Biology subjects

Nagamori, S.

Publications and source records attributed to Nagamori, S..

11 recordsLinked to original sources

bioRxiv2026

A reabsorption mechanism in Drosophila renal tubules maintains amino acid homeostasis

The kidney plays a central role in maintaining systemic homeostasis by regulating the excretion of metabolic compounds. In mammals, this is achieved through filtration followed by selective reabsorption, whereas insect Malpighian tubules (MTs) are thought to function primarily through active secretion. Although reabsorption of ions and water has been described in MTs, whether these tissues regulate metabolite balance through reabsorption remains unclear. Here, we uncover an amino acid (AA) reabsorption mechanism in Drosophila MTs. Quantification of AAs in excreta reveals that AA excretion changes rapidly and markedly in response to dietary protein levels, while systemic AA levels remain stable. Using ex vivo assays, we show that MTs actively transport AAs. We identify the AA transporter Slimfast (Slif), expressed in the lower tubule, as a key mediator required to maintain systemic levels of basic AAs. These findings reveal a previously unrecognised reabsorption-based strategy in insect renal tubules that functionally parallels mammalian kidney physiology, despite their distinct organisation. Our study demonstrates that AA excretion forms an additional layer to achieve metabolic homeostasis.

physiology↗

bioRxiv2024

Serine chirality guides metabolic flow between one-carbon metabolism and neuromodulator synthesis

O_SCPLOWLC_SCPLOW-serine serves as a central metabolic node that integrates glycolytic flux, lipid metabolism, and one-carbon metabolism. In the mature central nervous system, O_SCPLOWLC_SCPLOW-serine is actively stereo-converted to O_SCPLOWDC_SCPLOW-serine, which functions as a neurotransmitter. However, the role of O_SCPLOWDC_SCPLOW-serine in cellular metabolism remains unclear. Here, we show that O_SCPLOWDC_SCPLOW-serine competes with mitochondrial O_SCPLOWLC_SCPLOW-serine transport, thereby suppressing one-carbon metabolism. Metabolomic analysis revealed that O_SCPLOWDC_SCPLOW-serine reduces intracellular glycine and formate levels, indicating inhibition of the initial step of the one-carbon pathway. Molecular dynamics simulations and enzymatic assays revealed that O_SCPLOWDC_SCPLOW-serine has low affinity for serine hydroxymethyltransferase 2 (Shmt2), which catalyzes the first step in mitochondrial one-carbon metabolism, and does not directly inhibit its activity. Instead, membrane transport assays demonstrated that O_SCPLOWDC_SCPLOW-serine competes with mitochondrial O_SCPLOWLC_SCPLOW-serine transport, depleting the substrate of Shmt2. Functionally, under O_SCPLOWLC_SCPLOW-serine poor conditions in vitro and ex vivo, O_SCPLOWDC_SCPLOW-serine inhibited the proliferation of immature and undifferentiated neural cells including glioblastoma stem cells, which depend highly on one-carbon metabolism. Notably, endogenous O_SCPLOWDC_SCPLOW-serine levels were low during early neurodevelopment, but increased with maturation, coinciding with a shift in the transcriptional profiles of serine metabolic enzymes at the cellular level. Given that O_SCPLOWLC_SCPLOW-serine supports neurodevelopment and O_SCPLOWDC_SCPLOW-serine modulates neurotransmission, this developmental shift in serine enantiomer metabolism appears to align with the functional transitions of the maturing nervous system. Thus, our findings reveal that serine chirality can influence mitochondrial substrate availability and one-carbon flux, offering previously unappreciated insight into the stereoselective regulation of cellular metabolism.

developmental biology↗

bioRxiv2024

Lipogenic Lung Fibroblast-derived Extracellular Vesicles Mitigate Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease Pathologies through LAT1-mediated Alveolar Type II Cell Restoration

Emerging research has revealed specific cellular aberrations in Chronic Obstructive Pulmonary Disease (COPD), with a particular focus on alveolar type 2 (AT2) cells, which play a pivotal role in the restoration of damaged lung tissue and promotion of normal cellular differentiation. Lipofibroblasts (LipoFBs), which are stromal fibroblasts that house lipid droplets, have been identified in close proximity to AT2 cells and have been demonstrated to support AT2 function. In this study, we present a comprehensive investigation into the therapeutic potential of extracellular vesicles (EVs) derived from LipoFBs (LipoFB-EVs) in COPD treatment. They effectively mitigate key COPD pathologies such as cellular senescence and inflammatory responses in lung epithelial cells. This is achieved by reducing reactive oxygen species (ROS) levels and modulating DNA damage response pathways. Moreover, LipoFB-EVs demonstrate antifibrotic properties by inhibiting TGF-{beta}-induced myofibroblast differentiation, surpassing conventional antifibrotic drugs. They also aid in restoring impaired AT2 stem cells, which are crucial for lung homeostasis, by enhancing their viability, colony-forming ability, and proliferation. Furthermore, we identify the presence of L-type amino acid transporter 1 (LAT1) within LipoFB-EVs, which mediates amino acid uptake, particularly leucine transport, and contributes to the restoration of AT2 cell dysfunction. Importantly, the administration of LipoFB-EVs in murine models of COPD resulted in significant improvements in airway inflammation, remodeling, obstruction, cellular senescence, and alveolar emphysema induced by both short- and long-term CS exposure. Overall, our findings highlight the therapeutic potential of LipoFB-EVs as a novel regenerative therapy for COPD, offering promising avenues for future clinical interventions.

molecular biology↗

bioRxiv2023

Identification of three distinct cell populations for urate excretion in human kidney

In humans, uric acid is an end-product of purine metabolism. Urate excretion from human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here we show that renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all renal tubular cells expressed the same set of urate transporters. Only 32% of renal tubular cells were related to both reabsorption and secretion, while the remaining renal tubular cells were related to either reabsorption or secretion, at 5% and 63% respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on cell-units. Our findings also suggest that three different tubular cell populations cooperate to regulate urate excretion from human kidney. Highlight/Key pointsO_LIWe identified three distinct cell populations within the human renal anatomy that predict putative cellular transport mode, and our findings indicate cellular inhomogeneity with distinct roles such as urate secretion and reabsorption. C_LIO_LIOur model of physiological urate handling demonstrates the excretion dynamics in human kidney in terms of single cell-units. C_LIO_LIOur cellular urate transport analyses suggest the reversibility of some urate transporters even in certain physiological conditions. C_LIO_LIThe physiological function of SLC2A9 is not limited to urate reabsorption; it is also involved in urate secretion restriction. C_LIO_LIThis methodology can be applied to investigations of transport mechanisms in general, regardless of epithelial cell types, species, and substrates. C_LI

bioinformatics↗

bioRxiv2021

Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/ AP-2-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a unified model for the endocytosis of SV membranes at mammalian central synapses.

neuroscience↗

bioRxiv2021

Ca2+-mediated higher-order assembly of b0,+AT-rBAT is a key step for system b0,+ biogenesis and cystinuria

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, which causes recurrent kidney stones and occasionally severe kidney failure. Mutations of the two responsible proteins, rBAT and b0,+AT, which comprise system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Although recent structural insights into human b0,+AT-rBAT suggested a model for transport-inactivating mutations, the mechanisms by which type I mutations trigger trafficking deficiencies are not well understood. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+-mediated higher-order assembly of system b0,+ is the key to its trafficking on the cell surface. We show that Ca2+ stabilizes the interface between two rBAT molecules to mediate super-dimerization, and this in turn facilitates the N-glycan maturation of system b0,+. A common cystinuria mutant T216M and mutations that disrupt the Ca2+ site in rBAT cause the loss of higher-order assemblies, resulting in protein trafficking deficiency. Mutations at the super-dimer interface reproduce the mis-trafficking phenotype, demonstrating that super-dimerization is essential for cellular function. Cell-based transport assays confirmed the importance of the Ca2+ site and super-dimerization, and additionally suggested which residues are involved in cationic amino acid recognition. Taken together, our results provide the molecular basis of type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and trafficking diseases in general.

biochemistry↗

bioRxiv2020

D-Serine, an emerging biomarker of kidney diseases, is a hidden substrate of sodium-coupled monocarboxylate transporters

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While D-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal D-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a D-serine transporter previously uncharacterized in the kidney and discovered D-serine as a noncanonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal D-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

biochemistry↗

bioRxiv2019

Human URAT1/SLC22A12 gene promoter is regulated by 27-hydroxycholesterol through estrogen response elements

Elevated levels of uric acid, a metabolite of purine in humans, is related to various diseases, such as gout, atherosclerosis and renal dysfunction. The excretion and reabsorption of uric acid to/from urine is tightly regulated by uric acid transporters. The amino acid sequences of uric acid reabsorption transporters, URAT1/SLC22A12, OAT4/SLC22A11, and OAT10/SLC22A13, share closer phylogenic relationship, whereas the gene promoter sequences are distant phylogenic relationship. Through the single-cell RNA-sequencing analysis of an adult human kidney, we found that only a small number of cells express these transporters, despite their role in the regulation of serum uric acid levels. Transcriptional motif analysis on these transporter genes, revealed that the URAT1/SLC22A12 gene promoter displayed the most conserved estrogen response elements (EREs) among the three transporters. The endogenous selective estrogen receptor modulator (SERM) 27-hydroxycholesterol (27HC) had positive effects on the transcriptional activity of URAT1/SLC22A12. We also found that 27HC increased the protein and gene expression of URAT1/SLC22A12 in mouse kidneys and human kidney organoids, respectively. These results strongly suggest the role of 27HC for URAT1/SLC22A12 expression in renal proximal tubules and upregulation of serum uric acid levels and also show the relationship between cholesterol metabolism and serum uric acid regulation. Significance StatementThe elevated levels of serum uric acid cause various diseases, and the excretion/reabsorption of uric acid to/from urine is tightly regulated by the uric acid transporters. We found that despite the role in serum uric acid regulation, only a small number of cells express URAT1/SLC22A12. We also found that URAT1/SLC22A12 gene promoter region has effective estrogen response elements, and endogenous selective estrogen receptor (ER) modulator 27-hydroxycholesterol (27HC) increased URAT1/SLC22A12 expression in the mice kidneys and human kidney organoids. These suggest that 27HC increases URAT1/SLC22A12 expression and upregulate serum uric acid levels. Since 27HC connects cholesterol metabolism, our study indicates the important link between cholesterol metabolism and serum uric acid regulation, and also provides a novel therapeutic approach to hyperuricemia.

biochemistry↗

bioRxiv2019

Brainstem organoids from human pluripotent stem cells contain neural crest population

The brainstem controls heartbeat, blood pressure and respiration, which are life-sustaining functions, therefore, disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models have limitations, hampering the elucidation of diseases affecting specific components of the brain. Here, we developed a method to generate human brainstem organoids (hBSOs), containing neural crest stem cells as well as midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, and dopaminergic neurons, demonstrated by specific electrophysiological signatures. Single-cell RNA sequence analysis, together with proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem and neural crest, which raises the possibility of making use of hBSOs in grafting for transplantation, efficient drug screenings and modeling the neural crest diseases.

developmental biology↗

bioRxiv2019

Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target Kapβ2 and dysregulate phase separation of low-complexity domains

Low-complexity (LC) domains of proteins are found in about one fifth of human proteome, and a group of LC-domains form labile cross-{beta} polymers and liquid-like droplets. Polymers and droplets formed from LC-domains are dynamically regulated by posttranslational modifications and molecular chaperones including nuclear transport receptors. Repeat expansion in the first intron of a gene designated C9orf72, which is the most prevalent form of familial amyotrophic lateral sclerosis (ALS), causes nucleocytoplasmic transport deficit, however, the detailed mechanism remains unsolved. Here we show that the proline:arginine (PR) poly-dipeptides encoded by the C9orf72 repeat expansion bound nuclear transport receptor Kap{beta}2 through its nuclear localization signal (NLS) recognition motif, and inhibited the ability of Kap{beta}2 to melt fused in sarcoma (FUS) droplets by competing interaction with FUS. The findings in this study offer mechanistic insights as to how the C9orf72 repeat expansion disables nucleocytoplasmic transport and causes neurodegenerative diseases.

molecular biology↗

bioRxiv2019

Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc

The L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 [A] resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.

biochemistry↗