SUMMARY IDH1 and IDH2 mutations occur frequently in gliomas and acute myeloid leukemia, leading to simultaneous loss and gain of activities in the production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG), respectively. Here we demonstrate that 2-HG is a competitive inhibitor of multiple α-KG-dependent dioxygenases, including histone demethylases and the TET family of 5-methlycytosine (5mC) hydroxylases. 2-HG occupies the same space as α-KG does in the active site of histone demethylases. Ectopic expression of tumor-derived IDH1 and IDH2 mutants inhibits histone demethylation and 5mC hydroxylation. In glioma, IDH1 mutations are associated with increased histone methylation and decreased 5-hydroxylmethylcytosine (5hmC). Hence, tumor-derived IDH1 and IDH2 mutations reduce α-KG and accumulate an α-KG antagonist, 2-HG, leading to genome-wide histone and DNA methylation alterations.

SUMMARY Tumor cells commonly have increased glucose uptake and lactate accumulation. Lactate is produced from pyruvate by lactate dehydrogenase A (LDH-A), which is frequently overexpressed in tumor cells and is important for cell growth. Elevated transcription by c-Myc or HIF1α may contribute to increased LDH-A in some cancer types. Here, we show that LDH-A is acetylated at lysine 5 (K5) and that this acetylation inhibits LDH-A activity. Furthermore, the K5-acetylated LDH-A is recognized by the HSC70 chaperone and delivered to lysosomes for degradation. Replacement of endogenous LDH-A with an acetylation mimetic mutant decreases cell proliferation and migration. Importantly, K5 acetylation of LDH-A is reduced in human pancreatic cancers. Our study reveals a mechanism of LDH-A upregulation in pancreatic cancers.

Posttranslational modifications (PTMs) of proteins are responsible for sensing and transducing signals to regulate various cellular functions and signaling events. S-nitrosylation (SNO) is one of the most important and universal PTMs. With the avalanche of protein sequences generated in the post-genomic age, it is highly desired to develop computational methods for timely identifying the exact SNO sites in proteins because this kind of information is very useful for both basic research and drug development. Here, a new predictor, called iSNO-PseAAC, was developed for identifying the SNO sites in proteins by incorporating the position-specific amino acid propensity (PSAAP) into the general form of pseudo amino acid composition (PseAAC). The predictor was implemented using the conditional random field (CRF) algorithm. As a demonstration, a benchmark dataset was constructed that contains 731 SNO sites and 810 non-SNO sites. To reduce the homology bias, none of these sites were derived from the proteins that had pairwise sequence identity to any other. It was observed that the overall cross-validation success rate achieved by iSNO-PseAAC in identifying nitrosylated proteins on an independent dataset was over 90%, indicating that the new predictor is quite promising. Furthermore, a user-friendly web-server for iSNO-PseAAC was established at http://app.aporc.org/iSNO-PseAAC/, by which users can easily obtain the desired results without the need to follow the mathematical equations involved during the process of developing the prediction method. It is anticipated that iSNO-PseAAC may become a useful high throughput tool for identifying the SNO sites, or at the very least play a complementary role to the existing methods in this area.

Directional motility is a fundamental function of immune cells, which are recruited to sites of pathogen invasion or tissue damage by chemoattractant signals. To move, cells need to generate lamellipodial membrane protrusions at the front and retract the trailing end. These elementary events are initiated by Rho-family GTPases, which cycle between active GTP-bound and inactive GDP-bound states. How the activity of these “molecular switches” is spatially coordinated is only beginning to be understood. Here, we show that myosin IXb (Myo9b), a Rho GTPase-activating protein (RhoGAP) expressed in immune cells, is essential for coordinating the activity of Rho. We generated Myo9b-deficient mice and show that Myo9b −/− macrophages have strikingly defective spreading and polarization. Furthermore, Myo9b −/− macrophages fail to generate lamellipodia in response to a chemoattractant, and migration in a chemotactic gradient is severely impaired. Inhibition of Rho rescues the Myo9b −/− phenotype, but impairs tail retraction. We also found that Myo9b is important in vivo. Chemoattractant-induced monocyte recruitment to the peritoneal cavity is substantially reduced in Myo9b −/− mice. Thus, we identify the “motorized Rho inhibitor” Myo9b as a key molecular component required for spatially coordinated cell shape changes and motility.

BackgroundNeonatal hypoxic-ischemic encephalopathy (HIE) is an important cause of motor and cognitive impairment in children. Clinically, male infants are more vulnerable to ischemic insults and suffer more long-term deficits than females; however, the mechanisms underlying this sex difference remain elusive. Inflammatory processes initiated by microglial activation are fundamental in the pathophysiology of ischemia. Recent studies report a sexual dimorphism in microglia numbers and expression of activation markers in the neonatal brain under normal conditions. How these basal sex differences in microglia affect HIE remains largely unexplored. This study investigated sex differences in ischemic outcomes and inflammation triggered by HIE. We hypothesize that ischemia induces sex-specific brain injury in male and female neonates and that microglial activation and inflammatory responses play an important role in this sexual dimorphism.MethodsMale and female C57BL6 mice were subjected to 60-min Rice-Vanucci modeling (RVM) at post-natal day 10 (P10) to induce HIE. Stroke outcomes were measured 1, 3, 7, and 30 days after stroke. Microglial activation and inflammatory responses were evaluated by flow cytometry and cytokine analysis.ResultsOn day 1 of HIE, no difference in infarct volumes or seizure scores was seen between male and female neonates. However, female neonates exhibited significantly smaller infarct size and fewer seizures compared to males 3 days after HIE. Females also had less brain tissue loss and behavioral deficits compared to males at the chronic stage of HIE. Male animals demonstrated increased microglial activation and up-regulated inflammatory response compared to females at day 3.ConclusionsHIE leads to an equivalent primary brain injury in male and female neonates at the acute stage that develops into sexually dimorphic outcomes at later time points. An innate immune response secondary to the primary injury may contribute to sexual dimorphism in HIE.

As one of the most important and universal posttranslational modifications (PTMs) of proteins, S-nitrosylation (SNO) plays crucial roles in a variety of biological processes, including the regulation of cellular dynamics and many signaling events. Knowledge of SNO sites in proteins is very useful for drug development and basic research as well. Unfortunately, it is both time-consuming and costly to determine the SNO sites purely based on biological experiments. Facing the explosive protein sequence data generated in the post-genomic era, we are challenged to develop automated vehicles for timely and effectively determining the SNO sites for uncharacterized proteins. To address the challenge, a new predictor called iSNO-AAPair was developed by taking into account the coupling effects for all the pairs formed by the nearest residues and the pairs by the next nearest residues along protein chains. The cross-validation results on a state-of-the-art benchmark have shown that the new predictor outperformed the existing predictors. The same was true when tested by the independent proteins whose experimental SNO sites were known. A user-friendly web-server for iSNO-AAPair was established at , by which users can easily obtain their desired results without the need to follow the mathematical equations involved during its development.

Aging is a non modifiable risk factor for stroke. Since not all strokes can be prevented, a major emerging area of research is the development of effective strategies to enhance functional recovery after stroke. However, in the vast majority of pre-clinical stroke studies, the behavioral tests used to assess functional recovery have only been validated for use in young animals, or are designed for rats. Mice are increasingly utilized in stroke models but well validated behavioral tests designed for rats are not necessarily reproducible in mice. We examined a battery of behavioral tests to evaluate functional recovery in an aging murine model of stroke. We found that the vertical pole, hanging wire and open field can accurately assess acute behavioral impairments after stroke in both young and aging male mice, but animals recover rapidly on these tasks. The corner test can accurately and repeatedly differentiate stroke from sham animals up to 30 days post stroke and can be performed reliably in aging mice. Aging male mice had significantly worse behavioral impairment compared to young male mice in the first two weeks after stroke but eventually recovered to the same degree as young mice. In contrast, chronic infarct size, as measured by ipsilateral cerebral atrophy, was significantly lower in aging male mice compared to young male mice. Reactive gliosis, formation of glial scar, and an enhanced innate immune response was seen in the aging brain and may contribute to the delayed behavioral recovery seen in aged animals.

Stroke survivors often experience social isolation. Social interaction improves quality of life and decreases mortality after stroke. Male mice (20–25 g; C57BL/6N), all initially pair housed, were subjected to middle cerebral artery occlusion (MCAO). Mice were subsequently assigned into one of three housing conditions: (1) Isolated (SI); (2) Paired with their original cage mate who was also subjected to stroke (stroke partner (PH-SP)); or (3) Paired with their original cage mate who underwent sham surgery (healthy partner (PH-HP)). Infarct analysis was performed 72 h after stroke and chronic survival was assessed at day 30. Immediate post-stroke isolation led to a significant increase in infarct size and mortality. Interestingly, mice paired with a healthy partner had significantly lower mortality than mice paired with a stroke partner, despite equivalent infarct damage. To control for changes in infarct size induced by immediate post-stroke isolation, additional cohorts were assessed that remained pair housed for three days after stroke prior to randomization. Levels of brain-derived neurotrophic factor (BDNF) were assessed at 90 days and cell proliferation (in cohorts injected with 5-bromo-2′-deoxyuridine, BrdU) was evaluated at 8 and 90 days after stroke. All mice in the delayed housing protocol had equivalent infarct volumes (SI, PH-HP and PH-SP). Mice paired with a healthy partner showed enhanced behavioral recovery compared with either isolated mice or mice paired with a stroke partner. Behavioral improvements paralleled changes in BDNF levels and neurogenesis. These findings suggest that the social environment has an important role in recovery after ischemic brain injury.