Lysine residues can be post-translationally modified by various acyl modifications in bacteria and eukarya. Here, we showed that two major acyl modifications, acetylation and succinylation, were changed in response to the carbon source in the Gram-positive model bacterium Bacillus subtilis. Acetylation was more common when the cells were grown on glucose, glycerol, or pyruvate, whereas succinylation was upregulated when the cells were grown on citrate, reflecting the metabolic states that preferentially produce acetyl-CoA and succinyl-CoA, respectively. To identify and quantify changes in acetylation and succinylation in response to the carbon source, we performed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic analysis of cells grown on glucose or citrate. We identified 629 acetylated proteins with 1355 unique acetylation sites and 204 succinylated proteins with 327 unique succinylation sites. Acetylation targeted different metabolic pathways under the two growth conditions: branched-chain amino acid biosynthesis and purine metabolism in glucose and the citrate cycle in citrate. Succinylation preferentially targeted the citrate cycle in citrate. Acetylation and succinylation mostly targeted different lysine residues and showed a preference for different residues surrounding the modification sites, suggesting that the two modifications may depend on different factors such as characteristics of acyl-group donors, molecular environment of the lysine substrate, and/or the modifying enzymes. Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome. Mutations that modulate protein acylation affected B. subtilis growth. A mutation in acetate kinase (ackA) increased the global acetylation level, suggesting that acetyl phosphate-dependent acetylation is common in B. subtilis, just as it is in Escherichia coli. Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.

Macrophages are important participants in neovascularization. This study was designed to examine the role of the monocyte/macrophage chemotactic proteins, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) in a mouse model of oxygen-induced ischemic retinopathy and to determine whether the morphology and distribution of macrophages/microglia are concomitantly altered. The MCP-1, MIP-1alpha mRNA levels increased at 3 h after ischemia. MCP-1, MIP-1alpha, and vascular endothelial growth factor protein levels were also increased markedly and were maximal on days 1, 0.5, and 1, respectively, after ischemia. In situ hybridization showed that MCP-1 and MIP-1alpha were localized in the hypoxic inner retina. Immunostaining demonstrated that the macrophages/microglia in the retina had morphological changes with enlarged processes, and some were closely associated with neovascular tufts at postnatal day 17. Coadministration of the neutralizing antibodies against MCP-1 and MIP-1alpha inhibited retinal neovascularization by 30%. Our data suggest that MCP-1 and MIP-1alpha are involved in the induction of retinal neovascularization and play a role in the inflammation induced by the ischemic retinopathy, possibly by modulating or attracting macrophages/microglia.

1. Changes in membrane capacitance evoked by the rapid photolysis of a caged Ca2+ compound, DM‐nitrophen or nitrophenyl‐EGTA, were investigated in undifferentiated PC12 cells. They were interpreted as representing exocytosis and endocytosis. 2. The Ca2+ jumps evoked two components of exocytosis. Slow exocytosis was selectively evoked with small increases in intracellular Ca2+ concentration between 5 and 10 microM, while fast exocytosis preceded the slow one at [Ca2+]i greater than 10 microM. 3. The release rates of the two components of exocytosis depended steeply on [Ca2+]i. A half‐maximal release rate was achieved at 8 and 24 microM for the slow and fast exocytoses, respectively. 4. Prior Ca2+ rises did not augment the fast exocytosis. 5. The fast exocytosis was often followed by a rapid decrease in membrane capacitance, representing endocytosis, after a delay of 0.5‐2 s. The speed and delay in the fast endocytosis were Ca2+ dependent. Amounts of the fast endocytosis tended to balance with those of the fast exocytosis evoked by the same Ca2+ jumps. 6. The slow exocytosis was followed by a sluggish endocytosis that was associated with large capacitance steps indicative of secretory processes involving large dense‐core vesicles. The onset of the slow endocytosis exhibited a complex Ca2+ dependence. The amounts of the slow endocytosis appeared to parallel those of the slow exocytosis. Prior induction of the slow exocytosis gave rise to selective excess retrieval of membrane during the slow endocytosis. 7. These data indicate the existence of two distinct populations of secretory vesicles in PC12 cells. They seem to couple selectively with specific endocytotic mechanisms. Our data suggest that the two vesicles belong to two distinct secretory pathways.

Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine. Six of the resulting N-acetyl-l-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive toward l-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicumΔargR led to the accumulation of 34 mM l-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM l-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM l-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction.

The diterpene cyclase CotB2 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to the tricyclic cyclooctat-9-en-7-ol, which is characterized by a 5-8-5-fused ring skeleton. We have previously proposed a cyclization cascade involving a unique carbon-carbon bond rearrangement combined with multiple hydride shifts, all occurring at a single active site. Here, we report the first high-resolution X-ray crystal structure of CotB2 with bound substrate analog geranylgeranyl thiodiphosphate (GGSPP). In the GGSPP-bound form, GGSPP folds into a unique S-shaped conformation that probably reflects the substrate-bound state prior to ionization of the substrate GGPP. The folded framework of GGSPP is surrounded by hydrophobic residues and several aromatic and asparagine residues that are well-positioned to stabilize a series of reactive carbocation intermediates through a combination of cation-π and dipole charge interactions. The combined crystal structures and mutagenesis-based biochemical assays provide a structural basis for exquisite control of ring formation and stereochemistry during CotB2 catalysis.

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The results indicate that TNF-alpha is produced by activated macrophages/microglia and may participate in retinal neovascularization during post-ischemic inflammation through the induction of potent angiogenic factors in an autocrine or paracrine manner.

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