Cryo-EM structures of the plant anion channel SLAC1 from Arabidopsis thaliana suggest a combined activation model.

The anion channel SLAC1 functions as a crucial effector in the ABA signaling, leading to stomata closure. SLAC1 is activated by phosphorylation in its intracellular domains. Both a binding-activation model and an inhibition-release model for activation have been proposed based on only the closed structures of SLAC1, rendering the structure-based activation mechanism controversial. Here we report cryo-EM structures of Arabidopsis SLAC1 WT and its phosphomimetic mutants in open and closed states. Comparison of the open structure with the closed ones reveals the structural basis for opening of the conductance pore. Multiple phosphorylation of an intracellular domain (ICD) causes dissociation of ICD from the transmembrane domain. A conserved, positively-charged sequence motif in the intracellular loop 2 (ICL2) seems to be capable of sensing of the negatively charged phosphorylated ICD. Interactions between ICL2 and ICD drive drastic conformational changes, thereby widening the pore. From our results we propose that SLAC1 operates by a mechanism combining the binding-activation and inhibition-release models.

Mitochondria-derived peptide SHLP2 regulates energy homeostasis through the activation of hypothalamic neurons.

Small humanin-like peptide 2 (SHLP2) is a mitochondrial-derived peptide implicated in several biological processes such as aging and oxidative stress. However, its functional role in the regulation of energy homeostasis remains unclear, and its corresponding receptor is not identified. Hereby, we demonstrate that both systemic and intracerebroventricular (ICV) administrations of SHLP2 protected the male mice from high-fat diet (HFD)-induced obesity and improved insulin sensitivity. In addition, the activation of pro-opiomelanocortin (POMC) neurons by SHLP2 in the arcuate nucleus of the hypothalamus (ARC) is involved in the suppression of food intake and the promotion of thermogenesis. Through high-throughput structural complementation screening, we discovered that SHLP2 binds to and activates chemokine receptor 7 (CXCR7). Taken together, our study not only reveals the therapeutic potential of SHLP2 in metabolic disorders but also provides important mechanistic insights into how it exerts its effects on energy homeostasis.

Cytosolic domain regulates the calcium sensitivity and surface expression of BEST1 channels in the HEK293 cells.

BEST family is a class of Ca2+-activated Cl- channels evolutionary well conserved from bacteria to human. The human BEST paralogs (BEST1-BEST4) share significant amino acid sequence homology in the N-terminal region, which forms the transmembrane helicases and contains the direct calcium-binding site, Ca2+-clasp. But the cytosolic C-terminal region is less conserved in the paralogs. Interestingly, this domain-specific sequence conservation is also found in the BEST1 orthologs. However, the functional role of the C-terminal region in the BEST channels is still poorly understood. Thus, we aimed to understand the functional role of the C-terminal region in the human and mouse BEST1 channels by using electrophysiological recordings. We found that the calcium-dependent activation of BEST1 channels can be modulated by the C-terminal region. The C-terminal deletion hBEST1 reduced the Ca2+-dependent current activation and the hBEST1-mBEST1 chimera showed a significantly reduced calcium sensitivity to hBEST1 in the HEK293 cells. And the C-terminal domain could regulate cellular expression and plasma membrane targeting of BEST1 channels. Our results can provide a basis for understanding the C-terminal roles in the structure-function of BEST family proteins. [BMB Reports 2023; 56(3): 172-177].

Transmembrane topology and oligomeric nature of an astrocytic membrane protein, MLC1.

MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.

Plasma membrane localization of MLC1 regulates cellular morphology and motility.

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare form of infantile-onset leukodystrophy. The disorder is caused primarily by mutations of MLC1 that leads to a series of phenotypic outcomes including vacuolation of myelin and astrocytes, subcortical cysts, brain edema, and macrocephaly. Recent studies have indicated that functional interactions among MLC1, GlialCAM, and ClC-2 channels play key roles in the regulation of neuronal, glial and vascular homeostasis. However, the physiological role of MLC1 in cellular homeostatic communication remains poorly understood. In the present study, we investigated the cellular function of MLC1 and its effects on cell-cell interactions. METHODS: MLC1-dependent cellular morphology and motility were analyzed by using confocal and live cell imaging technique. Biochemical approaches such as immunoblotting, co-immunoprecipitation, and surface biotinylation were conducted to support data. RESULTS: We found that the altered MLC1 expression and localization led to a great alteration in cellular morphology and motility through actin remodeling. MLC1 overexpression induced filopodia formation and suppressed motility. And, MLC1 proteins expressed in patient-derived MLC1 mutants resulted in trapping in the ER although no changes in morphology or motility were observed. Interestingly knockdown of Mlc1 induced Arp3-Cortactin interaction, lamellipodia formation, and increased the membrane ruffling of the astrocytes. These data indicate that subcellular localization of expressed MLC1 at the plasma membrane is critical for changes in actin dynamics through ARP2/3 complex. Thus, our results suggest that misallocation of pathogenic mutant MLC1 may disturbs the stable cell-cell communication and the homeostatic regulation of astrocytes in patients with MLC.

RGS19 converts iron deprivation stress into a growth-inhibitory signal.

Iron chelation is a promising therapeutic strategy for cancer that works, in part, by inducing overexpression of N-myc downstream-regulated gene 1 protein (NDRG1), a known growth inhibitor and metastasis suppressor. However, details of the signaling cascades that convert physical stress into a biological response remain elusive. We investigated the role of RGS19, a regulator of G-protein signaling, in iron chelator-induced NDRG1 overexpression in HeLa cells. Knockdown of RGS19 diminished the expression of genes involved in desferrioxamine (DFO)-induced growth inhibition. Conversely, overexpression of RGS19 enhanced the expression of these genes. Moreover, overexpression of RGS19 reduced cell viability. Overexpression of G-protein alpha subunit i3 (Gαi3) repressed the induction of NDRG1 expression. Selective inhibition of downstream targets of Gαi3 abrogated DFO-induced overexpression of NDRG1. DFO protected RGS19 from proteolysis induced by GAIP interacting protein N terminus (GIPN); moreover, an iron-deficient RGS19 mutant was stable in the presence of GIPN and retained GTPase-activating protein activity. RGS19 was co-purified with iron and showed unique UV-absorption characteristics frequently observed in iron-binding proteins. This study demonstrates that RGS19 senses cellular iron availability and is stabilized under iron-depleted conditions, resulting in the induction of a growth-inhibitory signal.

Induction of apoptosis by obovatol as a novel therapeutic strategy for acute myeloid leukemia.

Obovatol, a compound isolated from the bark cortex of Magnolia officinalis (cortex Magnoliae officinalis; M. officinalis), has been studied for use in the treatment of solid cancers. However, the mechanisms of action and the effects of obovatol against acute myeloid leukemia (AML) remain unclear and require further investigation. Therefore, this study was conducted using a human AML cell line (MM6). Obovatol increased pro-apoptotic (Bax) and decreased anti-apoptotic (Bcl-2) protein expression, resulting in caspase-3 and caspase-9 activation measured by caspase-Glo 3/7 assay. Furthermore, obovatol activated the mitogen-activated protein kinase (MAPK) signaling pathway [c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38] and inhibited the activation of the nuclear factor-κB (NF-κB) signaling pathway analyzed by western blot analysis. Taken together, these findings provide evidence that obovatol inhibits cell proliferation in AML and induces apoptosis through the activation of the MAPK pathway in addition to the intrinsic apoptotic pathway. In addition, obovatol suppressed the expression of mixed-lineage leukemia (MLL) target genes by inhibiting the activation of the NF-κB pathway. Therefore, these results suggest that obovatol may have potential for use in the treatment of leukemia.

Regulation of HOXA9 activity by predominant expression of DACH1 against C/EBPα and GATA-1 in myeloid leukemia with MLL-AF9.

Although MLL-AF9 caused by the chromosomal translocation t(9;11) has a critical role in acute myeloid leukemia, the molecular pathogenesis is poorly understood. Here, we identified that the cell fate determination factor DACH1 is directly up-regulated by MLL-AF9. Recently we showed that the forced expression of DACH1 in myeloid cells induced p27(Kip1) and repressed p21(Cip1), which is a pivotal characteristic of the myeloid progenitor. Consistent with our previous study, ectopic expression of DACH1 contributed to the maintenance of colonogenic activity and blocked the differentiation of myeloid progenitors. Moreover, we here identified an endogenous HOXA9-DACH1 complex mediated by the carboxyl terminus of DACH1 in t(9;11) leukemia cells. qRT-PCR revealed that DACH1 has a stronger transcription-promoting activity with HOXA9 than does PBX2 with HOXA9. Furthermore, C/EBPα and GATA-1 can directly bind to the promoter of DACH1 and act as a transcriptional suppressor. Expression of DACH1 is down-regulated during myeloid differentiation and shows an inverse pattern compared to C/EBPα and GATA-1 expression. However, ectopic expression of C/EBPα and/or GATA-1 could not abrogate the over-expression of DACH1 induced by MLL-AF9. Therefore, we postulate that the inability of C/EBPα and GATA-1 to down-regulate DACH1 expression induced by MLL-AF9 during myeloid differentiation may contribute to t(9;11) leukemogenesis.

DACH1 regulates cell cycle progression of myeloid cells through the control of cyclin D, Cdk 4/6 and p21Cip1.

The cell-fate determination factor Dachshund, a component of the Retinal Determination Gene Network (RDGN), has a role in breast tumor proliferation through the repression of cyclin D1 and several key regulators of embryonic stem cell function, such as Nanog and Sox2. However, little is known about the role of DACH1 in a myeloid lineage as a cell cycle regulator. Here, we identified the differential expression levels of extensive cell cycle regulators controlled by DACH1 in myeloid progenitor cells. The forced expression of DACH1 induced p27(Kip1) and repressed p21(Cip1), which is a pivotal characteristic of the myeloid progenitor. Furthermore, DACH1 significantly increased the expression of cyclin D1, D3, F, and Cdk 1, 4, and 6 in myeloid progenitor cells. The knockdown of DACH1 blocked the cell cycle progression of HL-60 promyeloblastic cells through the decrease of cyclin D1, D3, F, and Cdk 1, 4, and 6 and increase in p21(Cip1), which in turn decreased the phosphorylation of the Rb protein. The expression of Sox2, Oct4, and Klf4 was significantly up-regulated by the forced expression of DACH1 in mouse myeloid progenitor cells.

Detection of low-abundant novel transcripts in mouse hematopoietic stem cells.

Gene expression profiles of hematopoietic stem cells (HSCs) provide clues for understanding molecular mechanisms of HSC behavior, including self-renewal and differentiation. We took advantage of serial analysis of gene expression (SAGE) to identify medium- and low-abundant transcripts expressed in HSCs/hematopoietic progenitor cells (HPCs). Among a total of 31,380 unique transcripts, 17,326 (55%) correspond to known genes and, 14,054 (45%) are low-copy transcripts that have no matches to currently known genes. Among the former class, 3,899 (23%) were alternatively spliced transcripts and 3,754 (22%) represent anti-sense transcripts from known genes. Mapping of the SAGE tags to the mouse genome showed that differences in gene expression exist among chromosomes. In addition, comparison of the HSCs/HPCs SAGE data to that of myeloid progenitor cells revealed that massive genetic reprogramming occurs in hematopoietic cell differentiation. Our results demonstrate a previously unrecognized complexity of gene expression in HSCs/HPCs, and indicate the need for further efforts to fully identify and characterize the transcripts expressed in this cell type.

Transcriptome analysis of the zebrafish mind bomb mutant.

Mind bomb (Mib) facilitates Notch signaling pathway by promoting the endocytosis of Notch ligand. The zebrafish mib ( ta52b ) mutant has a defect in its ubiquitin ligase activity which is necessary to inhibit the neurogenesis, resulting in a neuronal hyperplasia. Several genes regulated in the mib ( ta52b ) mutant have been well established, however, there were relatively few reports about the transcriptome profile. To identify the genes differentially expressed in the mib ( ta52b ) mutant, genome-wide analysis was performed using serial analysis of gene expression. Three hundred and thirty-five transcripts were identified whose expressions were significantly altered in the mib ( ta52b ) mutant as compared with the wild-type. Interestingly, it was suggested that the mib ( ta52b ) mutation may affect not only neurogenesis but also mesoderm development. These results provide new insights into Notch signaling pathway.

A comparison of gene expression profiles between primary human AML cells and AML cell line.

In acute myeloid leukemia (AML), hematologic malignancies are characterized by recurring chromosomal abnormalities. Chromosome translocation t(9;11)(p22;q23) is one of the most common genetic aberrations and results in the formation of the MLL-AF9 fusion gene that functions as a facilitator of cell growth directly. In order to study this type of AML, the cell lines with cytogenetically diagnosed t(9;11)(p22;q23), such as Mono Mac 6 (MM6), have been widely used. To examine whether there is any difference in gene expression between the primary human t(9;11) AML cells and MM6 cell line, genome-wide transcriptome analysis was performed on MM6 cell line using SAGE and the results were compared to the profile of primary human t(9;11) AML cells. 884 transcripts which were alternatively expressed between MM6 cells and primary human t(9;11) cells were identified through statistical analysis (P < 0.05) and 4-fold expression change. Of these transcripts, 830 (94%) matched to known genes or EST were classified by functional categories (http://david.abcc.ncifcrf.gov/). The majority of alternatively expressed genes in MM6 were involved in biosynthetic and metabolic processes, but HRAS, a protein that is known to be associated with leukemogenesis, was expressed only in MM6 cells and several other genes involved in Erk1/Erk2 MAPK pathway were also over-expressed in MM6. Therefore, since MM6 cell line has a similar expression profile to primary human t(9;11) AML in general and expresses uniquely a strong Erk1/Erk2 MAPK pathway including HRAS, it can be used as a model for HRAS-positive t(9;11) AML.

Stable transcriptional status in the apoptotic erythroid genome.

When a cell is destined for apoptosis, will its genome reprogram its transcriptional machinery to overcome the life-threatening challenge? To address this issue, we performed a genome-wide transcriptome analysis in EPO (erythropoietin) deprivation-induced apoptotic erythroid cells using the SAGE method. The results show that the transcript contents for the majority of the genes remain unchanged in the apoptotic cells, including the apoptotic genes and the heat shock genes. Of the small number of genes with an altered expression, they are mainly associated with cellular structure. Our study reveals that there is no genetic reprogramming for the transcriptional machinery in the apoptotic genome. Apoptosis, as defined by programmed cell death, is not a crisis but a peaceful physiological process.