Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum

The glyoxalase system catalyzes the conversion of toxic, metabolically produced α-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn(2+) and non-Zn(2+) activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni(2+)/Co(2+)-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn(2+)-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.     

Structural genomics of proteins from conserved biochemical pathways and processes

During the past year, X-ray crystallographers and solution NMR spectroscopists have made significant progress towards the complete structural characterization of conserved biochemical pathways and processes. Some of these advances were made in the context of nascent structural genomics programs, which promise to accelerate structural studies of biologically and medically important proteins. The results of high-throughput protein production, crystallization, structure determination, homology modeling and functional annotation published by two such programs have provided insight into the evolution and function of enzymes in the isoprenoid biosynthesis and ribulose monophosphate pathways.     

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution,new validation tools,and implications for the future

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.     

Contact Hypersensitivity to Oxazolone Provokes Vulvar Mechanical Hyperalgesia in Mice

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition.     

Mate choice decision rules: Trait synergisms and preference shifts

An important and understudied question in sexual selection is how females evaluate information from multiple secondary sexual traits (SSTs), particularly when expression of traits is phenotypically uncorrelated. We performed mate choice experiments on zebra finches (Taeniopygia guttata castanotis Gould) to evaluate two hypotheses: preference shifts (obstacles to choice using one trait increase chooser reliance on others) and trait synergisms (choice based on the sum/product of two or more independently varying traits). The first experiment, which employed males raised on diets that impact SST expression, supported the trait synergism hypothesis: overall, male pairing success was best predicted by synergisms involving beak color and cheek patch size. Results did not support the preference shift hypothesis. Results of a follow‐up experiment that included males reared on a single diet, and in which male beak color and cheek patch size were manipulated, were also consistent with the trait synergism hypothesis. Results have implications for understanding the long‐term persistence of multiple SSTs in populations and for the measurement of repeatability and heritability of mate preferences.     

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs II. The dimer-tetramer transition of apovitellenin I.

Further studies have been made of the physical properties of hen's apovitellenin I, the principal low-molecular-weight protein from the high-lipid low density lipoprotein of the yolk of hen's eggs. The methods used included chromatography, sedimentation, viscosity, optical rotation, and spin labelling; the solvents used were aqueous urea, and, for some experiments, aqueous formamide. It is concluded that a neutral pH the protein is present in these solvents as an aggregate of molecular weight 36000 corresponding to a tetramer. Below about pH 4-5 solutions of the tetramer increased greatly in viscosity; furthermore, a covalently bound spin label increased in mobility. These changes were reversible and were apparently the result of dissociation of the tetramer to a dimer. This disociation did not involve a change in the proportion of alpha-helix. In contrast to the results of previous experiments, it now seems probably that the apovitellenin I dimer is stabilized by an interchain disulphide bond.     

Regulatory effects of neurotransmitters on electroshock and pentylenetetrazol seizures

Evidence that biogenic amines and a neuroactive purine are involved in pathophysiological mechanisms of experimental seizures produced by electroshock or pentylenetetrazol is reviewed. Diminishing the influence of norepinephrine (NE) in the brain by depleting its content or by blockade of beta adrenoreceptors enhances seizure activity, and increasing its influence may suppress seizures. Serotonin appears to have action similar to that of NE, but dopamine has little effect in these seizure models. Adenosine also appears to suppress seizure activity. Thus NE, serotonin, and adenosine may be natural, endogenous substances that inhibit or confine seizure activity in the brain.