A Novel Interaction between MFN2/Marf and MARK4/PAR-1 Is Implicated in Synaptic Defects and Mitochondrial Dysfunction.

As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2 (MFN2)/mitochondrial assembly regulatory factor (Marf), plays a role in mitochondrial fusion, mutations of which are implicated in age-related human diseases, including several neurodegenerative disorders. However, the regulation of MFN2/Marf-mediated mitochondrial fusion, as well as the pathologic mechanism of neurodegeneration, is not clearly understood. Here, we identified a novel interaction between MFN2/Marf and microtubule affinity-regulating kinase 4 (MARK4)/PAR-1. In the Drosophila larval neuromuscular junction, muscle-specific overexpression of MFN2/Marf decreased the number of synaptic boutons, and the loss of MARK4/PAR-1 alleviated the synaptic defects of MFN2/Marf overexpression. Downregulation of MARK4/PAR-1 rescued the mitochondrial hyperfusion phenotype caused by MFN2/Marf overexpression in the Drosophila muscles as well as in the cultured cells. In addition, knockdown of MARK4/PAR-1 rescued the respiratory dysfunction of mitochondria induced by MFN2/Marf overexpression in mammalian cells. Together, our results indicate that the interaction between MFN2/Marf and MARK4/PAR-1 is fine-tuned to maintain synaptic integrity and mitochondrial homeostasis, and its dysregulation may be implicated in neurologic pathogenesis.

Real-Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label-Free Imaging with Optical Diffraction Tomography.

Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug-resistant bacteria. As a mimic of AMPs, peptoids with N-substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure-activity relationship (SAR) of a library of indole side chain-containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label-free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real-time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance-breaking novel antibiotic drug.

Neuronal growth regulator 1 promotes adipocyte lipid trafficking via interaction with CD36.

Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein associated with several human pathologies, including obesity, depression, and autism. Recently, significantly enlarged white adipose tissue, hepatic lipid accumulation, and decreased muscle capacity were reported in Negr1-deficient mice. However, the mechanism behind these phenotypes was not clear. In the present study, we found NEGR1 to interact with cluster of differentiation 36 (CD36), the major fatty acid translocase in the plasma membrane. Binding assays with a soluble form of NEGR1 and in situ proximal ligation assays indicated that NEGR1-CD36 interaction occurs at the outer leaflet of the cell membrane. Furthermore, we show that NEGR1 overexpression induced CD36 protein destabilization in vitro. Both mRNA and protein levels of CD36 were significantly elevated in the white adipose tissue and liver tissues of Negr1-/- mice. Accordingly, fatty acid uptake rate increased in NEGR1-deficient primary adipocytes. Finally, we demonstrated that Negr1-/- mouse embryonic fibroblasts showed elevated reactive oxygen species levels and decreased adenosine monophosphate-activated protein kinase activation compared with control mouse embryonic fibroblasts. Based on these results, we propose that NEGR1 regulates cellular fat content by controlling the expression of CD36.

Na/K-ATPase beta1-subunit associates with neuronal growth regulator 1 (NEGR1) to participate in intercellular interactions.

Neuronal growth regulator 1 (NEGR1) is a GPI-anchored membrane protein that is involved in neural cell adhesion and communication. Multiple genome wide association studies have found that NEGR1 is a generic risk factor for multiple human diseases, including obesity, autism, and depression. Recently, we reported that Negr1-/- mice showed a highly increased fat mass and affective behavior. In the present study, we identified Na/K-ATPase, beta1-subunit (ATP1B1) as an NEGR1 binding partner by yeast two-hybrid screening. NEGR1 and ATP1B1 were found to form a relatively stable complex in cells, at least partially co-localizing in membrane lipid rafts. We found that NEGR1 binds with ATP1B1 at its C-terminus, away from the binding site for the alpha subunit, and may contribute to intercellular interactions. Collectively, we report ATP1B1 as a novel NEGR1-interacting protein, which may help deciphering molecular networks underlying NEGR1-associated human diseases. [BMB Reports 2021; 54(3): 164-169].

MAM domain containing 2 is a potential breast cancer biomarker that exhibits tumour-suppressive activity.

OBJECTIVES: The aim of this study was to discover new potential biomarkers of breast cancer and investigate their cellular functions. MATERIALS AND METHODS: We analysed the gene expression profiles of matched pairs of breast tumour and normal tissues from 24 breast cancer patients. Tetracycline-inducible MAMDC2 expression system was established and used to evaluate cell proliferation in vitro and in vivo. MAMDC2-mediated signalling was determined using immunoblot analysis. RESULTS: We identified MAMDC2 as a down-regulated gene showing significant prognostic capability. Overexpression of MAMDC2 or treatment with MAMDC2-containing culture medium significantly inhibited the cell proliferation of T-47D cells. Furthermore, MAMDC2 expression reduced in vivo growth of T-47D xenograft tumours. MAMDC2 may exert its growth-inhibitory functions by attenuating the MAPK signalling pathway. CONCLUSION: We report that MAMDC2 has a tumour-suppressive role and, as a secretory protein, it might be useful as a biomarker for breast cancer treatment.

Actin-microtubule crosslinker Pod-1 tunes PAR-1 signaling to control synaptic development and tau-mediated synaptic toxicity.

Partitioning-defective 1 (PAR-1), a conserved cell polarity regulator, plays an important role in synaptic development, and its mutation affects the formation of synaptic boutons and localization of postsynaptic density protein Discs large (Dlg) at the neuromuscular junction (NMJ) in Drosophila. Drosophila PAR-1 and its human homolog, Microtubule affinity-regulating kinases (MARK), are also known to be implicated in Alzheimer's disease (AD) by controlling tau-mediated Aß toxicity. However, the molecular mechanisms of PAR-1 function remain incompletely understood. Here we identified Pod-1, an actin-microtubule crosslinker, which functionally and physically interacts with PAR-1 in Drosophila. Pod-1 prominently co-localizes with PAR-1 in the postsynaptic region and regulates PAR-1 activity at the NMJ. Synaptic defects, including the reduction of boutons and delocalization of Dlg caused by PAR-1 overexpression, were rescued by Pod-1 knockdown. Conversely, the reduction of synaptic boutons in PAR-1 overexpressed NMJ was synergistically enhanced by the overexpression of Pod-1. Furthermore, Pod-1 increases the PAR-1 dependent S262 phosphorylation of tau, which is known to contribute to tau-mediated Aß toxicity. In line with the change of tau phosphorylation, Pod-1 knockdown rescued tau-mediated synaptic toxicity at the NMJ. Our results suggest that Pod-1 may act as a modulator of PAR-1 in synaptic development and tau-mediated toxicity.

Label-Free Tomographic Imaging of Lipid Droplets in Foam Cells for Machine-Learning-Assisted Therapeutic Evaluation of Targeted Nanodrugs.

Lipid droplet (LD) accumulation, a key feature of foam cells, constitutes an attractive target for therapeutic intervention in atherosclerosis. However, despite advances in cellular imaging techniques, current noninvasive and quantitative methods have limited application in living foam cells. Here, using optical diffraction tomography (ODT), we performed quantitative morphological and biophysical analysis of living foam cells in a label-free manner. We identified LDs in foam cells by verifying the specific refractive index using correlative imaging comprising ODT integrated with three-dimensional fluorescence imaging. Through time-lapse monitoring of three-dimensional dynamics of label-free living foam cells, we precisely and quantitatively evaluated the therapeutic effects of a nanodrug (mannose-polyethylene glycol-glycol chitosan-fluorescein isothiocyanate-lobeglitazone; MMR-Lobe) designed to affect the targeted delivery of lobeglitazone to foam cells based on high mannose receptor specificity. Furthermore, by exploiting machine-learning-based image analysis, we further demonstrated therapeutic evaluation at the single-cell level. These findings suggest that refractive index measurement is a promising tool to explore new drugs against LD-related metabolic diseases.

Two major alternative splice variants of beta-TrCP1 interact with CENP-W with different binding preferences.

Beta-transducin repeat containing protein 1 (ß-TrCP1) is a versatile F-box protein that is responsible for substrate recognition of SCFß-TrCP1 ubiquitin ligase. In human cells, two major alternatively spliced isoforms (b and f) of ß-TrCP1 were found. Recently, we identified that CENP-W interacts with the ß-TrCP1 and regulates the cellular distribution of ß-TrCP1. In this study, we examined whether CENP-W, a new kinetochore component, may differentially regulate the two major isoforms of human ß-TrCP1 (b and f), especially in the cytoplasmic-nuclear shuttling of ß-TrCP1. An in vivo binding assay was performed to examine whether CENP-W binds differently to the two isoforms of ß-TrCP1. EGFP-conjugated ß-TrCP1 isoforms were co-transfected with NLS-defective mutant CENP-W and their cellular distribution were observed using a fluorescence microscopy. Although CENP-W interacts with both b and f isoforms, it has a greater affinity for the b isoform rather than f isoform. Moreover, CENP-W effectively regulates the nuclear-cytoplasmic shuttling of these two ß-TrCP1 isoforms, but with a slight preference towards the b isoform. The Elongin C-binding motif existing in the b isoform may be involved in their specific association. CENP-W showed a higher affinity toward the ß-TrCP1 b isoform, and translocated isoform b more efficiently than isoform f, which may allow a fine regulation of of ß-TrCP1 in the cells.

CENP-W inhibits CDC25A degradation by destabilizing the SCFß-TrCP-1 complex at G2/M.

Skp, Cullin, F-box (SCF)ß-TrCP-1 ubiquitin ligases play a central role in cell cycle regulation and tumorigenesis via proteolytic cleavage of many essential cell cycle regulators. In this study, we propose that centromere protein (CENP)-W, a newly identified kinetochore component, is a novel negative regulator of the SCFß-TrCP-1 complex. CENP-W interacts with Cullin (CUL)-1 and ß-Transducin repeat-containing protein (ß-TrCP)-1 through highly overlapped binding sites with S-phase kinase-associated protein (SKP)-1. CENP-W is incorporated into the SCFß-TrCP-1 complex to promote complex disassembly. Unlike other known regulators that increase SCFß-TrCP-1 ubiquitin ligase activity by promoting complex reassociation, CENP-W-mediated complex disorganization induced ß-TrCP1 degradation and consequently decreased its activity. The association between CENP-W and the SCFß-TrCP-1 complex was prominent during the G2/M transition in the nucleus. Especially, CENP-W knockdown decreased the cell division cycle-25A protein level, leading to a delay in mitotic progression. We propose that CENP-W participates in cell cycle regulation by modulating SCFß-TrCP-1 ubiquitin ligase activity.-Cheon, Y., Lee, S. CENP-W inhibits CDC25A degradation by destabilizing the SCFß-TrCP-1 complex at G2/M.

Centromere protein W interacts with beta-transducin repeat-containing protein 1 and modulates its subcellular localization.

Beta-transducin repeat-containing protein 1 (ß-TrCP1) is a substrate-recognition module of SCFß-TrCP1 ubiquitin ligases and its subcellular distribution is known to be critical for target specificity. Heterogeneous nuclear ribonucleoprotein (hnRNP) U, an abundant nuclear protein, is known to be a unique regulator of ß-TrCP1 shuttling between the cytoplasm and the nucleus. In this study, we report that centromere protein W (CENP-W), which is frequently overexpressed in a variety of human cancers, may also contribute to ß-TrCP1 shuttling. Although hnRNP U and CENP-W can interact with ß-TrCP1 and transport it independently, these proteins do not compete for ß-TrCP1 binding, but rather cooperate to form a stable shuttling complex. Intriguingly, we found that overexpression of CENP-W leads to accumulation of ß-TrCP1 in the nucleus. Thus, we propose that CENP-W may function as a booster of ß-TrCP1 nuclear import to increase the oncogenicity of ß-TrCP1.

New centromeric component CENP-W is an RNA-associated nuclear matrix protein that interacts with nucleophosmin/B23 protein.

CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.

Newly identified tumor-associated role of human Sharpin.

In order to discover previously unidentified cancer-associated genes, we analyzed genome-wide differences in gene expression between tumor biopsies and normal tissues. Among those differentially regulated genes, we identified Sharpin (Shank-associated RH domain-interacting protein) as a commonly up-regulated gene in multiple human cancer types. Although rat Sharpin is reported to interact with Shank1, a multidomain scaffold protein localized in postsynaptic densities, its exact roles are unknown. Whereas human Sharpin homologue was primarily localized in the cytosol of cultured cells, they were detected in both cytosol and nucleus of the cells from ovarian and liver cancer tissues using immunohistochemical staining. In addition, Chinese ovary hamster cells over-expressing Sharpin exhibited enhanced cancer-specific phenotypes in multiple in vitro tumor assays. Taken together, the results suggest that Sharpin is not an inert scaffold protein, but may play tumor-associated roles during cancer biogenesis.