ULK1-dependent phosphorylation of PKM2 antagonizes O-GlcNAcylation and regulates the Warburg effect in breast cancer.

Pyruvate kinase M2 (PKM2) is a central metabolic enzyme driving the Warburg effect in tumor growth. Previous investigations have demonstrated that PKM2 is subject to O-linked ß-N-acetylglucosamine (O-GlcNAc) modification, which is a nutrient-sensitive post-translational modification. Here we found that unc-51 like autophagy activating kinase 1 (ULK1), a glucose-sensitive kinase, interacts with PKM2 and phosphorylates PKM2 at Ser333. Ser333 phosphorylation antagonizes PKM2 O-GlcNAcylation, promotes its tetramer formation and enzymatic activity, and decreases its nuclear localization. As PKM2 is known to have a nuclear role in regulating c-Myc, we also show that PKM2-S333 phosphorylation inhibits c-Myc expression. By downregulating glucose consumption and lactate production, PKM2 pS333 attenuates the Warburg effect. Through mouse xenograft assays, we demonstrate that the phospho-deficient PKM2-S333A mutant promotes tumor growth in vivo. In conclusion, we identified a ULK1-PKM2-c-Myc axis in inhibiting breast cancer, and a glucose-sensitive phosphorylation of PKM2 in modulating the Warburg effect.

Four novel compound heterozygous mutations in C5orf42 gene in patients with pure and mild Joubert syndrome.

Joubert syndrome (JS) is a rare clinically and genetically heterogeneous disease. Using whole or targeted exome sequencing, we identified four novel compound heterozygous mutations in chromosome 5 open reading frame 42 gene (C5orf42), including c.2876C>T (missense mutation) and c.3921+1G>A (splicing mutation), c.2292 -2delA (splicing mutation) and c.4067C>T (missense mutation), c.6997_6998insT (frameshift mutation) and c.8710C>T (nonsense mutation), c.3981G>C (nonsense mutation) and c.230 _233del (frameshift mutation), in four Chinese JS families. They were all inherited from their heterozygosis parents in the autosomal recessive inheritance mode. Pure JS clinical manifestations and mild neuroimaging findings were found in these patients. These verified the previous findings that C5orf42 mutations generally resulted in a purely neurological Joubert phenotype, and neuroimaging findings were mild in JS with C5orf42 mutations. Our report analyzed these C5orf42 mutations-associated phenotypes and neuroimaging findings in JS and updated the genetic variation spectrum of JS caused by C5orf42.These will help clinicians and geneticists reach a more accurate diagnosis for JS.

Centrosomes: Til O-GlcNAc Do Us Apart.

The centrosome apparatus is vital for spindle assembly and chromosome segregation during mitotic divisions. Its replication, disjunction and separation have to be fine-tuned in space and time. A multitude of post-translational modifications (PTMs) have been implicated in centrosome modulation, including phosphorylation, ubiquitination and acetylation. Among them is the emerging O-linked N-acetylglucosamine (O-GlcNAc) modification. This quintessential PTM has a sole writer, O-GlcNAc transferase (OGT), and the only eraser, O-GlcNAcase (OGA). O-GlcNAc couples glucose metabolism with signal transduction and forms a yin-yang relationship with phosphorylation. Evidence from proteomic studies as well as single protein investigations has pinpointed a role of O-GlcNAc in centrosome number and separation, centriole number and distribution, as well as the cilia machinery emanating from the centrosomes. Herein we review our current understanding of the sweet modification embedded in centrosome dynamics and speculate that more molecular details will be unveiled in the future.

[Association of TLR3-1377C/T gene polymorphisms and expression with susceptibility to enterovirus 71 encephalitis in children].

OBJECTIVE: To investigate the association of gene polymorphisms of Toll-like receptor 3 (TLR3)-1377C/T and expression of TLR3 with the susceptibility to enterovirus 71 (EV71) encephalitis in children. METHODS: A total of 187 children with EV71 infection (59 children in the encephalitis group and 128 in the non-encephalitis group) and 232 children who underwent physical examination were enrolled in the case-control study. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the TLR3-1377C/T gene polymorphisms. ELISA was used to measure the serum level of TLR3. RESULTS: There were no significant differences in the genotype and allele frequencies of TLR3-1377C/T between the non-encephalitis group and the encephalitis group. Compared with the control group, the encephalitis group and the non-encephalitis group had significant increases in the serum level of TLR3 (P<0.05), and the non-encephalitis group had the highest level (P<0.05). The encephalitis group had a significantly higher EV71 viral load than the non-encephalitis group (P<0.01). The children aged <1 year or ≥1 year in the encephalitis group and the non-encephalitis group had significant increases in the serum level of TLR3 compared with their counterparts in the control group (P<0.05), and the children aged <1 year or ≥1 year in the non-encephalitis group had a significantly higher serum level of TLR3 than those in the encephalitis group (P<0.05). In the encephalitis group, the children aged ≥1 year had a significantly higher TLR3 concentration than those aged <1 year (P<0.05), and there were no significant differences in the TLR3 concentration between the children aged ≥1 year and <1 year in the non-encephalitis group and the control group. In the encephalitis group, the proportion of children aged <1 year was significantly higher than those aged ≥1 year (P<0.05). CONCLUSIONS: The TLR3-1377C/T gene polymorphisms are not significantly associated with the development of EV71 encephalitis. Low expression of TLR3 might weaken the inhibitory effect on virus replication and promote the development of EV71 encephalitis. The deficiency in the expression of TLR3 in serum after EV71 infection might be an important factor for the development of encephalitis in infants.

Association of interleukin-6-572C/G gene polymorphism and serum or cerebrospinal fluid interleukin-6 level with enterovirus 71 encephalitis in Chinese Han patients with hand, foot, and mouth disease.

Interleukin-6 (IL-6), as one of pro-inflammatory cytokines, plays a key role in Enterovirus 71 (EV71) encephalitis. We investigated the association of IL-6-572C/G polymorphism and serum or cerebrospinal fluid (CSF) IL-6 level with EV71 encephalitis in patients with hand, foot, and mouth disease (HFMD). This study was carried out in 59 Chinese Han patients with EV71 encephalitis, 128 EV71-related HFMD without complications, and 232 controls. The IL-6-572C/G polymorphism was detected by polymerase chain reaction-restricted fragment length polymorphism gene analysis. Serum or CSF IL-6 levels were determined using a commercial enzyme-linked immunosorbent assay. The patients with EV71 encephalitis had a higher frequency of IL-6-572GG/GC genotype compared to the patients with EV71-related HFMD without encephalitis complications (40.7 vs. 15.6 %, odds ratio (OR)=3.70, 95 % confidence interval (CI)=1.83-7.50, p=0.001). Similarly, the frequency of IL-6-572 G allele among the patients with EV71 encephalitis was also higher than that of patients with EV71-related HFMD without encephalitis complications (23.7 vs. 8.6 %, OR=3.31, 95 % CI=1.80-6.08, p<0.001). Serum IL-6 levels in G carries (CG + GG) (195.1 ± 11.8 pg/ml) elevated significantly compared to CC homozygotes (167.7 ± 6.7 pg/ml) in EV71-infected patients (p<0.001), but no significant differences were observed in CSF IL-6 levels among different genotypes in patients with EV71 encephalitis. Furthermore, G carriers (GG + GC) (10.6 ±.29 mg/l) had significantly higher blood CRP levels compared to CC homozygotes (9.31 ± 1.93 mg/l) in patients with EV71 encephalitis (p=0.005). These findings suggested that IL-6-572 G allele was significantly associated with the susceptibility to EV71 encephalitis in Chinese Han patients, and IL-6-572 G allele might elevate the risk to EV71 encephalitis.

[Differences of apoptotic-regulating gene expression in the hippocampus in rats with different ages after status convulsion].

OBJECTIVE: To explore the molecular mechanism of brain protection against convulsive brain damage in premature brains by observing the changes of apoptotic-regulating genes of bcl-2 and c-Jun expression in the hippocampus in Wistar rats with different ages after status convulsion (SC). METHODS: SC was induced in infant Wistar rats (IRs) and adult Wistar rats (ARs) by intraperitoneal injection of lithium-pilocarpine. The rats were sacrificed at 3 hrs, 6 hrs, 12 hrs, 1 day, 3 days and 7 days after SC (n=8). Bcl-2 and c-Jun protein and mRNA levels were measured using immunocytochemistry, RT-PCR and in situ hybridization. RESULTS: c-Jun protein levels increased significantly at 3 hrs and reached the peak at 6 hrs after SC in both IRs and ARs compared to those in the normal control group (P<0.01). c-Jun protein levels started to decrease 12 hrs after SC in both IRs and ARs. The expression of c-Jun protein in IRs returned to the basal level 1 day after SC, while remained higher in ARs than in the normal control group by 7 days after SC. The expression of c-Jun protein in ARs was much higher than that in IRs from 6 hrs to 7 days after SC (P<0.05). c-Jun mRNA level was in parallel with the protein level as mentioned in IRs and ARs after SC. There were no changes observed in both bcl-2 protein and bcl-2 mRNA levels after SC in IRs and ARs. CONCLUSIONS: SC may induce an up-regulation of proapoptotic gene c-Jun in the hippocampus after SC, with a less strong extent and shorter duration in IRs compared to that in ARs. This might be one mechanism of brain protection against convulsive brain damage in IRs. The expression of bcl-2 remains unchanged after SC and is not affected by age in both IRs and ARs.

[Effect of duration of convulsion state on neuronal apoptosis and early apoptotic events in hippocampus of rats].

OBJECTIVE: To explore the influence of duration of convulsion state (SC) on neuronal apoptosis, mitochondrial membrane potential (Deltapsim) and cytochrome C (cyt C) release in hippocampus in Wistar rats after SC. METHODS: SC lasting for 30 minutes or 3 hours was induced by intraperitoneal injection of lithium chloride and pilocarpine. Rats were sacrificed at 3, 6, 12 hours and on 1 day after 30 minutes SC and at 3, 6 hours and on 1 day after 3 hours SC. The apoptosis, mitochondrial Deltapsim and intracellular cyt C were determined with flow cytometry, and the correlation with SC duration was compared. RESULTS: The proportion of apoptotic cells, the decrease in mitochondrial Deltapsim and the release of intracellular cyt C significantly changed at 30 minutes after SC. The peak level of apoptosis was seen at 12 th hour after SC and that of mitochondrial peaked at 6 th hour after SC in apoptosis and the two early apoptotic events, respectively. Compared with the same time point after 30 minutes SC, the levels of apoptosis and the two early apoptotic events after 3 hours SC were much higher. The neuronal apoptosis and the two early apoptotic events in hippocampus after SC showed positive correlation with the duration of SC in partial correlation analysis (all P<0.05). CONCLUSION: Severe seizure could induce the changes in neuronal apoptosis and the early apoptotic events in hippocampus after SC. The longer the duration of SC is, the more serious change in apoptosis and early apoptotic events are.