JMJD5 cleaves monomethylated histone H3 N-tail under DNA damaging stress.

The histone H3 N-terminal protein domain (N-tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N-tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N-tail at the carboxyl side of monomethyl-lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl-lysine (Kme2), trimethyl-lysine (Kme3), or unmethyl-lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N-tail cleavage in gene expression regulation.

Brain-selective kinase 2 (BRSK2) phosphorylation on PCTAIRE1 negatively regulates glucose-stimulated insulin secretion in pancreatic ß-cells.

Brain-selective kinase 2 (BRSK2) has been shown to play an essential role in neuronal polarization. In the present study, we show that BRSK2 is also abundantly expressed in pancreatic islets and MIN6 ß-cell line. Yeast two-hybrid screening, GST fusion protein pull-down, and co-immunoprecipitation assays reveal that BRSK2 interacts with CDK-related protein kinase PCTAIRE1, a kinase involved in neurite outgrowth and neurotransmitter release. In MIN6 cells, BRSK2 co-localizes with PCTAIRE1 in the cytoplasm and phosphorylates one of its serine residues, Ser-12. Phosphorylation of PCTAIRE1 by BRSK2 reduces glucose-stimulated insulin secretion (GSIS) in MIN6 cells. Conversely, knockdown of BRSK2 by siRNA increases serum insulin levels in mice. Our results reveal a novel function of BRSK2 in the regulation of GSIS in ß-cells via a PCTAIRE1-dependent mechanism and suggest that BRSK2 is an attractive target for developing novel diabetic drugs.

Value of magnetic resonance imaging and sialography of the parotid gland for diagnosis of primary Sjögren syndrome.

AIM: To evaluate the utility of magnetic resonance imaging (MRI) and magnetic resonance sialography (MRS) for diagnosis of primary Sjögren syndrome (pSS) singly or integrated with 2016 American College of Rheumatology (ACR)/European League Against Rheumatic Diseases (EULAR) classification criteria. METHODS: The diagnostic efficiencies of MRI, MRS, and labial salivary gland biopsy (LSGB) were evaluated. The prediction model was established by multivariate analysis. Finally, performance of the ACR/EULAR criteria was evaluated after addition of MRI + MRS or replacement of original items by MRI + MRS. RESULTS: The combined use of LSGB + MRI + MRS provided the greatest diagnostic value. MRI and MRS grade had positive correlations with disease duration and pathological grade of the labial gland (both P < 0.001). MRI and MRS grade had positive correlations with xerostomia severity and negative correlations with unstimulated salivary flow rate (both P < 0.001). The consistency of MRI grade and MRS grade in the diagnosis of parotid gland lesions was poor (κ = 0.253, P < 0.001). The diagnostic efficiency of our prediction model (AUC 0.906) was similar to that of criteria from the ACR/EULAR (AUC 0.930). Adding MRI + MRS to the ACR/EULAR criteria improved the sensitivity (92.3% vs 90.8%), whereas the specificity remained the same (88.9% vs 89.1%). Replacing LSGB by MRI + MRS in the ACR/EULAR criteria decreased both sensitivity and specificity (88.1% vs 90.8% and 86.4% vs 89.1%, respectively). CONCLUSION: The combined application of MRI and MRS has ideal clinical application value in the diagnosis of early-stage pSS. Validity of the ACR/EULAR criteria remains high after incorporation of MRI + MRS.

Jab1 interacts with brain-specific kinase 2 (BRSK2) and promotes its degradation in the ubiquitin-proteasome pathway.

Brain-specific kinase 2 (BRSK2) was classified as an AMP-activated protein kinase (AMPK)-related kinase and one of the substrates of LKB1. Studies on homologs of BRSK2 in mice, SADA and SADB, implied that it might be involved in the regulation of cell polarity and cell cycle. However, physiological functions and molecular regulatory mechanisms of BRSK2 are incompletely understood. In this study, we isolated a novel BRSK2-interacting protein, c-Jun activation domain-binding protein-1 (Jab1), which was reported to mediate degradation of multiple proteins and positively regulate cell cycle progression. GST pull-down and immunoprecipitation assays revealed the direct interaction between BRSK2 and Jab1 in vitro and in vivo, respectively. The co-localization between Jab1 and BRSK2 in the perinuclear region was observed. Intriguingly, Jab1 promoted the ubiquitination and proteasome-dependent degradation of BRSK2. Silencing of endogenous Jab1 increased the cellular BRSK2 protein level. Consistent with this, BRSK2-mediated cell cycle arrest at the G2/M phase in mammalian cells was reversed by exogenous Jab1. Taken together, our findings provide a novel regulatory mechanism of BRSK2 through direct interaction with Jab1.

Neurotransmitter inactivation is important for the origin of nerve system in animal early evolution: a suggestion from genomic comparison.

Metazoans possess complicated multicellular structure among the multicellularities of eukaryotes. One evolutionary pressure that permits such complexity relates to the directed and precise informational transmission performed by numerous synapses in neuron system. Neurotransmitter inactivations play essential roles in the termination of synaptic transmission and are thus crucial for precise synaptic transmission. Here, we performed a genomic comparison among 11 eukaryotic organisms including five bilaterian species and six pan-unicellular eukaryotes to search for genes related to metazoan multicellular function. The result showed that the majority of genes related to neurotransmitter inactivation in the synaptic cleft endured high and stable selective pressure and were specifically present in bilaterians, whereas genes related to transmitter release and postsynaptic transmitter receptors did not show these properties. From these data we conclude that neurotransmitter inaction may play a critical role in the origin of the nerve system encountered in the early evolution of metazoan. In addition, we suggest that neurotransmitter inactivation probably participates in the formation or refinement of the synapse, following the concept of "ontogeny recapitulates phylogeny." Further experimental evidence is needed to support the suggestion and to explain the importance of neurotransmitter inactivation to metazoan multicellular function.

Identification of a novel human lysophosphatidic acid acyltransferase, LPAAT-theta, which activates mTOR pathway.

Lysophosphatidic acid acyltransferase (LPAAT) is an intrinsic membrane protein that catalyzes the synthesis of phosphatidic acid (PA) from lysophosphatidic acid (LPA). It is well known that LPAAT is involved in lipid biosynthesis, while its role in tumour progression has been of emerging interest in the last few years. To date, seven members of the LPAAT gene family have been found in human. Here we report a novel LPAAT member, designated as LPAAT-theta, which was 2728 base pairs in length and contained an open reading frame (ORF) encoding 434 amino acids. The LPAAT-theta gene consisted of 12 exons and 11 introns, and mapped to chromosome 4q21.23. LPAAT-theta was ubiquitously expressed in 18 human tissues by RT-PCR analysis. Subcellular localization of LPAAT-theta-EGFP fusion protein revealed that LPAAT-theta was distributed primarily in the endoplasmic reticulum (ER) of COS-7 cells. Furthermore, we found that the overexpression of LPAAT-theta can induce mTOR-dependent p70S6K phosphorylation on Thr389 and 4EBP1 phosphorylation on Ser65 in HEK293T cells.

APC/C(Cdh1) targets brain-specific kinase 2 (BRSK2) for degradation via the ubiquitin-proteasome pathway.

Studies of brain-specific kinase 2 (BRSK2), an AMP-activated protein kinase (AMPK)-related kinase, and its homologs suggest that they are multifunctional regulators of cell-cycle progression. BRSK2, which contains a ubiquitin-associated (UBA) domain, is polyubiquitinated in cells. However, the regulatory mechanisms and exact biological function of BRSK2 remain unclear. Herein, we show that BRSK2 co-localizes with the centrosomes during mitosis. We also demonstrate that BRSK2 protein levels fluctuate during the cell cycle, peaking during mitosis and declining in G1 phase. Furthermore, Cdh1, rather than Cdc20, promotes the degradation of BRSK2 in vivo. Consistent with this finding, knock-down of endogenous Cdh1 blocks BRSK2 degradation during the G1 phase. The conserved KEN box of BRSK2 is required for anaphase-promoting complex/cyclosome-Cdh1 (APC/C(Cdh1))-dependent degradation. Additionally, overexpression of either BRSK2(WT) or BRSK2(ΔKEN) increases the percentage of cells in G2/M. Thus, our results provide the first evidence that BRSK2 regulates cell-cycle progression controlled by APC/C(Cdh1) through the ubiquitin-proteasome pathway.

Identification of a novel human lactate dehydrogenase gene LDHAL6A, which activates transcriptional activities of AP1(PMA).

L-lactate dehydrogenase is a crucial enzyme in the process of glycolysis. Here we report the cloning and characterization of another novel lactate dehydrogenase gene, named as LDHAL6A (lactate dehydrogenase A-like 6A), which encodes a 332-amino-acid protein. The LDHAL6A gene consists of seven exons, and is mapped to 11p15.1 by searching the UCSC genomic database. By RT-PCR analysis in various tissues, LDHAL6A was found to be exclusively expressed in human testis. Subcellular localization demonstrated that LDHAL6A protein was located in the cytoplasm when overexpressed in COS7 cells. Furthermore, we found that the recombinant protein GST-LDHAL6A can catalyze the pyruvate convert into the lactate with NADH as its coenzyme. And in the dual luciferase reporter system, expression of LDHAL6A was able to activate transcriptional activities of AP1(PMA).

Cloning and characterization of the human gene RAP2C, a novel member of Ras family, which activates transcriptional activities of SRE.

The Ras family regulates a wide variety of cellular functions that include cell growth, differentiation, and apoptosis. In this study, we identified a novel human gene named RAP2C, isolated from human testis cDNA library, and mapped to Xq26.2 by searching the UCSC genomic database. The RAP2C cDNA contains an open reading frame of 552 bp, encoding a putative protein of 183 amino acid residues. The predicted protein contains a RAS domain. By RT-PCR analysis in various tissues, RAP2C was found to be principally expressed in the liver, skeletal muscle, prostate, uterus, rectum, stomach, and bladder and to a less extent in brain, kidney, pancreas, and bone marrow. RAP2C protein was located in cytoplasm when overexpressed in COS-7 cells. Reporter gene assays showed that overexpression of RAP2C in HEK293T cells activated the transcriptional activities of serum response element (SRE). These results indicate that RAP2C is a novel member of the Ras family, belonging to the Rap branch of small GTPase proteins and may be involved in SRE-mediated gene transcription.

Cloning and characterization of the human gene DERP6, which activates transcriptional activities of p53.

We report here the cloning and characterization of the human gene DERP6, isolated from human testis cDNA library, and mapped to 17p13.1 by searching the UCSC genomic database. The DERP6 cDNA consists of 1486 nucleotides and has a 316-amino acids open reading frame. The Northern hybridization analysis showed that DERP6 was ubiquitously expressed in all the 16 adult tissues, especially highly expressed in heart, brain, liver, skeletal muscle and testis. DERP6 protein was located in cytoplasm when overexpressed in cultured cells. Reporter gene assays showed that overexpression of DERP6 in cells activated the transcriptional activities of p53. These results indicate that the human gene DERP6 may be involved in p53-mediated gene transcription.

BRSK2 is activated by cyclic AMP-dependent protein kinase A through phosphorylation at Thr260.

Brain selective kinase 2 (BRSK2) has been identified as a member of AMPK related kinases. LKB1 can phosphorylate the Thr174 of BRSK2, increasing its activity >50-fold. In this study, we identified cAMP-dependent protein kinase A (PKA) as another upstream kinase of BRSK2, which can phosphorylate BRSK2 at Thr260. The association between these two proteins was confirmed by GST pull-down. Furthermore, our study indicated that the kinase activity of BRSK2 can be increased through phosphorylation by PKA.