Joon Bum Kim

Migrasomes, the newly discovered cellular organelles that form large vesicle-like structures on the retraction fibers of migrating cells, are thought to be involved in communication between neighboring cells, cellular content transfer, unwanted material shedding, and information integration. Although their formation has been described previously, the molecular mechanisms of migrasome biogenesis are largely unknown. Here, we developed a cell line that overexpresses GFP-tetraspanin4, enabling observation of migrasomes. To identify compounds that regulate migrasome activity in retinal pigment epithelial (RPE) cells, we screened a fecal chemical library and identified cadaverine, a biogenic amine, as a potent migrasome formation inducer. Compared with normal migrating cells, those treated with cadaverine had significantly more migrasomes. Putrescine, another biogenic amine, also increased migrasome formation. Trace amine-associated receptor 8 (TAAR8) depletion inhibited migrasome increase in cadaverine-treated RPE cells, and cadaverine also inhibited protein kinase A phosphorylation. In RPE cells, cadaverine triggers migrasome formation via a TAAR8-mediated protein kinase A signaling pathway.

A specialized membrane-bound organelle, named the melanosome, is central to the storage and transport of melanin as well as melanin synthesis in melanocytes. Although previous studies have linked melanosomal degradation to autophagy, the precise mechanisms remain elusive. Autophagy, a complex catabolic process involving autophagosomes and lysosomes, plays a vital role in cellular constituent degradation. In this study, the role of autophagy in melanosomal degradation was explored, employing a cell-based screening system designed to unveil key pathway regulators. We identified specific dipeptidyl peptidase-4 inhibitors, such as teneligliptin hydrobromide and retagliptin phosphate, as novel agents inducing melanophagy through a comprehensive screening of a ubiquitination-related chemical library. We found that treatment with teneligliptin hydrobromide or retagliptin phosphate not only diminishes melanin content elevated by alpha-melanocyte-stimulating hormone (α-MSH) but also triggers autophagy activation within B16F1 cells. In addition, the targeted inhibition of unc-51-like kinase (ULK1) significantly attenuated both the anti-pigmentation effects and autophagy induced by teneligliptin hydrobromide and retagliptin phosphate in α-MSH-treated cells. Collectively, our data demonstrate a new frontier in understanding melanosomal degradation, identifying teneligliptin hydrobromide and retagliptin phosphate as promising inducers of melanophagy via autophagy activation. This study contributes essential insights into cellular degradation mechanisms and offers potential therapeutic avenues in the regulation of pigmentation.

Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel–Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.

Selective autophagy controls cellular homeostasis by degrading unnecessary or damaged cellular components. Melanosomes are specialized organelles that regulate the biogenesis, storage, and transport of melanin in melanocytes. However, the mechanisms underlying melanosomal autophagy, known as the melanophagy pathway, are poorly understood. To better understand the mechanism of melanophagy, we screened an endocrine-hormone chemical library and identified nalfurafine hydrochlorides, a κ-opioid receptor agonist, as a potent inducer of melanophagy. Treatment with nalfurafine hydrochloride increased autophagy and reduced melanin content in alpha-melanocyte-stimulating hormone (α-MSH)-treated cells. Furthermore, inhibition of autophagy blocked melanosomal degradation and reversed the nalfurafine hydrochloride-induced decrease in melanin content in α-MSH-treated cells. Consistently, treatment with other κ-opioid receptor agonists, such as MCOPPB or mianserin, inhibited excessive melanin production but induced autophagy in B16F1 cells. Furthermore, nalfurafine hydrochloride inhibited protein kinase A (PKA) activation, which was notably restored by forskolin, a PKA activator. Additionally, forskolin treatment further suppressed melanosomal degradation as well as the anti-pigmentation activity of nalfurafine hydrochloride in α-MSH-treated cells. Collectively, our data suggest that stimulation of κ-opioid receptors induces melanophagy by inhibiting PKA activation in α-MSH-treated B16F1 cells.

Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely unknown. In this study, we identified several BRD4 inhibitors, including molibresib, a novel pexophagy inducer, via chemical library screening. Treatment with molibresib promotes loss of peroxisomes selectively, but not mitochondria, ER, or Golgi apparatus in HeLa cells. Consistently, depletion of BRD4 expression also induced pexophagy in RPE cells. In addition, the inhibition of BRD4 by molibresib increased autophagic degradation of peroxisome ATG7-dependency. We further found that molibresib produced reactive oxygen species (ROS), which potentiates ATM activation. Inhibition of ROS or ATM suppressed the loss of peroxisomes in molibresib-treated cells. Taken together, our data suggest that inhibition of BRD4 promotes pexophagy by increasing ROS and ATM activation.

Primary cilia help to maintain cellular homeostasis by sensing conditions in the extracellular environment, including growth factors, nutrients, and hormones that are involved in various signaling pathways. Recently, we have shown that enhanced primary ciliogenesis in dopamine neurons promotes neuronal survival in a Parkinson’s disease model. Moreover, we performed fecal metabolite screening in order to identify several candidates for improving primary ciliogenesis, including L-carnitine and acetyl-L-carnitine. However, the role of carnitine in primary ciliogenesis has remained unclear. In addition, the relationship between primary cilia and neurodegenerative diseases has remained unclear. In this study, we have evaluated the effects of carnitine on primary ciliogenesis in 1-methyl-4-phenylpyridinium ion (MPP⁺)-treated cells. We found that both L-carnitine and acetyl-L-carnitine promoted primary ciliogenesis in SH-SY5Y cells. In addition, the enhancement of ciliogenesis by carnitine suppressed MPP⁺-induced mitochondrial reactive oxygen species overproduction and mitochondrial fragmentation in SH-SY5Y cells. Moreover, carnitine inhibited the production of pro-inflammatory cytokines in MPP⁺-treated SH-SY5Y cells. Taken together, our findings suggest that enhanced ciliogenesis regulates MPP⁺-induced neurotoxicity and inflammation.

Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.

Primary cilia mediate the interactions between cells and external stresses. Thus, dysregulation of primary cilia is implicated in various ciliopathies, e.g., degeneration of the retina caused by dysregulation of the photoreceptor primary cilium. Particulate matter (PM) can cause epithelium injury and endothelial dysfunction by increasing oxidative stress and inflammatory responses. Previously, we showed that PM disrupts the formation of primary cilia in retinal pigment epithelium (RPE) cells. In the present study, we identified 2-isopropylmalic acid (2-IPMA) as a novel inducer of primary ciliogenesis from a metabolite library screening. Both ciliated cells and primary cilium length were increased in 2-IPMA-treated RPE cells. Notably, 2-IPMA strongly promoted primary ciliogenesis and restored PM2.5-induced dysgenesis of primary cilia in RPE cells. Both excessive reactive oxygen species (ROS) generation and activation of a stress kinase, JNK, by PM2.5 were reduced by 2-IPMA. Moreover, 2-IPMA inhibited proinflammatory cytokine production, i.e., IL-6 and TNF-α, induced by PM2.5 in RPE cells. Taken together, our data suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in RPE cells.

Mitochondrial dynamics control mitochondrial morphology and function, and aberrations in these are associated with various neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. To identify novel regulators of mitochondrial dynamics, we screened a phytochemical library and identified liquiritigenin as a potent inducer of mitochondrial fusion. Treatment with liquiritigenin induced an elongated mitochondrial morphology in SK-N-MC cells. In addition, liquiritigenin rescued mitochondrial fragmentation induced by knockout of mitochondrial fusion mediators such as Mfn1, Mfn2, and Opa1. Furthermore, we found that treatment with liquiritigenin notably inhibited mitochondrial fragmentation and cytotoxicity induced by Aβ in SK-N-MC cells.