TMCO1 Is an ER Ca(2+) Load-Activated Ca(2+) Channel.

Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading and disassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions.

MARCH1 negatively regulates TBK1-mTOR signaling pathway by ubiquitinating TBK1.

BACKGROUND: TBK1 positively regulates the growth factor-mediated mTOR signaling pathway by phosphorylating mTOR. However, it remains unclear how the TBK1-mTOR signaling pathway is regulated. Considering that STING not only interacts with TBK1 but also with MARCH1, we speculated that MARCH1 might regulate the mTOR signaling pathway by targeting TBK1. The aim of this study was to determine whether MARCH1 regulates the mTOR signaling pathway by targeting TBK1. METHODS: The co-immunoprecipitation (Co-IP) assay was used to verify the interaction between MARCH1 with STING or TBK1. The ubiquitination of STING or TBK1 was analyzed using denatured co-immunoprecipitation. The level of proteins detected in the co-immunoprecipitation or denatured co-immunoprecipitation samples were determined by Western blotting. Stable knocked-down cells were constructed by infecting lentivirus bearing the related shRNA sequences. Scratch wound healing and clonogenic cell survival assays were used to detect the migration and proliferation of breast cancer cells. RESULTS: We showed that MARCH1 played an important role in growth factor-induced the TBK1- mTOR signaling pathway. MARCH1 overexpression attenuated the growth factor-induced activation of mTOR signaling pathway, whereas its deficiency resulted in the opposite effect. Mechanistically, MARCH1 interacted with and promoted the K63-linked ubiquitination of TBK1. This ubiquitination of TBK1 then attenuated its interaction with mTOR, thereby inhibiting the growth factor-induced mTOR signaling pathway. Importantly, faster proliferation induced by MARCH1 deficiency was weakened by mTOR, STING, or TBK1 inhibition. CONCLUSION: MARCH1 suppressed growth factors mediated the mTOR signaling pathway by targeting the STING-TBK1-mTOR axis.

Genome-wide loss of 5-hmC is a novel epigenetic feature of Huntington's disease.

5-Hydroxymethylcytosine (5-hmC) may represent a new epigenetic modification of cytosine. While the dynamics of 5-hmC during neurodevelopment have recently been reported, little is known about its genomic distribution and function(s) in neurodegenerative diseases such as Huntington's disease (HD). We here observed a marked reduction of the 5-hmC signal in YAC128 (yeast artificial chromosome transgene with 128 CAG repeats) HD mouse brain tissues when compared with age-matched wild-type (WT) mice, suggesting a deficiency of 5-hmC reconstruction in HD brains during postnatal development. Genome-wide distribution analysis of 5-hmC further confirmed the diminishment of the 5-hmC signal in striatum and cortex in YAC128 HD mice. General genomic features of 5-hmC are highly conserved, not being affected by either disease or brain regions. Intriguingly, we have identified disease-specific (YAC128 versus WT) differentially hydroxymethylated regions (DhMRs), and found that acquisition of DhmRs in gene body is a positive epigenetic regulator for gene expression. Ingenuity pathway analysis (IPA) of genotype-specific DhMR-annotated genes revealed that alternation of a number of canonical pathways involving neuronal development/differentiation (Wnt/ß-catenin/Sox pathway, axonal guidance signaling pathway) and neuronal function/survival (glutamate receptor/calcium/CREB, GABA receptor signaling, dopamine-DARPP32 feedback pathway, etc.) could be important for the onset of HD. Our results indicate that loss of the 5-hmC marker is a novel epigenetic feature in HD, and that this aberrant epigenetic regulation may impair the neurogenesis, neuronal function and survival in HD brain. Our study also opens a new avenue for HD treatment; re-establishing the native 5-hmC landscape may have the potential to slow/halt the progression of HD.

Dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic DNA damage in Huntington disease.

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Indications of oxidative stress are apparent in brain tissues from both HD patients and HD mouse models; however, the origin of this oxidant stress remains a mystery. Here, we used a yeast artificial chromosome transgenic mouse model of HD (YAC128) to investigate the potential connections between dysregulation of cytosolic Ca(2+) signaling and mitochondrial oxidative damage in HD cells. We found that YAC128 mouse embryonic fibroblasts exhibit a strikingly higher level of mitochondrial matrix Ca(2+) loading and elevated superoxide generation compared with WT cells, indicating that both mitochondrial Ca(2+) signaling and superoxide generation are dysregulated in HD cells. The excessive mitochondrial oxidant stress is critically dependent on mitochondrial Ca(2+) loading in HD cells, because blocking mitochondrial Ca(2+) uptake abolished elevated superoxide generation. Similar results were obtained using neurons from HD model mice and fibroblast cells from HD patients. More importantly, mitochondrial Ca(2+) loading in HD cells caused a 2-fold higher level of mitochondrial genomic DNA (mtDNA) damage due to the excessive oxidant generation. This study provides strong evidence to support a new causal link between dysregulated mitochondrial Ca(2+) signaling, elevated mitochondrial oxidant stress, and mtDNA damage in HD. Our results also indicate that reducing mitochondrial Ca(2+) uptake could be a therapeutic strategy for HD.

SPOP negatively regulates mTORC1 activity by ubiquitinating Sec13.

The mammalian target of rapamycin complex1 (mTORC1) can response to amino acid to regulate metabolism and cell growth. GATOR2 act as important role in amino acid mediated mTORC1 signaling pathway by repressing GTPase activity (GAP) of GATOR1. However, it is still unclear how GATOR2 regulates mTORC1 signaling pathway. Here, we found that K63-ubiquitination of Sce13, one component of GATOR2, suppresses the mTORC1 activity by lessening the inter-interaction of GATOR2. Mechanistically, the ubiquitination of Sec13 was mediated by SPOP. Subsequently, the ubiquitination of Sec13 attenuated its interaction with the other component of GATOR2, thus suppressing the activity of mTORC1. Importantly, the deficiency of SPOP promoted the faster proliferation and migration of breast cancer cells, which was attenuated by knocking down of Sec13. Therefore, SPOP can act as a tumor suppressor gene by negatively regulating mTORC1 signaling pathway.

Cyclin-dependent kinase 5 negatively regulates antiviral immune response by disrupting myeloid differentiation primary response protein 88 self-association.

As a member of the pattern recognition receptors (PRRs) involving in the innate immune system, Toll-like receptors (TLRs) can sense a wide range of microbial pathogens and combat infections by producing antimicrobial products, inflammatory cytokines, and chemokines. All TLRs, with the exception of TLR3, activate a signalling cascade via the myeloid differentiation primary response gene 88 (MyD88). Therefore, the activation of MyD88-dependent signalling pathway must be finely controlled. Herein, we identified that cyclin-dependent kinase 5 (CDK5) negatively regulated TLR-MyD88 signalling pathway by targeting MyD88. Overexpression of CDK5 reduced the production of interferons (IFNs), while a deficiency in CDK5 increased the expression of IFNs in response to vesicular stomatitis virus (VSV) infection. Mechanistically, CDK5 suppressed the formation of MyD88 homodimers, resulting in the attenuated production of IFNs induced by VSV infection. Surprisingly, its kinase activity does not play a role in this process. Therefore, CDK5 can act as an internal regulator to prevent excessive production of IFNs by restricting TLR-MyD88-induced activation of antiviral innate immunity in A549 cells.

Miro2 supplies a platform for Parkin translocation to damaged mitochondria.

PINK1/Parkin-mediated mitophagy is an important process in selective removal of damaged mitochondria, in which translocation of Parkin to damaged mitochondria is recognized as an initiation step. At present, how the damaged mitochondria are selectively recognized and targeted by Parkin is not fully understood. Here we show that Miro2, an outer mitochondrial membrane protein, undergoes demultimerization from a tetramer to a monomer and alteration in mitochondrial localization upon CCCP treatment, suggesting a CCCP-induced realignment of Miro2. The realignment of Miro2 is tightly regulated by PINK1-mediated phosphorylation at Ser325/Ser430 and by Ca2+ binding to EF2 domain, which are both essential for the subsequent Parkin translocation. Interestingly, ablation of Miro2 in mouse causes delayed reticulocyte maturation, lactic acidosis and cardiac disorders. Furthermore, several Miro2 mutations found in the congenital lactic acidosis patients also disable its realignment and Parkin translocation. These findings reveal an important role of Miro2 to mediate Parkin translocation by sensing both depolarization and Ca2+ release from damaged mitochondria to ensure the accuracy of mitophagy.

A computational network analysis based on targets of antipsychotic agents.

Currently, numerous antipsychotic agents have been developed in the area of pharmacological treatment of schizophrenia. However, the molecular mechanism underlying multi targets of antipsychotics were yet to be explored. In this study we performed a computational network analysis based on targets of antipsychotic agents. We retrieved a total of 96 targets from 56 antipsychotic agents. By expression enrichment analysis, we identified that the expressions of antipsychotic target genes were significantly enriched in liver, brain, blood and corpus striatum. By protein-protein interaction (PPI) network analysis, a PPI network with 77 significantly interconnected target genes was generated. By historeceptomics analysis, significant brain region specific target-drug interactions were identified in targets of dopamine receptors (DRD1-Olanzapine in caudate nucleus and pons (P-value<0.005), DRD2-Bifeprunox in caudate nucleus and pituitary (P-value<0.0005), DRD4-Loxapine in Pineal (P-value<0.00001)) and 5-hydroxytryptamine receptor (HTR2A-Risperidone in occipital lobe, prefrontal cortex and subthalamic nucleus (P-value<0.0001)). By pathway grouped network analysis, 34 significant pathways were identified and significantly grouped into 6 sub networks related with drug metabolism, Calcium signaling, GABA receptors, dopamine receptors, Bile secretion and Gap junction. Our results may provide biological explanation for antipsychotic targets and insights for molecular mechanism of antipsychotic agents.

TMCO1 is essential for ovarian follicle development by regulating ER Ca2+ store of granulosa cells.

TMCO1 (transmembrane and coiled-coil domains 1) is an endoplasmic reticulum (ER) transmembrane protein that actively prevents Ca2+ stores from overfilling. To characterize its physiological function(s), we generated Tmco1-/- knockout (KO) mice. In addition to the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, Tmco1-/- females manifest gradual loss of ovarian follicles, impaired ovarian follicle development, and subfertility with a phenotype analogous to the premature ovarian failure (POF) in women. In line with the role of TMCO1 as a Ca2+ load-activated Ca2+ channel, we have detected a supernormal Ca2+ signaling in Tmco1-/- granulosa cells (GCs). Interestingly, although spontaneous Ca2+ oscillation pattern was altered, ER Ca2+ stores of germinal vesicle (GV) stage oocytes and metaphase II (MII) arrested eggs were normal upon Tmco1 ablation. Combined with RNA-sequencing analysis, we also detected increased ER stress-mediated apoptosis and enhanced reactive oxygen species (ROS) level in Tmco1-/- GCs, indicating the dysfunctions of GCs upon TMCO1 deficiency. Taken together, these results reveal that TMCO1 is essential for ovarian follicle development and female fertility by maintaining ER Ca2+ homeostasis of GCs, disruption of which causes ER stress-mediated apoptosis and increased cellular ROS level in GCs and thus leads to impaired ovarian follicle development.

Convergent evidence from systematic analysis of GWAS revealed genetic basis of esophageal cancer.

Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with risk of esophageal cancer (EC). However, investigation of genetic basis from the perspective of systematic biology and integrative genomics remains scarce.In this study, we explored genetic basis of EC based on GWAS data and implemented a series of bioinformatics methods including functional annotation, expression quantitative trait loci (eQTL) analysis, pathway enrichment analysis and pathway grouped network analysis.Two hundred and thirteen risk SNPs were identified, in which 44 SNPs were found to have significantly differential gene expression in esophageal tissues by eQTL analysis. By pathway enrichment analysis, 170 risk genes mapped by risk SNPs were enriched into 38 significant GO terms and 17 significant KEGG pathways, which were significantly grouped into 9 sub-networks by pathway grouped network analysis. The 9 groups of interconnected pathways were mainly involved with muscle cell proliferation, cellular response to interleukin-6, cell adhesion molecules, and ethanol oxidation, which might participate in the development of EC.Our findings provide genetic evidence and new insight for exploring the molecular mechanisms of EC.

Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy.

Mitochondrial sequestration by autophagosomes is a key step in mitophagy while the mechanisms mediating this process are not fully understood. It has been reported that Endophilin B1 (EB1) promotes mitochondrial sequestration by binding and shaping membrane. However, the role of EB1 homolog Endophilin B2 (EB2) in mitophagy remains unclear. Here we report that EB2 plays an indispensable role in mitochondria sequestration and inner mitochondrial membrane (IMM) protein degradation during mitophagy. Similar to EB1, EB2 aggregates into foci and then translocates to damaged mitochondria. Loss of either EB2 and/or EB1 significantly enervates the foci translocation to fragmented mitochondria and IMM degradation, and the EB1/EB2 heterodimer formed by EB1/EB2 interaction promotes the above process. We noticed that, it is the dimer domain of EB2 but not that of EB1 mediating the heterodimer formation, manifesting the importance of EB2 in mitophagy. Furthermore, we demonstrate that the EB foci formation is closely regulated by the PINK1-Parkin signaling pathway. From these results, we propose that EB1/EB2 heterodimers may serve as linkers between damaged mitochondria and phagophores during mitophagy.

Adult neural progenitor cells from Huntington's disease mouse brain exhibit increased proliferation and migration due to enhanced calcium and ROS signals.

OBJECTIVES: Huntington's disease (HD) is an inherited human neurodegenerative disorder characterized by uncontrollable movement, psychiatric disturbance and cognitive decline. Impaired proliferative/differentiational potentials of adult neural progenitor cells (ANPCs) have been thought to be a pathogenic mechanism involved in it. In this study, we aimed to elucidate intrinsic properties of ANPCs subjected to neurodegenerative condition in YAC128 HD mice. MATERIALS AND METHODS: ANPCs were isolated from the SVZ regions of 4-month-old WT and YAC128 mice. Cell proliferation, migration and neuronal differentiation in vitro were compared between these two genotypes with/without Ca(2+) inhibitors or ROS scavenger treatments. Differences in ANPC proliferation and differentiation capabilities in vivo between the two genotypes were evaluated using Ki-67 and Doublecortin (DCX) immunofluorescence respectively. RESULTS: Compared to WT ANPCs, YAC128 ANPCs had significantly enhanced cell proliferation, migration and neuronal differentiation in vitro, accompanied by increased Ca(2+) and ROS signals. Raised proliferation and migration in YAC128 ANPCs were abolished by Ca(2+) signalling antagonists and ROS scavenging. However, in vivo, HD ANPCs failed to show any elevated proliferation or differentiation. CONCLUSIONS: Increased Ca(2+) signalling and higher level of ROS conferred HD ANPC enhancement of proliferation and migration potentials. However, the in vivo micro-environment did not support endogenous ANPCs to respond appropriately to neuronal loss in these YAC128 mouse brains.