Biology subjects

Yamada, Y.

Publications and source records attributed to Yamada, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Cold adaptation in Upper Paleolithic hunter-gatherers of eastern Eurasia

Previous genomic studies understanding the dispersal of Homo sapiens have suggested that present-day East Eurasians and Native Americans can trace their ancestry to migrations from Southeast Asia. However, ineluctable adaptations during the Last Glacial Maximum (LGM) remain unclear. By analyzing 42 genomes of up to 30-fold coverage from prehistoric hunter-gatherers, Jomon, we reveal their descent from Upper Paleolithic (UP) foragers who migrated to and isolated in the Japanese archipelago during Late Pleistocene. We provide compelling evidence suggesting that these UP people underwent positive selection for cold environments, aiding their survival through the LGM facilitated by non-shivering thermogenesis and detecting it polygenically across multiple loci in the Jomon lineage. Our study pioneers the close estimation of the physiological adaptation of ancient humans by the paleogenomic approach.

evolutionary biology↗

bioRxiv2024

Sildenafil amplifies calcium influx and insulin secretion in pancreatic β cells

Sildenafil, a phosphodiesterase-5 (PDE5) inhibitor, has been shown to improve insulin sensitivity in animal models and prediabetic patients. However, its other metabolic effects remain poorly investigated. This study examines the impact of sildenafil on insulin secretion in MIN6-K8 mouse clonal {beta} cells. Sildenafil is shown to amplify insulin secretion by enhancing Ca2+ influx, an effect that requires other depolarizing stimuli in MIN6-K8 cells but not in KATP channel-deficient {beta} cells, which are already depolarized. These results indicate that the action of sildenafil is dependent on depolarization and is independent of KATP channels. Furthermore, sildenafil-amplified insulin secretion is not inhibited by nifedipine or PDE5 knockdown. Thus, sildenafil stimulates Ca2+ influx independently of L-type voltage-dependent Ca2+ channels (VDCCs) and PDE5, a mechanism that differs from the known pharmacology of sildenafil and conventional insulin secretory pathways. Our results reposition sildenafil as an insulinotropic agent that can be used as a potential anti-diabetic medicine or a tool to elucidate the molecular mechanism of insulin secretion.

cell biology↗

bioRxiv2023

Heart generation via blastocyst complementation in Mesp1/2-deficient mice

Heart transplantation is the only curative option available for patients with advanced heart failure. However, donor organ shortage and graft rejection remain critical challenges in heart transplantation. Blastocyst complementation using pluripotent stem cells (PSCs) can be used to generate organs such as the pancreas and kidneys in animal models with abnormalities in essential developmental genes. Nonetheless, whether functional adult hearts can be generated with blastocyst complementation remains unclear, and it is unknown if parenchyma, blood vessels, and stroma can be generated concomitantly from PSCs using blastocyst complementation to avoid graft rejection. Here, we show the generation of functional adult hearts in acardiac Mesp1 and Mesp2 double-knockout (Mesp1/2-DKO) mice via blastocyst complementation using mouse PSCs. Our result shows that the generated hearts were structurally and functionally normal and restored embryonic lethality in Mesp1/2-DKO mice. All four cardiovascular lineages, including cardiomyocytes, vascular endothelial cells, smooth muscle cells, and cardiac fibroblasts, were virtually entirely derived from exogenous PSCs in the myocardium. Exogenous rat PSCs also generated rat-derived xenogeneic hearts in Mesp1/2-DKO mice via interspecies blastocyst complementation. Thus, blastocyst complementation is a viable technique for generating hearts derived from PSCs and may represent significant progress toward generating rejection-free hearts.

cell biology↗

bioRxiv2023

CTRP7 as a Molecular Biomarker for Predicting Responsiveness to Pulmonary Vasodilators: Insights from Human and Animal Studies in Pulmonary Arterial Hypertension

BackgroundPulmonary arterial hypertension (PAH) is a life-threatening condition. Although pulmonary vasodilators have shown promise in managing PAH, the overall prognosis improvement is modest, partly because of the absence of a biomarker for guiding their selection. This study aimed to identify the molecular-based predictors of responsiveness to pulmonary vasodilators by combining human and basic studies. Methods and ResultsRNA sequencing was conducted on cultured pulmonary artery smooth muscle cells (PASMCs) from patients with and without PAH, identifying variations in 2,076 genes. The candidates were narrowed down to established clinical biomarkers, and their plasma levels were assessed in PAH (n = 123) and non-PAH patients (n = 25). C1q/TNF-related protein 7 (CTRP7) exhibited elevated expression in PASMCs (5.37 Log2 fold change, p < 0.01), and the plasma of patients with PAH compared to non-PAH (28.2 (9.3- 101.2) vs. 9.32 (4.6-18.6) ng/ml, p < 0.01). A plasma assessment revealed a significant correlation between CTRP7 and interleukin 6 (IL-6) levels (r = 0.544, p < 0.001). Chromatin immunoprecipitation demonstrated that IL-6 upregulated CTRP7 expression in PASMCs. Among pivotal factors, CTRP7 reduced prostacyclin analog receptor (PTGIR) expression through Rab5a-mediated internalization, resulting in diminished responsiveness to selexipag (a prostacyclin analog). Incorporating the human study, hypoxic PH mice demonstrated reduced PTGIR expression in the pulmonary arteries, which correlated with limited responses to selexipag treatment (low cardiac output and persistent pulmonary artery resistance), which was mitigated by silencing CTRP7 expression in the pulmonary arteries using adeno-associated virus 6. ConclusionsAmong the candidates with increased expression in patients with PAH based on RNA sequencing of PASMCs, CTRP7 demonstrated elevated plasma levels compared to non-PAH. CTRP7 regulated PTGIR internalization via inflammation and Rab5a, influencing responsiveness to selexipag. CTRP7 emerged as a compelling biomarker for predicting responsiveness to prostacyclin analogs, potentially advancing the development of innovative PAH treatment strategies. Novel and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIPulmonary arterial hypertension (PAH) is a life-threatening vascular disease with limited treatment options. C_LIO_LIAlthough pulmonary vasodilators have shown promise in managing PAH, the overall improvement in prognosis is modest. C_LIO_LIFurther research is needed to understand the molecular features of pulmonary vasodilators and identify biomarkers for appropriate selection. C_LI What New Information Does This Article Contribute?O_LIC1q/TNF-related protein 7 (CTRP7) was found to be elevated in the pulmonary artery smooth muscle cells (PASMCs) and plasma of patients with pulmonary arterial hypertension (PAH). C_LIO_LIThis study established a connection between inflammation and pulmonary vascular responsiveness via CTRP7-mediated internalization and degradation of the prostacyclin analog receptor. C_LIO_LIThis study indicated the possibility of using plasma CTRP levels as a clinical biomarker to monitor pulmonary responsiveness, which can contribute to the development of novel treatment strategies for patients with PAH. C_LI Pulmonary arterial hypertension (PAH) is a life-threatening and enigmatic lung vascular disorder characterized by progressive pulmonary vascular remodeling and vasoconstriction. Despite significant advancements in the treatment of PAH using pulmonary vasodilators, some individuals display limited responses, primarily due to a lack of understanding of the molecular features of each pulmonary vasodilator and the absence of a biomarker for appropriate selection. The present study aimed to identify promising biomarkers for predicting a patients responsiveness to pulmonary vasodilators through a two-step approach, involving RNA sequencing of PASMCs and the measurement of plasma levels. CTRP7 emerged as a prominent biomarker, displaying increased expression in both PASMCs and plasma obtained from patients with PAH. Mechanistically, IL-6 transcriptionally modulated CTPR7, leading to the internalization and degradation of PTGIR, resulting in a reduced response to selexipag (prostacyclin analog) in PASMCs. Integrating the human studies, a hypoxic mouse model exhibited lower PTGIR expression in pulmonary arteries, correlating with limited responses to selexipag treatment, which were ameliorated by silencing CTRP7 expression in pulmonary arteries using adeno-associated virus 6 transduction. This study collectively demonstrates that CTRP7 emerges as a compelling biomarker for predicting responsiveness to prostacyclin analogs, with the potential to advance the development of innovative PAH treatment strategies.

molecular biology↗

bioRxiv2023

Neonics and mites can interfere with extending the bee-colony longevity during wintering

A mathematical model that can estimate the apparent longevity of bee-colony proposed by the authors to clarify that the longevity normally changes with seasons as follows: The longevity maintains almost constant from at 20 to 30 days from late spring to late autumn, thereafter, it continues to extend till late spring up to 160 to 200 days. Immediately after overwintering, the longevity is shortened almost vertically from one sixth to one tenth. Such normal seasonal changes in longevity are shown when a pesticide-free food is administered to the bee-colony and when pesticide-containing sugar syrup is. However, abnormal seasonal changes in longevity, which does not extend even if winter approaches, are shown for the bee-colony ingesting neonicotinoid-containing pollen and for the colony infested with Varroa mites. Judging from the fact that pollen is the main food for the bee-brood, that mites parasitize larvae and pupae, and that the vital functions and organs of honeybees are created during the larval and pupal stages, it can be inferred that a neonicotinoid-containing pollen paste and parasitic mites cause serious damage to the bee ability to detect the arrival of winter. Such dysfunction in the larval and pupal stages probably interferes with extending the longevity of adult bees even as winter approaches.

zoology↗

bioRxiv2023

NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, which is typically considered too large for NMR studies, we successfully assigned the main chain using an amino acid-selective unlabeling method. Because we found that the Zn2+-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by mutating the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

biochemistry↗

bioRxiv2022

Disruption of lactate metabolism in the peripheral nervous system leads to motor-selective deficits

Schwann cells (SCs) myelinate and provide trophic support to axons in the peripheral nervous system (PNS) and disruption of SC cellular metabolism leads to demyelination and axon degeneration, both symptoms of peripheral neuropathies. The lactate shuttle hypothesis proposes that glycolytic support cells supply lactate to adjacent axons to sustain their high metabolic demands, a process that requires the interconversion of lactate and pyruvate via lactate dehydrogenase (LDH) in both SCs and neurons. To test this hypothesis in the PNS, we selectively knocked out the genes for both LDH enzymes, LDHA and LDHB, in motor neurons (MNs), sensory neurons (SNs), or SCs. Interestingly, motor axons and their synapses progressively degenerate when LDH is deleted from either MNs or SCs; however, defects in sensory axons or their terminals were not observed when LDH was excised from either SNs or SCs. Deletion of LDH in SCs also leads to a decrease in total ATP levels in peripheral nerves despite a marked accumulation of pyruvate and glycolytic intermediates, consistent with the failure of pyruvate to lactate conversion in SCs leading to energetic deficits in axons. These results support a model in which motor axons are more dependent on SC-derived lactate than are sensory axons, a specific dependency that suggests LDH and lactate shuttling influence the course of motor-dominated neuropathies such as ALS.

neuroscience↗

bioRxiv2022

SARM1 promotes axonal, synaptic, and mitochondrial pathologies in Charcot-Marie-Tooth disease type 2A

Charcot-Marie-Tooth disease (CMT) type 2A is an axonal neuropathy caused by mutations in the mitofusin 2 (MFN2) gene. MFN2 mutations result in profound mitochondrial abnormalities, but the mechanism underlying axonal pathology is unknown. SARM1, the central executioner of axon degeneration, can induce neuropathy and is activated by dysfunctional mitochondria. We tested the role of SARM1 in a rat model carrying a dominant CMT2A mutation (Mfn2H361Y) that exhibits progressive dying-back axonal degeneration, NMJ abnormalities, muscle atrophy, and mitochondrial abnormalities, all hallmarks of the human disease. We generated Sarm1 knockout and Mfn2H361Y, Sarm1 double mutant rats and find that deletion of SARM1 rescues axonal, synaptic, and muscle phenotypes, demonstrating that SARM1 induces much of the neuropathology in this model. Despite the presence of mutant Mfn2 protein in these double mutant rats, loss of SARM1 also dramatically suppressed many mitochondrial defects, including the number, size, and cristae density defects of synaptic mitochondria. This surprising finding indicates that dysfunctional mitochondria activate SARM1, and activated SARM1 feeds back on mitochondria to exacerbate mitochondrial pathology. As such, this work identifies SARM1 inhibition as an exciting therapeutic candidate for the treatment of CMT2A and other neurodegenerative diseases with prominent mitochondrial pathology.

neuroscience↗

bioRxiv2022

Loss of Stathmin-2, a hallmark of TDP-43-associated ALS, causes motor neuropathy

TDP-43 mediates proper Stathmin-2 (STMN2) mRNA splicing, and STMN2 protein is reduced in the spinal cord of most ALS patients. To test the hypothesis that STMN2 loss contributes to ALS pathogenesis, we generated constitutive and conditional STMN2 knockout mice. Constitutive STMN2 loss results in early-onset sensory and motor neuropathy featuring impaired motor behavior and dramatic distal neuromuscular junction (NMJ) denervation of fast-fatigable motor units, which are selectively vulnerable in ALS, without axon or motoneuron degeneration. Selective excision of STMN2 in motoneurons leads to similar NMJ pathology. STMN2 KO heterozygous mice, which better model the partial loss of STMN2 protein found in ALS patients, display a slowly progressive, motor-selective neuropathy with functional deficits and NMJ denervation. Thus, our findings strongly support the hypothesis that STMN2 reduction due to TDP-43 pathology contributes to ALS pathogenesis.

neuroscience↗

bioRxiv2022

Macrophage depletion blocks congenital SARM1-dependent neuropathy

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified two rare NMNAT2 missense variants in two brothers afflicted with a progressive neuropathy syndrome. The polymorphisms result in amino acid substitutions, V98M and R232Q, which reduce NMNAT2 NAD+-synthetase activity. We generated a mouse model of the human syndrome and found that Nmnat2V98M/Nmnat2R232Q compound-heterozygous CRISPR mice survive to adulthood but develop progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes are all SARM1-dependent. Remarkably, macrophage depletion therapy blocks and reverses neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induces an inflammatory neuropathy and highlight the potential of immune therapy to treat this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

neuroscience↗

bioRxiv2021

Theoretical investigation of active listening behavior based on the echolocation of CF-FM bats

Bats perceive the three-dimensional (3D) environment by emitting ultrasound pulses from their nose or mouth and receiving echoes through both ears. To detect the position of a target object, it is necessary to know the distance and direction of the target. Certain bat species synchronize the movement of their pinnae with pulse emission, and it is this behavior that enables 3D direction detection. However, the significance of bats ear motions remains unclear. In this study, we construct a model of an active listening system including the motion of the ears, and conduct mathematical investigations to clarify the importance of ear motion in 3D direction detection. The theory suggests that only certain ear motions, namely three-axis rotation, accomplish accurate and robust 3D direction detection. Our theoretical analysis also strongly supports the behavior whereby bats move their pinnae in the antiphase mode. In addition, we provide the conditions for ear motions to ensure accurate and robust direction detection, suggesting that simple shaped hearing directionality and well-selected uncomplicated ear motions are sufficient to achieve precise and robust 3D direction detection. Our findings and mathematical approach have the potential to be used in the design of active sensing systems in various engineering fields. Author SummaryMany mammals use visual sensing for primary perception of their surroundings, whereas bats accomplish spatial perception by active acoustic sensing. In particular, by emitting ultrasound pulses and listening to the echoes, bats localize reflective objects, a process known as echolocation. Certain bat species move both of their ears while receiving the echoes, but the essential theory behind this ear movement remains unclear. This paper describes a simple mathematical model for investigating the active listening strategy employed by bats. The theory suggests that the ear motions employed by bats enables highly accurate direction detection that is robust to observation errors. In addition, we determine what kind of ear motions are optimal for 3D direction detection. This study not only reveals the significance of pinnae motions in bats, but also opens up the possibility of engineering applications for active listening systems.

animal behavior and cognition↗

bioRxiv2021

Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted scientists to study their cancer resistance mechanisms in order to provide clues for human cancer prevention. Although cancer resistance in NMRs has been intensively investigated at the cellular level, it is still unknown how strongly resistant NMR individuals are to carcinogenesis and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration and reduced induction of inflammatory genes. NMRs harbor loss-of-function mutations in receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. A necroptosis-inducing stimulus did not increase death of NMR cells. After carcinogenic insults, leakage of the HMGB1, a marker of necrotic cell death, was not increased in NMR skin. In mice, inhibition or knockout of RIPK3 reduced immune cell infiltration and delayed the onset of chemical carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs. Further in vivo study of the unusual tissue immune system and carcinogenesis resistance of NMRs may lead to the development of new strategies to prevent carcinogenesis in humans. Significance StatementIn contrast with intensive studies of cancer resistance mechanisms in naked mole-rats (NMRs) at the cellular level, little is known about how NMR individuals respond to carcinogenesis induction, despite the fact that cell-to-cell interactions in tissues regulate carcinogenesis in vivo. Here, we demonstrate that NMRs are remarkably resistant to chemical carcinogenesis induction and characteristically have attenuated tissue inflammatory responses to carcinogenic insults. NMRs have loss-of-function mutations in RIPK3 and MLKL genes and thus cannot activate necroptosis, a type of inflammation-inducing cell death. RIPK3 inhibition in mice reduced immune cell infiltration in response to carcinogenic insults and delayed the onset of chemical-induced carcinogenesis. Our results highlight the importance of studies on dampened tissue inflammatory responses to understand cancer resistance of NMRs.

cancer biology↗

bioRxiv2021

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might also activate SARM1. Here we show that the nicotinamide analogue 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration and neuronal death. Systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to peripheral nerve induces SARM1-dependent axon degeneration. We also identify a related pyridine derivative, 2-aminopyridine, as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity, and furthermore, suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

neuroscience↗

bioRxiv2021

Tandem mass spectrum similarity-based network analysis using 13C-labeled and non-labeled metabolome data to identify the biosynthesis pathway of the blood pressure-lowering asparagus metabolite asparaptine A

Asparaptine, a conjugate of arginine and asparagusic acid, was found in asparagus (Asparagus officinalis) as a naturally occurring inhibitor of angiotensin-converting enzyme (ACE) in vitro. The biosynthetic pathway to asparaptine is largely unknown; however, it is suggested that asparagusic acid may be biosynthesized from valine. To determine which metabolites are involved in the asparaptine biosynthetic pathway, we performed tandem mass spectrometry similarity-based metabolome network analysis using 13C labeled and non-labeled valine-fed asparagus calluses. We determined that valine is used as a starting material, S(2-carboxy-n-propyl)-cysteine as an intermediate, and two new metabolites as asparaptine analogs, lysine- and histidine-type conjugates, are involved in the pathway. Asparaptine was therefore renamed asparaptine A (arginine type), and the two analogs were named asparaptines B (lysine type) and C (histidine type). Oral feeding of asparaptine A to a hypertensive mouse species showed that this metabolite lowers both blood pressure and heart rate within two hours and both of which were back to normal two days later. These results suggest that asparaptine A may not only have effects as an ACE inhibitor, but also has {beta}-antagonistic effects, which are well-known to be preventive for cardiovascular diseases.

plant biology↗

bioRxiv2021

Transport engineering for improving production and secretion of valuable alkaloids in Escherichia coli

Metabolic engineering of microorganisms to produce specialized plant metabolites has been established. However, these methods are limited by low productivity and the intracellular accumulation of metabolites. Here, we aimed to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. We established a reticuline-producing Escherichia coli strain and introduced a multidrug and toxic compound extrusion transporter, Arabidopsis AtDTX1, into it. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 significantly enhanced reticuline production by 11-fold; produced reticuline was secreted into the medium. AtDTX1 expression conferred high plasmid stability, and up- or downregulated genes associated with biological processes including metabolic pathways for reticuline biosynthesis, leading to a high production and secretion of reticuline. The successful application of a transporter for alkaloid production suggests that the transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

synthetic biology↗

bioRxiv2021

Multi-metabolomics using imaging mass spectrometry and liquid chromatography-tandem mass spectrometry for spatially characterizing monoterpene indole alkaloids secreted from roots

Plants release specialized (secondary) metabolites from their roots to communicate with other organisms, including soil microorganisms. The spatial behavior of such metabolites around these roots can help us understand roles for the communication; however, currently they are unclear because soil-based studies are complex. Here, we established a multi-metabolomics approach using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially assign metabolites under laboratory conditions using agar. In a case study using Catharanthus roseus, we showed that 58 nitrogen (N)-containing metabolites are released from the roots into the agar. For the metabolite assignment, we used 15N-labeled and nonlabeled LC-MS/MS data, previously reported. Four metabolite ions were identified using authentic standard compounds as derived from monoterpene indole alkaloids (MIAs) such as ajmalicine, catharanthine, serpentine, and yohimbine. An alkaloid network analysis using dot products and spinglass methods characterized five clusters to which the 58 ions belong. The analysis clustered ions from the indolic skeleton-type MIAs to a cluster, suggesting that other communities may represent distinct metabolite groups. For future chemical assignments of the serpentine community, key fragmentation patterns were characterized using the 15N-labeled and nonlabeled MS/MS spectra.

plant biology↗

bioRxiv2020

Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear. Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

biophysics↗

bioRxiv2020

Size matters: optimal mask diameter and box size for single-particle cryogenic electron microscopy

Recently it has been demonstrated that single-particle cryogenic electron microscopy (cryo-EM) at 200 keV is capable of determining protein structures, including those smaller than 100 kDa, at sub-3.0 [A] resolutions, without using significant defocus or a phase plate. However, the majority of near-atomic resolution cryo-EM structures has been determined using 300 keV. Consequently, many typical parameter settings for the cryo-EM computational image processing steps, especially those associated with the contrast transfer function, are based on the accumulated experience of 300 kV cryo-EM. We have therefore revised these parameters, established theoretical bases for criteria to find an optimal mask diameter and box size for a given dataset irrespective of acceleration voltage or protein size, and proposed a protocol. Considering the defocus distributions of the datasets, merely optimizing the mask diameters and box sizes yielded meaningful resolution improvements for the reconstruction of < 200 kDa proteins using 200 kV cryo-EM.

molecular biology↗