallography, we found that these compounds have enhanced binding affinities due to their greater shape and charge complementarity with the surface hydrophobic pockets of ClpP. N-terminome profiling of cancer cells upon treatment with one of these compounds revealed the global proteomic changes that arise and identified the structural motifs preferred for protein cleavage by compound-activated ClpP. Together, our studies provide the structural and molecular basis by which dysregulated ClpP affects cancer cell viability and proliferation.

Uncoupling Proteins and Regulated Proton Leak in Mitochondria.

Higher concentration of protons in the mitochondrial intermembrane space compared to the matrix results in an electrochemical potential causing the back flux of protons to the matrix. This proton transport can take place through ATP synthase complex (leading to formation of ATP) or can occur via proton transporters of the mitochondrial carrier superfamily and/or membrane lipids. Some mitochondrial proton transporters, such as uncoupling proteins (UCPs), transport protons as their general regulating function; while others are symporters or antiporters, which use the proton gradient as a driving force to co-transport other substrates across the mitochondrial inner membrane (such as phosphate carrier, a symporter; or aspartate/glutamate transporter, an antiporter). Passage (or leakage) of protons across the inner membrane to matrix from any route other than ATP synthase negatively impacts ATP synthesis. The focus of this review is on regulated proton transport by UCPs. Recent findings on the structure and function of UCPs, and the related research methodologies, are also critically reviewed. Due to structural similarity of members of the mitochondrial carrier superfamily, several of the known structural features are potentially expandable to all members. Overall, this report provides a brief, yet comprehensive, overview of the current knowledge in the field.

Biphasic Proton Transport Mechanism for Uncoupling Proteins.

It has been suggested that uncoupling proteins (UCPs) transport protons via interconversion between two conformational states: one in the "cytoplasmic state" and the other in the "matrix state". Matrix and cytoplasmic salt-bridge networks are key controllers of these states. This study proposes a mechanism for proton transport in tetrameric UCP2, with focus on the role of the matrix network. Eleven mutants were prepared to disrupt (K → Q or D → N mutations) or alter (K → D and D → K mutations) the salt-bridges in the matrix network. Proteins were recombinantly expressed in Escherichia coli membrane, reconstituted in model lipid membranes, and their structures and functions were analyzed by gel electrophoresis, circular dichroism spectroscopy, fluorescence assays, as well as molecular dynamics simulations. It is shown that the UCP2 matrix network contains five salt-bridges (rather than the previously reported three), and the matrix network can regulate the proton transport by holding the protein's transmembrane helices in close proximity, limiting the movement of the activator fatty acid(s). A biphasic two-state molecular model is proposed for proton transport in tetrameric (a dimer of stable dimers) UCP2, in which all the monomers are functional, and monomers in each dimer are in the same transport mode. Purine nucleotide (e.g., ATP) can occlude the internal pore of the monomeric units of UCP tetramers via interacting with positive residues at or in the proximity of the matrix network (K38, K141, K239, R88, R185, and R279) and prevent switching between cytoplasmic and matrix states, thus inhibiting the proton transport. This study provides new insights into the mechanism of proton transport and regulation in UCPs.

Functional Oligomeric Forms of Uncoupling Protein 2: Strong Evidence for Asymmetry in Protein and Lipid Bilayer Systems.

Stoichiometry of uncoupling proteins (UCPs) and their coexistence as functional monomeric and associated forms in lipid membranes remain intriguing open questions. In this study, tertiary and quaternary structures of UCP2 were analyzed experimentally and through molecular dynamics (MD) simulations. UCP2 was overexpressed in the inner membrane of Escherichia coli, then purified and reconstituted in lipid vesicles. Structure and proton transport function of UCP2 were characterized by circular dichroism (CD) spectroscopy and fluorescence methods. Findings suggest a tetrameric functional form for UCP2. MD simulations conclude that tetrameric UCP2 is a dimer of dimers, is more stable than its monomeric and dimeric forms, is asymmetrical and induces asymmetry in the membrane's lipid structure, and a biphasic on-off switch between the dimeric units is its possible mode of transport. MD simulations also show that the water density inside the UCP2 monomer is asymmetric, with the cytoplasmic side having a higher water density and a wider radius. In contrast, the structurally comparable adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier (AAC1) did not form tetramers, implying that tetramerization cannot be generalized to all mitochondrial carriers.

New coumarin derivatives from Ferula pseudalliacea with antibacterial activity.

In this study, the antibacterial activity of disesquiterpene coumarin and sesquiterpene coumarins obtained from Ferula pseudalliacea roots was evaluated by determination of minimum inhibitory concentration using the broth micro-dilution method against seven pathogenic bacterial strains (Staphylococcus aureus ATCC 25,923, vancomycin resistant clinical strain of Enterococcus faecium, Bacillus cereus PTCC 1015, Escherichia coli ATCC 25,922, Pseudomonas aeruginosa PTCC 1430, clinical strain of Klebsiella pneumoniae and a clinical strain of Helicobacter pylori). The overall inhibitory activities of the compounds were higher against Gram positive tested bacteria. Sanandajin and ethyl galbanate demonstrated significant activity against H. pylori strain, as well as S. aureus strain in concentration of 64 µg/ml. Methyl galbanate inhibited vancomycin resistant strain of E. faecium in concentration of 64 µg/ml. The results of the present investigation indicated that disesquiterpene and sesquiterpene coumarins isolated from F. pseudalliacea root extract can be considered as potent antibacterial agents for pharmaceutical and food industries.