Biology subjects

Moriya, H.

Publications and source records attributed to Moriya, H..

10 recordsLinked to original sources

bioRxiv2026

Limit-pushing overexpression reveals constraints on protein abundance

Proteins are often classified as toxic or non-toxic without measuring the abundance reached, leaving constraints on tolerable protein abundance unresolved. We established a limit-pushing approach in Saccharomyces cerevisiae combining strong inducible expression with gTOW-mediated high-copy selection to counteract copy-number compensation while measuring protein abundance and growth. Nearly all of approximately 80 chromosome I proteins severely inhibited growth or reduced viability at sufficiently high abundance. We established IE50, the expression level associated with a 50% reduction in growth rate, to quantify their widely varying overexpression tolerance. IE50 was positively associated with predicted structural order and cytoplasmic localization propensity and negatively associated with sulphur content. Single-cell imaging linked higher tolerance to proteins remaining cytoplasmic without becoming aggregation-positive and revealed abundance-dependent changes in localization and organelle morphology. At extreme abundance, Fun12, Nup60, and Pex22 generated distinct large-scale intracellular states through specific sequence regions. These findings establish overexpression toxicity as a quantitative property linked to protein characteristics and reveal both constraints on tolerable abundance and sequence-dependent capacities for intracellular organization.

systems biology↗

bioRxiv2025

Systematic overexpression reveals a conserved spectrum of harmful and beneficial amino acid homorepeats shaped by evolution

Proteins are composed of combinations of 20 amino acids, yet the principles governing their sequence compatibility within living cells remain poorly understood. Here, we evaluated the "neutrality"--an integrated measure of harmful versus beneficial properties--of amino acid homorepeats (PolyX) in Saccharomyces cerevisiae. Using the genetic tug-of-war (gTOW) method to quantify maximum expression limits, we found that neutrality is highly amino-acid-specific and intrinsically linked to physicochemical properties. Hydrophobic repeats (I, V, W, F, Y) were universally harmful and aggregation-prone, while hydrophilic repeats (E, S, N, Q) not only exhibited high neutrality but also actively suppressed the proteotoxic stress and aggregation of host proteins. These experimental neutrality values show a striking correlation with the occurrence and length of homorepeats across diverse eukaryotic proteomes. Our findings suggest that PolyX neutrality defines the "compatible sequence space" of proteins, where certain sequences like Poly10E act as evolutionary buffers to maintain proteostasis. This study provides a quantitative framework for understanding how physical constraints on protein solubility shape the evolution of proteomes and offers a new metric for rational protein design.

systems biology↗

bioRxiv2024

Radular teeth matrix protein 1 directs iron oxide deposition in chiton teeth

Chitons deposit magnetite, a type of iron oxide, on the cusps of their radular teeth. The molecules that control the deposition of iron oxide on chiton teeth have not been identified. Using immunofluorescence staining, we found that chiton-specific radular teeth matrix protein 1 (RTMP1) is highly expressed in tooth epithelial cells prior to iron oxide deposition and preexists in the cusp at the site of iron oxide deposition. RTMP1 was found to be secreted from the elongated epithelial cells covering teeth and transported into teeth through microvilli. Moreover, recombinant RTMP1 exhibited the ability to form iron oxide. These results indicate that RTMP1 is a key molecule for iron oxide deposition in chiton teeth.

molecular biology↗

bioRxiv2024

Impact of Maximal Overexpression of a Non-toxic Protein on Yeast Cell Physiology

While it is recognized that excess expression of non-essential proteins burdens cell growth, the physiological state of cells under such stress is largely unknown. This is because it is challenging to distinguish between adverse effects arising from the properties of the expressed excess protein (cytotoxicity) and those caused solely by protein overexpression. In this study, we attempted to identify the model protein with the lowest cytotoxicity in yeast cells by introducing a new neutrality index. We found that a non-fluorescent fluorescent protein (mox-YG) and an inactive glycolytic enzyme (Gpm1- CCmut) showed the lowest cytotoxicity. These proteins can be expressed at levels exceeding 40% of total protein while maintaining yeast growth. The transcriptome of cells expressing mox-YG to the limit indicated that the cells were in a nitrogen source requirement state. Proteome analysis revealed increased mitochondrial protein and decreased ribosome abundance, similar to the inactivated state of the TORC1 pathway. The decrease in ribosome abundance was presumably due to defective nucleolus formation, partially rescued by a mutation in the nuclear exosome. These findings suggest that massive overexpression of excess protein, termed protein burden, causes nitrogen source starvation, a metabolic shift toward more energy-efficient respiration, and a ribosomal biosynthesis defect due to an imbalance between ribosomal protein and rRNA synthesis in the nucleolus.

cell biology↗

bioRxiv2024

Improving the Z3EV promoter system to create the strongest yeast promoter

Promoters for artificial control of gene expression are central tools in genetic engineering. In the budding yeast S. cerevisiae, a variety of constitutive and controllable promoters with different strengths have been constructed using endogenous gene promoters, synthetic transcription factors and their binding sequences, and artificial sequences. However, there have been few attempts to construct the highest-strength promoter in yeast cells. In this study, by incrementally increasing the binding sequences of the synthetic transcription factor Z3EV, we were able to construct a promoter (P36) with approximately 1.4 times the strength of the TDH3 promoter. This is stronger than any previously reported promoter. Although the P36 promoter exhibits some leakage in the absence of induction, the expression induction by {beta}-estradiol is maintained. When combined with a multicopy plasmid, it can express up to approximately 50% of total protein as a heterologous protein. This promoter system can be used to gain knowledge about the cell physiology resulting from the ultimate overexpression of excess proteins and is expected to be a useful tool for heterologous protein expression in yeast.

molecular biology↗

bioRxiv2024

Toxicity of the model protein 3xGFP arises from degradation overload, not from aggregate formation

While protein aggregation can cause cytotoxicity, it also forms to mitigate cytotoxicity from misfolded proteins, though the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein linked protein (3xGFP) in yeast cells induces giant aggregates, and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3xGFP-op using non-aggregative 3xMOX-op as a control. The 3xGFP aggregates were induced by misfolding, and 3xGFP-op had higher cytotoxicity than 3xMOX-op because it perturbs the ubiquitin-proteasome system. Static aggregates formed by 3xGFP-op dynamically trapped Hsp70, causing the heat shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3xGFP-op did not cause critical Hsp70 depletion and that the formation of aggregates functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3xGFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3xGFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat shock response while preventing an overload to the degradation system.

cell biology↗

bioRxiv2022

Overexpression profiling reveals cellular requirements in context of genetic backgrounds and environments

Overexpression due to copy number variation, promoter mutation, or aneuploidy is often observed, but its adaptive role is not clearly understood. Using a novel "overexpression profiling" method designated ADOPT, we systematically obtained genes whose overexpression was functionally adaptive (GOFAs) under stress conditions in budding yeast to elucidate the nature of adaptive overexpression. GOFAs obtained under heat, salt, and oxidative stress were unique genes that differed from known stress response genes. GOFAs under salt (NaCl) stress were genes involved in calcium homeostasis, reflecting the calcium deficiency of the medium. GOFAs from different genetic backgrounds and co-overexpressing strains revealed that calcium and potassium requirements in salt stress tolerance differ among strains, which is reflected. Profiling of the knockout collection suggested that the effect of calcium was to prevent mitochondrial outbursts. Mitochondria-enhancing GOFAs were adaptive only when calcium was sufficient and conversely non-adaptive in calcium deficiency, supporting the above hypothesis. Adaptive overexpression, thus, reflects the cellular requirements for maximizing the organisms adaptive capacity within a given environmental and genetic context.

systems biology↗

bioRxiv2021

EEG-based neurofeedback with network components extraction: data-driven approach by multilayer ICA extension and simultaneous EEG-fMRI measurements

Several studies have reported advanced treatments for depressive symptoms, such as real-time neurofeedback (NF) with functional MRI (fMRI) and/or electroencephalogram (EEG). NF focusing on a regularization of brain activity associated with the amygdala or functional connectivity (FC) between the executive control network (ECN) and default mode network (DMN) has been applied to reduce depressive symptoms. However, it is practically difficult to install the fMRI-NF system and to consistently provide treatment, because of high cost. Additionally, no practical signal processing techniques have been developed extracting FC-related features from EEG signals, particularly when no physical forward models are available. In this regard, stacked pooling and linear components estimation (SPLICE), recently proposed as a multilayer extension of independent component analysis (ICA) and related independent subspace analysis (ISA), can be a promising alternative. The resting-state EEG network features can be correlated with fMRI network activity corresponding to the DMN or ECN. This may enable the modulation of the target FC-related features in EEG-based NF. In this study, we developed a real-time EEG NF system for improving depressive symptoms by using the SPLICE. Utilizing information from the fMRI biomarkers, we evaluated our paradigm for effectiveness with regard to upregulation of the dorsolateral prefrontal cortex /middle frontal gyrus or downregulation of the precuneus/posterior cingulate cortex. We conducted an NF experiment in participants with subclinical depression; the participants were divided into the NF group (n=8) and the sham group (n=9). We found a significant reduction and a large effect size in the rumination response scale (RRS) score (reflection) in the NF group, compared to the sham group. However, we did not find a significant relationship between the training score and difference in symptoms. This suggests that increased controllability of the EEG signals did not directly reduce the RRS reflection score. This could be due to various reasons such as improper feature extraction, individual differences, and the targeted brain regions. In this paper, we also discuss the possible ways to modify our NF protocol including the design of the experiment, sample size, and online processing. We then discuss way to improve the NF training, based on our results.

neuroscience↗

bioRxiv2020

Genetic Profiling of Protein Burden and Nuclear Export Overload

Overproduction (op) of proteins triggers cellular defects. One of the defined consequences of protein overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion mutants of nonessential genes and temperature-sensitive mutants of essential genes, in the budding yeast Saccharomyces cerevisiae. To dissect interactions specific to the protein burden, we also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, probably through the formation of intracellular aggregates, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.

genomics↗

bioRxiv2019

Exploring the Complexity of Protein-Level Dosage Compensation that Fine-Tunes Stoichiometry of Multiprotein Complexes

Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how dominant this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 10 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent compensation. Our study reveals the complexity and robustness of the stoichiometry control system.

genetics↗