Proton gradient-driven import of the 16 kDa oxygen-evolving complex protein as the full precursor protein by isolated thylakoids

The biogenesis of the lumenal 16 kDa protein of the photosynthetic oxygen-evolving complex was analysed using an assay for the import of proteins by isolated thylakoids. The precursor protein is imported with high efficiency in the light in both the presence and absence of stromal extract. Import is almost completely blocked in the dark or if the uncoupler nigericin is present in the light. The data indicate that transport across the thylakoid membrane is driven by a proton motive force in which the proton gradient is the dominant component, and that the full precursor protein can be transported across the thylakoid membrane without prior cleavage by the stromal processing peptidase.     

Building Better Barrels – β-barrel Biogenesis and Insertion in Bacteria and Mitochondria

β-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial β-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial β-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.     

Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-Dependent Mitophagy

Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18 h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and “outside–in” directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.     

Novel protein science enabled by total chemical synthesis

Chemical synthesis is a well‐established method for the preparation in the research laboratory of multiple‐tens‐of‐milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d ‐protein and l ‐protein enantiomers facilitate crystallization and determination of protein structures by X‐ray diffraction. d ‐Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d ‐protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear‐loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time‐resolved FTIR for the elucidation of protein structure–activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein‐inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d ‐protein forms of polymerase enzymes.     

Spinal and peripheral modulation of gastric acid secretion and arterial pressure by neuropeptide Y, peptide YY, and pancreatic polypeptide in rats

Spinal and peripheral modulation of pentagastrin-stimulated gastric acid secretion by the pancreatic polypeptide-fold (PP-fold) peptides, neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), in urethane-anesthetized rats was evaluated. Neuropeptide Y, PYY, and PP (400 pmol) were administered via intravenous (IV) and intrathecal (IT) injections. The ₂ antagonist, yohimbine, was used to evaluate the role of the ₂ adrenergic receptors in the modulation of pentagastrin-stimulated gastric acid secretion by NPY, PYY, and PP. Peptide YY and PP (IV) rapidly increased pentagastrin-stimulated gastric acid secretion. Peptide YY and PP (IT) increased pentagastrin-stimulated gastric acid secretion following administration into the thoracic (T8–T10) region of the spinal cord. The ₂ adrenergic receptor antagonist, yohimbine, did not modify the increases in pentagastrin-stimulated gastric acid secretion following PYY and PP (IV or IT) administration. Neuropeptide Y (IT) decreased pentagastrin-stimulated gastric acid secretion. However, in the presence of ₂ adrenergic receptor blockade, pentagastrin-stimulated gastric acid secretion was potentiated by NPY (IT) administration. Therefore, the inhibitory effect of NPY (IT) on pentagastrin-stimulated gastric acid secretion required the activation of ₂ adrenergic receptors in the spinal cord of rats. Mean arterial blood pressure (MAP) was increased immediately following NPY and PYY (IV) administration. During the same time period, PP (IV) decreased MAP in anesthetized rats. Mean arterial blood pressure was rapidly increased by NPY and PYY (IT) in anesthetized rats. The increase in MAP following PYY (IT) was partially attenuated in the presence of yohimbine. The modulation of MAP and gastric acid secretion by the PP-fold peptides occurred by independent mechanisms at spinal and peripheral sites in the rat. The modulation of pentagastrin-stimulated gastric acid secretion by PYY and PP in rats differed from that of the third member of the PP-fold family, NPY, following spinal and peripheral administration.     

Confocal laser scanning and electron-microscopic analyses of the relationship between VIP-like and GnRH-like-immunoreactive neurons in the lateral septal-preoptic area of the pigeon

The lateral septum and the preoptic area of birds comprise neurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH). By use of immunohistochemical single- and double-labeling techniques, we have investigated the distribution and the connections of these two types of peptidergic neurons in the lateral septal-preoptic area of the pigeon at both the light- and electron-microscopic levels. An accumulation of VIP-like-ir neurons, some of which are cerebrospinal fluid-contacting neurons, is found in the area adjacent to the ventromedial walls of the lateral ventricles in the lateral septum corresponding to the medial part of the lateral septal organ. VIP-like-ir terminals are scattered throughout the lateral septal-preoptic area, which also contains GnRH-like-ir cell bodies. The number of GnRH-like-ir cell bodies in the lateral septum is smaller than that of the VIP-like-ir neurons. GnRH-like-ir cells have a simple bipolar or multipolar shape and a beaded axon that emerges from the soma or one of the proximal dendrites. Confocal laser scanning microscopy has shown VIP-like-ir terminals in close apposition to GnRH-like-ir cell bodies in the lateral septal-preoptic area. Furthermore, the electron-microscopic double-immunolabeling has revealed synaptic contacts between VIP-like-ir axon terminals and GnRH-like-ir cell bodies or dendrites. These contacts, however, do not show synaptic specializations. The present results suggest that functional interactions take place between VIP and GnRH neurons in the lateral septal-preoptic area of the pigeon and that these interactions are involved in mediating photoperiodic responses. Received: 14 November 1997 / Accepted: 19 December 1997     

Determinants of sweetness in proteins: a topological approach

Sweet taste in mammals is accounted for by a single receptor that shares homology with a metabotropic glutamate receptor. Most sweeteners are small molecular weight molecules that interact with small cavities in the so-called Venus Flytrap domains of the sweet receptor. The mechanism of action of larger molecules such as sweet proteins is, however, more difficult to interpret. The first and still the only general mechanism proposed for the action of sweet proteins, the "wedge model," hypothesizes that proteins bind to an external binding site of the active conformation of the sweet receptor. Here, I have extended the concept that inspired the wedge model using a combination of structural analysis, bioinformatics tools, and a relatively large dataset of mutations of the two most extensively studied sweet proteins, monellin and brazzein. I show here that it is possible to single out, among the ensemble yielded by low-resolution docking, a unique complex that satisfies simple topological constraints. These models of the complexes of monellin and brazzein are fully consistent with experimental evidence, thus providing predicting power for further validation of the wedge model.     

Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas L.)

Three hundred and ninety‐four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co‐infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co‐infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3‐expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.