Biology subjects

Shibata, H.

Publications and source records attributed to Shibata, H..

9 recordsLinked to original sources

bioRxiv2026

An ITK - SLP-76 Y173 Axis Regulates TCR Signal Persistence and Thymic Development

The TCR-induced tyrosine kinase cascade results in ITK-mediated phosphorylation of at least two substrates: PLC-{gamma}1 Y783, an essential mediator of TCR signaling, and SLP-76 Y173. We previously provided evidence that SLP-76 Y173 supports proximal TCR and Fc{varepsilon}RI signaling; but its role in thymocyte development was never explored. Here we demonstrate that SLP-76 Y173 was required for TCR/CD28-induced degradation of I{kappa}B; moreover, both ITK and SLP-76 Y173 were required for persistent TCR-induced phosphorylation of PLC-{gamma}1, ERK and p38. To explore whether SLP-76 Y173 may mediate ITK-dependent events in vivo, we replaced SLP 76 Y173 with phenylalanine in a mouse model. Like ITK-deficient thymocytes, SLP-76 Y173F thymocytes exhibited reduced thymic cellularity, reduced CD4/CD8 ratios, and reduced surface expression of CD5. In ex vivo-stimulated thymocytes, mutation of SLP-76 Y173 or inhibition of ITK reduced the persistence of TCR-induced calcium flux; moreover, TCR-induced phosphorylation of ERK and p38 depended on SLP-76 Y173. Like ITK-deficient thymi, SLP-76 Y173F thymocytes exhibited decreased production of CD4- iNKT1 thymocytes, coupled with increased production of innate-like CD4+ and CD8+ SP thymocytes. Our results provide strong evidence that the ITK substrate site, SLP-76 Y173, promotes TCR signal persistence that is required to mediate the thymic developmental effects of ITK.

immunology↗

bioRxiv2026

Pre-movement respiration-action coupling is specific to self-initiated action: Evidence for action alignment with ongoing breathing

Voluntary actions are coupled with the respiratory cycle, yet whether this coupling reflects action timing aligning with breathing or breathing adjusting to anticipated action remains unclear. This pre-registered study compared respiration-action coupling across three conditions using a modified Libet clock task. Thirty participants performed key presses under Self-initiated (freely chosen timing), Delayed (predictable timing after one full clock rotation), and Immediate (speeded response to stimulus onset) conditions. Circular uniformity tests revealed significant respiratory phase concentration at key press exclusively in the Self-initiated condition, and time-resolved phase consistency analysis showed that coupling emerged before movement onset only in this condition. These findings suggest that action timing aligns with ongoing breathing rhythms, and that temporal freedom, rather than preparation time or predictability, is critical for this coupling to emerge. Additionally, actions performed during inhalation were followed by shorter and deeper subsequent breaths across all conditions, indicating greater perturbation of ongoing breathing when acting during inhalation. Participants reported a subjective preference for pressing during exhalation, which correlated with individual differences in exhalation bias during self-initiated actions. Body awareness, but not interoceptive awareness or trait anxiety, predicted coupling strength. These results suggest that pre-movement respiration-action coupling reflects an implicit process whereby self-chosen action timing exploits ongoing respiratory states, potentially minimizing action-related respiratory perturbation.

neuroscience↗

bioRxiv2024

Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

The mechanistic target of rapamycin complex 1 (mTORC1) functions as a master regulator of cell growth and proliferation. We previously demonstrated that intracellular calcium ion (Ca2+) concentration modulates the mTORC1 pathway via binding of the Ca2+ sensor protein calmodulin (CaM) to tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTORC1. However, the precise molecular mechanism by which Ca2+/CaM modulates mTORC1 activity remains unclear. Here, we performed a binding assay based on nano-luciferase reconstitution, a method for detecting weak interactions between TSC2 and its target, Ras homolog enriched in brain (Rheb), an activator of mTORC1. CaM inhibited the binding of TSC2 to Rheb in a Ca2+-dependent manner. Live-cell imaging analysis indicated increased interaction between the CaM-binding region of TSC2 and CaM in response to elevated intracellular Ca2+ levels. Furthermore, treatment with carbachol, an acetylcholine analog, elevated intracellular Ca2+ levels, and activated mTORC1. Notably, carbachol-induced activation of mTORC1 was inhibited by CaM inhibitors, corroborating the role of Ca2+/CaM in promoting the mTORC1 pathway. Consistent with the effect of Ca2+/CaM on the TSC2-Rheb interaction, increased intracellular Ca2+ concentration promoted the dissociation of TSC2 from lysosomes without affecting Akt-dependent phosphorylation of TSC2, suggesting that the regulatory mechanism of TSC2 by Ca2+/CaM is distinct from the previously established action mechanism of TSC2. Collectively, our findings offer mechanistic insights into TSC2-Rheb regulation mediated by Ca2+/CaM, which links Ca2+ signaling to mTORC1 activation.

biochemistry↗

bioRxiv2024

ZIP13 regulates lipid metabolism by changing intracellular iron and zinc balance

Metabolic diseases are caused by a prolonged energy imbalance, and adipose tissue is known to be the main contributor. We previously reported that ZIP13, an Slc39a transporter whose deficiency causes Ehlers-Danlos syndrome spondylocheirodysplastic type 3 associated with lipoatrophy, inhibits the adipocyte browning pathway by modulating intracellular zinc status. The precise mechanisms of how ZIP13 regulates the homeostasis of adipose tissue remain unclear and therefore, we investigated the role of ZIP13 in mature adipocytes using adipocyte-specific Zip13-deficient mice. We herein demonstrate that these mice show accelerated lipolysis and reduced respiratory exchange ratio. In addition, abundance of iron and zinc balance were altered during differentiation in normal adipocytes, whereas iron distribution was substantially affected in Zip13-deficient adipocytes, which downregulated PDE activity and enhanced {beta}-adrenergic receptor signaling pathways. Importantly, we confirmed that ZIP13 could transport both zinc and iron, using the Xenopus oocyte transport system and in silico structural dynamics simulations, and that the defect in iron distribution perturbs proper lipolysis. Together, these results illustrate that ZIP13 acts as a key regulator for lipolysis in adipocytes via the proper use of metals, and that the ZIP13-iron axis plays an important role in regulation of lipid metabolism.

cell biology↗

bioRxiv2023

Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks

Although previous studies have reported that pre-mRNA splicing factors (SFs) are involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), their exact role in promoting HR remains poorly understood. Here, we showed that SART1, an SF upregulated in several types of cancer, promotes DSB end resection, which is an essential step in HR, without affecting the expression of HR factors. In the promotion of resection, SART1 is epistatic with BRCA1, an HR factor known to promote resection. Mechanistically, SART1 cooperates with BRCA1 to counteract the resection blockade posed by 53BP1 and RIF1. Moreover, SART1 is required for recruitment of the HR-promoting portion of BRCA1 to DSBs. Laser microirradiation experiments revealed that SART1 is recruited to DSBs in a BRCA1-dependent manner, and that the arginine/serine (RS)-rich domain is required for SART1 recruitment to DSBs. We also obtained evidence that SART1 is involved in transcription-associated HR together with BRCA1. Furthermore, chromosome analysis demonstrated that SART1 and BRCA1 epistatically suppressed genomic alterations caused by DSB misrepair in the G2 phase. Collectively, these results indicate that SART1 and BRCA1 cooperatively facilitate resection of DSBs arising in transcriptionally active genomic regions, thereby promoting faithful HR repair of such DSBs and suppressing genome instability.

cell biology↗

bioRxiv2021

Aberrant localization of CDC42 C-terminal variants to the Golgi apparatus drives pyrin inflammasome-dependent autoinflammation

Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Elevated levels of serum IL-18 in patients and effectiveness of IL-1{beta}-blocking therapy indicate that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1{beta} in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but no that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave way for elucidating the mechanism of pyrin inflammasome formation.

immunology↗

bioRxiv2019

Resurrecting the ancient glow

The colour of firefly bioluminescence is primarily determined by the structure of the enzyme luciferase1. To date, firefly luciferase genes have been isolated from over 30 extant species producing light ranging in colour from deep-green to orange-yellow. We have reconstructed ancestral firefly luciferase genes and characterised the enzymatic properties of the recombinant proteins in order to predict ancestral firefly light emission. Results showed that the synthetic luciferase for the last common firefly ancestor exhibited green light. All known firefly species are bioluminescent in the larval stages2, with a common shared ancestor arising approximately 100 Mya3. Combined, our findings propose within the Cretaceous forest the common ancestor of contemporary fireflies emitted green light, most likely for aposematic display from nocturnal predation.

evolutionary biology↗

bioRxiv2019

Jomon genome sheds light on East Asian population history

Anatomical modern humans reached East Asia by >40,000 years ago (kya). However, key questions still remain elusive with regard to the route(s) and the number of wave(s) in the dispersal into East Eurasia. Ancient genomes at the edge of East Eurasia may shed light on the detail picture of peopling to East Eurasia. Here, we analyze the whole-genome sequence of a 2.5 kya individual (IK002) characterized with a typical Jomon culture that started in the Japanese archipelago >16 kya. The phylogenetic analyses support multiple waves of migration, with IK002 forming a lineage basal to the rest of the ancient/present-day East Eurasians examined, likely to represent some of the earliest-wave migrants who went north toward East Asia from Southeast Asia. Furthermore, IK002 has the extra genetic affinity with the indigenous Taiwan aborigines, which may support a coastal route of the Jomon-ancestry migration from Southeast Asia to the Japanese archipelago. This study highlight the power of ancient genomics with the isolated population to provide new insights into complex history in East Eurasia.

genomics↗

bioRxiv2018

A new targeted capture method using bacterial artificial chromosome (BAC) libraries as baits for sequencing relatively large genes

To analyze a specific genome region using next-generation sequencing technologies, the enrichment of DNA libraries with targeted capture methods has been standardized. For enrichment of mitochondrial genome, a previous study developed an original targeted capture method that use baits constructed from long-range polymerase chain reaction (PCR) amplicons, common laboratory reagents, and equipment. In this study, a new targeted capture method is presented, that of bacterial artificial chromosome (BAC) double capture (BDC), modifying the previous method, but using BAC libraries as baits for sequencing a relatively large gene. We applied the BDC approach for the 214 kb autosomal region, ring finger protein 213, which is the susceptibility gene of moyamoya disease (MMD). To evaluate the reliability of BDC, cost and data quality were compared with those of a commercial kit. While the ratio of duplicate reads was higher, the cost was less than that of the commercial kit. The data quality was sufficiently the same as that of the kit. Thus, BDC can be an easy, low-cost, and useful method for analyzing individual genome region with substantial length.

genomics↗