Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein–associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Unbiased proteomics with acyl resin-assisted capture reveals diverse novel substrates of the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) at the synapse, with potential implications for the pathogenesis of neuronal ceroid lipofuscinosis, disulfide bond formation, synaptic adhesion and additional critical synaptic functions.     

Purification and characteristics of cytosolic chitinase from Piromyces communis OTS1

Abstract A chitinase was purified from the cytosolic fraction of the anaerobic rumen fungus Piromyces communis OTS1 by affinity chromatography using regenerated chitin, gel filtration and chromatofocusing. The chitinase was most active at pH 6.2 and at 60 °C in a 20-min assay. The molecular mass of the purified protein was estimated by SDS-PAGE to be 42 kDa and its pI was 4.9. The enzyme activity, which was of the 'endo' type, was inhibited by A⁺, Hg²⁺ and allosamidin. N -Acetyl- β -glucosaminidase and 'exo' type chitinase activity were absent from the purified preparation.     

Powdery mildew (Sphaerotheca fuliginea) resistance in melon is selectable at the haploid level

The major cause of powdery mildew in melons (Cucumis melo L.) is the fungus Sphaerotheca fuliginea. There are several cultivar- and season-specific races of this fungus. In order to control powdery mildew, it is important to introduce resistance to fungal infection into new cultivars during melon breeding. Haploid breeding is a powerful tool for the production of pure lines. In this study, it was investigated whether powdery mildew resistance could be manifested at the haploid level from two disease-resistant melon lines, PMR 45 and WMR 29. the effects of various races of S. fuliginea on diploid and haploid plants of PMR 45 and WMR 29 and of a disease-susceptible line, Fuyu 3 were measured. The responses of haploid and diploid plants to powdery mildew were identical. In addition, haploids that were generated from hybrids between Fuyu 3 and disease-resistant lines were examined. Seven out of 13 haploids from a Fuyu 3xPMR 45 cross and 10 out of 12 haploids from a Fuyu 3xWMR 29 cross were classified as resistant plants because they showed the same responses as their disease-resistant diploid parents to the various fungal races. These results indicate that resistance in PMR 45 and WMR 29 is selectable at the haploid level. All of the plant responses were observed by microscopy. A possible mechanism for generating powdery mildew resistance in two different melon lines is discussed.     

Crystal and molecular structure of 6,2'-anhydro-1-beta-D-arabinofuranosylctyosine.

The crystal and molecular structure of the title compound has been determined by X-ray diffraction method. The compound crystallizes in monoclinic system with the space group P2₁ and Z=2; the unit cell dimensions are a=10.491, b=7.255, c=6.858 A and β=103.55°. The structure was refined to an R-index of 0.051. The glycosyl torsion angle X_CN is 111.4° (syn-anti) and the arabinose ring forms an exo-conformation, in which C(4′) is displaced by 0.61 A out of the plane of remaining four atoms. The orientation of the C(5′)O(5′) bond is the gauche-gauche as similar as that found frequently in many nucleosides.     

Detection of 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein Cm-HMGR during fruit development in melon ( Cucumis melo L.)

The fruit size of melon (Cucumis melo L. reticulatus) is determined by the amount of cell proliferation in the pericarp during early fruit development. During this stage, expression and activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene is required for fruit growth. In this study, we performed a detailed analysis of the correlation between the expression of melon HMGR (Cm-HMGR) protein and cell division in the pericarp. Flow cytometric analysis revealed that the length of the cell division stage was correlated with the fruit size. Western gel blotting and tissue printing illustrated the temporal and spatial accumulation pattern of Cm-HMGR protein during fruit development. The accumulation of Cm-HMGR transiently increased at the beginning of the cell division stage in the pericarp, where active cell division occurred. The amount of Cm-HMGR was correlated with the length of the cell division period. These results strongly suggest that the expression of Cm-HMGR is involved in the determination of melon fruit size by regulating cell division during early fruit development.     

H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate

A mutant of Corynebacterim glutamicum ('Brevibacterium flayum') ATCC14067 with a reduced H+-ATPase activity, F172-8, was obtained as a spontaneous neomycin-resistant mutant. The ATPase activity of strain F172-8 was reduced to about 25% of that of the parental strain. Strain F172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor. It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent. The respiration rate per cell of the mutant also increased to twice as much as that of the parent. However, the growth rate of the mutant was lower than that of the parent. Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid. Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose. Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant. On the other hand, the parent accumulated alpha-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products. It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172-8, because a revertant of strain F172-8, R2-1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent. Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172-8.     

Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart

Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined.     

Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer

The Schizosaccharomyces pombe Ku70–Ku80 heterodimer is required for telomere length regulation. Lack of pku70⁺ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80⁺ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70⁺ increased the single-stranded overhang in both G₂ and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.     

Aligning LC peaks by converting gradient retention times to retention index of peptides in proteomic experiments

MOTIVATION: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool in proteomics studies, but when peptide retention information is used for identification purposes, it remains challenging to compare multiple LC-MS/MS runs or to match observed and predicted retention times, because small changes of LC conditions unavoidably lead to variability in retention times. In addition, non-contiguous retention data obtained with different LC-MS instruments or in different laboratories must be aligned to confirm and utilize rapidly accumulating published proteomics data. RESULTS: We have developed a new alignment method for peptide retention times based on linear solvent strength (LSS) theory. We found that log k(0) (logarithm of retention factor for a given organic solvent) in the LSS theory can be utilized as a 'universal' retention index of peptides (RIP) that is independent of LC gradients, and depends solely on the constituents of the mobile phase and the stationary phases. We introduced a machine learning-based scheme to optimize the conversion function of gradient retention times (t(g)) to log k(0). Using the optimized function, t(g) values obtained with different LC-MS systems can be directly compared with each other on the RIP scale. In an examination of Arabidopsis proteomic data, the vast majority of retention time variability was removed, and five datasets obtained with various LC-MS systems were successfully aligned on the RIP scale.