Yusuke Hirata

Mg2+-dependent Interactions of ATP with the Cystathionine-β-Synthase (CBS) Domains of a Magnesium Transporter

Background: CNNMs are evolutionarily conserved Mg2+ transporters that possess CBS domains. Results: CBS domains are essential for molecular function, and they bind to ATP in a Mg2+-dependent manner. Conclusion: The unique binding feature with ATP may contribute to Mg2+ transport by CNNMs. Significance: This is the first report showing the Mg2+-dependent and specific interactions of ATP with CBS domains. Ancient conserved domain protein/cyclin M (CNNM) family proteins are evolutionarily conserved Mg2+ transporters. However, their biochemical mechanism of action remains unknown. Here, we show the functional importance of the commonly conserved cystathionine-β-synthase (CBS) domains and reveal their unique binding ability to ATP. Deletion mutants of CNNM2 and CNNM4, lacking the CBS domains, are unable to promote Mg2+ efflux. Furthermore, the substitution of one amino acid residue in the CBS domains of CNNM2, which is associated with human hereditary hypomagnesemia, abrogates Mg2+ efflux. Binding analyses reveal that the CBS domains of CNNM2 bind directly to ATP and not AMP in a manner dependent on the presence of Mg2+, which is inhibited in a similar pattern by the disease-associated amino acid substitution. The requirement of Mg2+ for these interactions is a unique feature among CBS domains, which can be explained by the presence of highly electronegative surface potentials around the ATP binding site on CNNM2. These results demonstrate that the CBS domains play essential roles in Mg2+ efflux, probably through interactions with ATP. Interactions with ATP, which mostly forms complexes with Mg2+ in cells, may account for the rapid Mg2+ transport by CNNM family proteins.

Post-Translational Modifications of the TAK1-TAB Complex

Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family that is activated by growth factors and cytokines such as TGF-β, IL-1β, and TNF-α, and mediates a wide range of biological processes through activation of the nuclear factor-κB (NF-κB) and the mitogen-activated protein (MAP) kinase signaling pathways. It is well established that activation status of TAK1 is tightly regulated by forming a complex with its binding partners, TAK1-binding proteins (TAB1, TAB2, and TAB3). Interestingly, recent evidence indicates the importance of post-translational modifications (PTMs) of TAK1 and TABs in the regulation of TAK1 activation. To date, a number of PTMs of TAK1 and TABs have been revealed, and these PTMs appear to fine-tune and coordinate TAK1 activities depending on the cellular context. This review therefore focuses on recent advances in the understanding of the PTMs of the TAK1-TAB complex.

Identification of small subunit of serine palmitoyltransferase a as a lysophosphatidylinositol acyltransferase 1‐interacting protein

Lysophosphatidylinositol acyltransferase 1 (LPIAT1), also known as MBOAT7, is a phospholipid acyltransferase that selectively incorporates arachidonic acid (AA) into the sn‐2 position of phosphatidylinositol (PI). We previously demonstrated that LPIAT1 regulates AA content in PI and plays a crucial role in brain development in mice. However, how LPIAT1 is regulated and which proteins function cooperatively with LPIAT1 are unknown. In this study, using a split‐ubiquitin membrane yeast two‐hybrid system, we identified the small subunit of serine palmitoyltransferase a (ssSPTa) as an LPIAT1‐interacting protein. ssSPTa co‐immunoprecipitated and colocalized with LPIAT1 in cultured mammalian cells. Knockdown of ssSPTa decreased the LPIAT1‐dependent incorporation of exogenous AA into PI but did not affect the in vitro enzyme activity of LPIAT1 in the microsomal fraction. Interestingly, knockdown of ssSPTa decreased the protein level of LPIAT1 in the crude mitochondrial fraction but not in total homogenate or the microsomal fraction. LPIAT1 was localized to the mitochondria‐associated membrane (MAM), where AA‐selective acyl‐CoA synthetase is enriched. These results suggest that ssSPTa plays a role in fatty acid remodeling of PI, probably by facilitating the MAM localization of LPIAT1.

Mg2+ Extrusion from Intestinal Epithelia by CNNM Proteins Is Essential for Gonadogenesis via AMPK-TORC1 Signaling in Caenorhabditis elegans

Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK.

Trans-fatty acids promote proinflammatory signaling and cell death by stimulating the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway

Food-borne trans-fatty acids (TFAs) are mainly produced as byproducts during food manufacture. Recent epidemiological studies have revealed that TFA consumption is a major risk factor for various disorders, including atherosclerosis. However, the underlying mechanisms in this disease etiology are largely unknown. Here we have shown that TFAs potentiate activation of apoptosis signal-regulating kinase 1 (ASK1) induced by extracellular ATP, a damage-associated molecular pattern leaked from injured cells. Major food-associated TFAs such as elaidic acid (EA), linoelaidic acid, and trans-vaccenic acid, but not their corresponding cis isomers, dramatically enhanced extracellular ATP-induced apoptosis, accompanied by elevated activation of the ASK1-p38 pathway in a macrophage-like cell line, RAW264.7. Moreover, knocking out the ASK1-encoding gene abolished EA-mediated enhancement of apoptosis. We have reported previously that extracellular ATP induces apoptosis through the ASK1-p38 pathway activated by reactive oxygen species generated downstream of the P2X purinoceptor 7 (P2X7). However, here we show that EA did not increase ATP-induced reactive oxygen species generation but, rather, augmented the effects of calcium/calmodulin-dependent kinase II-dependent ASK1 activation. These results demonstrate that TFAs promote extracellular ATP-induced apoptosis by targeting ASK1 and indicate novel TFA-associated pathways leading to inflammatory signal transduction and cell death that underlie the pathogenesis and progression of TFA-induced atherosclerosis. Our study thus provides insight into the pathogenic mechanisms of and proposes potential therapeutic targets for these TFA-related disorders.

TRIM48 Promotes ASK1 Activation and Cell Death through Ubiquitination-Dependent Degradation of the ASK1-Negative Regulator PRMT1

Apoptosis signal-regulating kinase 1 (ASK1) is an oxidative stress-responsive kinase that is regulated by various interacting molecules and post-translational modifications. However, how these molecules and modifications cooperatively regulate ASK1 activity remains largely unknown. Here, we showed that tripartite motif 48 (TRIM48) orchestrates the regulation of oxidative stress-induced ASK1 activation. A pull-down screen identified a TRIM48-interacting partner, protein arginine methyltransferase 1 (PRMT1), which negatively regulates ASK1 activation by enhancing its interaction with thioredoxin (Trx), another ASK1-negative regulator. TRIM48 facilitates ASK1 activation by promoting K48-linked polyubiquitination and degradation of PRMT1. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death, whereas forced expression promoted cancer cell death in mouse xenograft model. These results indicate that TRIM48 facilitates oxidative stress-induced ASK1 activation and cell death through ubiquitination-dependent degradation of PRMT1. This study provides a cell death mechanism fine-tuned by the crosstalk between enzymes that engage various types of post-translational modifications.

Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor 1 (BIG1) Governs the Recruitment of Tumor Necrosis Factor Receptor-Associated Factor 2 (TRAF2) to Tumor Necrosis Factor Receptor 1 (TNFR1) Signaling Complexes.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a critical mediator of tumor necrosis factor-α (TNF-α) signaling. However, the regulatory mechanisms of TRAF2 are not fully understood. Here we show evidence that TRAF2 requires brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) to be recruited into TNF receptor 1 (TNFR1) signaling complexes. In BIG1 knockdown cells, TNF-α-induced c-Jun N-terminal kinase (JNK) activation was attenuated and the sensitivity to TNF-α-induced apoptosis was increased. Since these trends correlated well with those of TRAF2 deficient cells as previously demonstrated, we tested whether BIG1 functions as an upstream regulator of TRAF2 in TNFR1 signaling. As expected, we found that knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2 that is required for JNK activation, and impaired the recruitment of TRAF2 to the TNFR1 signaling complex (complex I). Moreover, we found that the recruitment of TRAF2 to the death-inducing signaling complex termed complex II was also impaired in BIG1 knockdown cells. These results suggest that BIG1 is a key component of the machinery that drives TRAF2 to the signaling complexes formed after TNFR1 activation. Thus, our data demonstrate a novel and unexpected function of BIG1 that regulates TNFR1 signaling by targeting TRAF2.

Multidisciplinary treatment for prepubertal juvenile myasthenia gravis with crisis.

The management of juvenile myasthenia gravis (MG) remains controversial. We report herein the case of a 12‐year‐old girl with prepubertal juvenile MG with respiratory crisis who underwent thymectomy following methylprednisolone pulse therapy. The patient initially developed progressively worsening fatigability, eyelid ptosis, and diplopia, followed by worsening generalized weakness, dysphagia, and dyspnea. Even after i.v. immunoglobulin, the patient presented with rapid onset of severe dyspnea requiring respiratory support with mechanical ventilation and was graded as Myasthenia Gravis Foundation of America class V. After a course of i.v. methylprednisolone pulse therapy, successful control of respiratory crisis was achieved, and trans‐sternal thymectomy was performed. Partial remission was achieved postoperatively with oral pyridostigmine without immunosuppressive agents such as steroids or calcineurin inhibitors for 18 months after thymectomy. Early thymectomy following induction methylprednisolone pulse therapy might be a treatment option for prepubertal juvenile MG with severe respiratory crisis.

Basolateral sorting of the Mg2+ transporter CNNM4 requires interaction with AP-1A and AP-1B

Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of μ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the μ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with μ1A and μ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.