Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics.

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis.

In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.

CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle.

Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

The combination of brentuximab vedotin and chidamide synergistically suppresses the proliferation of T-cell lymphoma cells through the enhancement of apoptosis.

PURPOSE: Peripheral T-cell lymphoma (PTCL) is an aggressive disease with a poor prognosis. Brentuximab vedotin (BV), an anti-CD30 monoclonal antibody linked to a microtubule-disrupting agent, has been approved for the treatment of PTCL. We evaluated a new effective combination partner of BV using non-clinical approaches that could potentially identify agents capable of improving survival times for patients with PTCL. METHODS: A high-throughput screening test was used to select the most synergistic partner of BV from 14 candidate drugs that were under development or available in clinical practice for PTCL. HH cells, originating from an aggressive cutaneous T-cell lymphoma, were used as an experimental model of PTCL. Apoptotic effects of the synergistic partner of BV were further investigated in vitro and in vivo using HH-cell xenograft mice. RESULTS: Chidamide (tucidinostat), a novel histone deacetylase inhibitor, was found to have the greatest synergistic effect with BV on HH cells. The combined effects of chidamide and BV were demonstrated in a study of HH-cell xenograft mice; mean tumor size following combined treatment was 22% of that observed in the control group, compared with 71% and 58% following chidamide and BV monotherapy, respectively. Further investigations in vitro and in vivo revealed that the levels of an anti-apoptotic protein, Bcl-2, and a rate-limiting factor of DNA replication, CDC45, were reduced in HH cells treated with chidamide combined with BV compared with the control group. CONCLUSION: The use of chidamide in conjunction with BV may positively affect and enhance T-cellular apoptotic pathways without offsetting each other.

Heme-dependent recognition of 5-aminolevulinate synthase by the human mitochondrial molecular chaperone ClpX.

The caseinolytic mitochondrial matrix peptidase chaperone subunit (ClpX) plays an important role in the heme-dependent regulation of 5-aminolevulinate synthase (ALAS1), a key enzyme in heme biosynthesis. However, the mechanisms underlying the role of ClpX in this process remain unclear. In this in vitro study, we confirmed the direct binding between ALAS1 and ClpX in a heme-dependent manner. The substitution of C108 P109 [CP motif 3 (CP3)] with A108 A109 in ALAS1 resulted in a loss of ability to bind ClpX. Computational disorder analyses revealed that CP3 was located in a potential intrinsically disordered protein region (IDPR). Thus, we propose that conditional disorder-to-order transitions in the IDPRs of ALAS1 may represent key mechanisms underlying the heme-dependent recognition of ALAS1 by ClpX.

Selective serotonin reuptake inhibitors and raft inhibitors shorten the period of Period1-driven circadian bioluminescence rhythms in rat-1 fibroblasts.

Alterations in circadian rhythm generation may be related to the development of mood disorders. Although it has been reported that the most popular antidepressant, selective serotonin reuptake inhibitors (SSRIs) affect circadian phase, no data are available that describe the effects of SSRIs on other circadian parameters (period, amplitude and damping rate) in dissociated cells. In the present study we used real-time monitoring of bioluminescence in rat-1 fibroblasts expressing the Period1-luciferase transgene, and that in Period1-luciferase transgenic mouse suprachiasmatic nucleus (SCN) explants, in order to characterize the effects of SSRI on circadian oscillator function in vitro. We found that mRNA of the serotonin transporter (SERT), a target of SSRIs, was expressed in rat-1 fibroblasts. Sertraline, fluoxetine, fluvoxamine, citalopram and paroxetine all significantly shortened the period of Period1-bioluminescence rhythms in rat-1 fibroblasts. The amplitude was reduced by sertraline, and the damping rate was decreased by sertraline, fluoxetine, flvoxamine and paroxetine. The effect of sertraline was dose-dependent, and it also shortened the circadian period in the SCN. SERT is associated with lipid microdomains, which are required for efficient SERT activity. Indeed, cholesterol chelating reagent methyl-beta-cyclodextrin significantly reduced the period and the amplitude in rat-1 fibroblasts. Furthermore, lipid binding reagent xylazine significantly reduced the period. In summary our data present evidence that SSRIs affect circadian rhythmicity. The action of SSRIs is likely mediated by suppression of SERT activity. A better understanding of the relationship between mental illness and biological timing may yield new insight into disease etiology and avenues for treatment.

Establishment of a cell model of X-linked sideroblastic anemia using genome editing.

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome-editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, ß-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared with wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, whereas the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency in combination with increased iron import into mitochondria during erythroid differentiation results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro.

MAP kinase additively activates the mouse Per1 gene promoter with CaM kinase II.

In a previous study, we showed that the Ca2+/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta) activates the mouse Per1 (mPer1) promoter through a 5'-GAGGGG-3' motif near exon1B. Here we use luciferase reporter gene assays to document additive activation of the mPer1 promoter by CaMKIIdelta and mitogen-activated protein kinase (MAPK) pathways. Transfection of constitutively active MEKK markedly increased mPer1 promoter activity in NB2A cells. Experiments using MAPK inhibitors and dominant-negative c-Jun NH2-terminal kinase 1 (JNK1) showed that extracellular signal-regulated kinase (ERK) accounts for MEKK-induced mPer1 gene activation. We next defined the ERK-responsive region in the mPer1 promoter. A region from -1735 to -1721 was required for ERK-induced promoter activation. We also identified a CaMKII-responsive element near exon 1B. Although mutation of the CaMKII-responsive element has no effect on the ERK responsiveness, elimination of a GC-rich sequence downstream of the CaMKII-responsive region totally abolished ERK responsiveness. Finally, ERK-induced promoter activation was additively potentiated by co-transfection with active CaMKIIdelta. These results suggest that additive activation by ERK and CaMKII, most likely as a result of photic stimulation in the suprachiasmatic nucleus, plays a critical role in activating the mPer1 gene promoter.

The two actin-interacting protein 1 genes have overlapping and essential function for embryonic development in Caenorhabditis elegans.

Disassembly of actin filaments by actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1) is a conserved mechanism to promote reorganization of the actin cytoskeleton. We previously reported that unc-78, an AIP1 gene in the nematode Caenorhabditis elegans, is required for organized assembly of sarcomeric actin filaments in the body wall muscle. unc-78 functions in larval and adult muscle, and an unc-78-null mutant is homozygous viable and shows only weak phenotypes in embryos. Here we report that a second AIP1 gene, aipl-1 (AIP1-like gene-1), has overlapping function with unc-78, and that depletion of the two AIP1 isoforms causes embryonic lethality. A single aipl-1-null mutation did not cause a detectable phenotype. However, depletion of both unc-78 and aipl-1 arrested development at late embryonic stages due to severe disorganization of sarcomeric actin filaments in body wall muscle. In vitro, both AIPL-1 and UNC-78 preferentially cooperated with UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament disassembly but not with UNC-60A, a nonmuscle ADF/cofilin. AIPL-1 is expressed in embryonic muscle, and forced expression of AIPL-1 in adult muscle compensated for the function of UNC-78. Thus our results suggest that enhancement of actin filament disassembly by ADF/cofilin and AIP1 proteins is critical for embryogenesis.

Differential subcellular distribution of Ca2+/calmodulin-dependent protein kinase II isoforms in the striatum and NG108-15 cells.

Four subunits of Ca2+/calmodulin-dependent protein kinase II (CaM KII) have several isoforms, which differ in the variable domain. We previously reported that all subunits were highly expressed in rat striatal neurons. To examine intracellular distributions of CaM KII subunits in the rat striatal neurons, we performed immunoblot analysis with antibodies specific to each subunit in cell extracts from the rat striatum after continuous sucrose density gradient fractionation. The alpha subunit, but not the beta, gamma, or delta subunits, was colocalized with synapsin I, and each subunit showed a distinct distribution pattern in the fractions. To examine further the intracellular distributions of CaM KII isoforms in the same subunit, we established NG108-15 cells stably expressing delta1, delta3, and delta4 isoforms and examined distributions of the delta and gamma isoforms in these cell lines after fractionation. Each of the overexpressed exogenous delta isoforms showed a distinct distribution pattern. The endogenous delta2 was colocalized with the overexpressed delta1, delta3, and delta4 isoforms. However, the endogenous gammaB/gammaC isoforms were not colocalized with the overexpressed delta isoforms. Furthermore, the endogenous delta1 was concentrated in the microsomal fraction from the rat striatum. With the results taken together, it is suggested that CaM KII forms oligomers between isoforms in the same subunit but not in different subunits. The variable domain of CaM KII isoforms might possibly be responsible for targeting to certain intracellular compartments.

Involvement of calcium/calmodulin-dependent protein kinase II in the induction of mPer1.

Recent studies suggest that CaM kinase II is involved in light-induced phase delays and induction of Per1 and Per2 genes in the hamster suprachiasmatic nucleus (SCN) (Yokota et al.,2001). We focused on intracellular mechanisms of the CaM kinase II-induced mPer1 gene expression. Immunoblotting and immunohistochemical analyses with isoform-specific antibodies against different isoforms of CaM kinase II and CaM kinase IV showed abundant expression of the delta isoform of CaM kinase II without significant expression of CaM kinase IV in the lateral ventral region of the rat SCN. We next defined the CaM kinase II-responsive region on the mPer1 promoter using a luciferase reporter gene assay. Transfection of the constitutively-active CaM kinase IIdelta greatly increased mPer1 promoter activity in NG108-15 cells and increased activity slightly but significantly in NB2A and C6 glioma cells. Similarly, transfection of a constitutively-active MEKK, an upstream kinase of mitogen-activated protein kinase (MAPK), greatly increased promoter activity in NB2A cells. Deletion and mutation analyses of the mPer1 promoter revealed that a 5'-GAGGGG-3' sequence motif near exon 1B, in which several zinc finger proteins seem to bind, was essential for the CaM kinase II-induced activation of the mPer1 promoter. These results suggest that CaM kinase IIdelta but not CaM kinase IV plays an essential role for mPer1 expression through the 5'-GAGGGG-3' motif on the mPer1 promoter.