Biology subjects

Iwai, K.

Publications and source records attributed to Iwai, K..

7 recordsLinked to original sources

bioRxiv2024

An atypical E3 ligase safeguards the ribosome during nutrient stress

Metabolic stress must be effectively mitigated for the survival of cells and organisms. Ribosomes have emerged as signaling hubs that sense metabolic perturbations and coordinate responses that either restore homeostasis or trigger cell death. As yet, the mechanisms governing these cell fate decisions are not well understood. Here, we report an unexpected role for the atypical E3 ligase HOIL-1 in safeguarding the ribosome. We find HOIL-1 mutations associated with cardiomyopathy broadly sensitize cells to nutrient and translational stress. These signals converge on the ribotoxic stress sentinel ZAK. Mechanistically, mutant HOIL-1 excludes a ribosome quality control E3 ligase from its functional complex and remodels the ribosome ubiquitin landscape. This quality control failure renders glucose starvation ribotoxic, precipitating a ZAK-ATF4-xCT-driven noncanonical cell death. We further show HOIL-1 loss exacerbates cardiac dysfunction under pressure overload. These data reveal an unrecognized ribosome signaling axis and a molecular circuit controlling cell fate during nutrient stress.

cell biology↗

bioRxiv2024

Engineering of highly active and diverse nuclease enzymes by combining machine learning and ultra-high-throughput screening

Optimizing enzymes to function in novel chemical environments is a central goal of synthetic biology, but optimization is often hindered by a rugged, expansive protein search space and costly experiments. In this work, we present TeleProt, an ML framework that blends evolutionary and experimental data to design diverse protein variant libraries, and employ it to improve the catalytic activity of a nuclease enzyme that degrades biofilms that accumulate on chronic wounds. After multiple rounds of high-throughput experiments using both TeleProt and standard directed evolution (DE) approaches in parallel, we find that our approach found a significantly better top-performing enzyme variant than DE, had a better hit rate at finding diverse, high-activity variants, and was even able to design a high-performance initial library using no prior experimental data. We have released a dataset of 55K nuclease variants, one of the most extensive genotype-phenotype enzyme activity landscapes to date, to drive further progress in ML-guided design.

bioinformatics↗

bioRxiv2023

HOIL-1L deficiency induces cell cycle alteration which causes immaturity of myocyte and fibrogenesis

HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop have not been clearly elucidated. Here, we sought to elucidate these mechanisms using the murine myoblast cell line C2C12 and disease-specific human induced pluripotent stem cells (hiPSCs). Myotubes were differentiated from control and HOIL-1L-KO C2C12 cells. Cardiomyocytes (CMs) were differentiated from control and patient-derived hiPSCs. We investigated the impact of HOIL-1L on differentiation of myotubes and CMs. Myotubes differentiated from HOIL-1L-KO C2C12 cells exhibited deteriorated differentiation and mitotic cell accumulation. CMs differentiaed from patient-derived hiPSCs had an abnormal morphology with a larger size and were excessively multinucleated compared with CMs differentiaed from control hiPSCs. Further analysis of hiPSC- derived CMs showed that HOIL-1L deficiency caused cell cycle alteration and mitotic cell accumulation. These results were supported by RNA sequencing of C2C12 cell-derived myotubes. In addition, SerpinE2, a cardiac fibrogenesis gene, was significantly upregulated in CMs differentiaed from patient-derived hiPSCs. These results demonstrate that abnormal cell maturation and fibrosis possibly contribute to the development of DCM and myopathy. In conclusion, HOIL-1L is an important intrinsic regulator of cell cycle-related myotube and CM maturation and cell proliferation.

cell biology↗

bioRxiv2023

HOIL1 regulates group 3 innate lymphoid cell numbers in the colon and protects against systemic dissemination, colonic ulceration, and lethality from Citrobacter rodentium infection

HOIL1-deficient patients experience chronic intestinal inflammation and diarrhea as well as increased susceptibility to certain bacterial infections. HOIL1 is a component of the linear ubiquitin chain assembly complex (LUBAC) that regulates immune signaling pathways including NF-{kappa}B-activating pathways. We have shown previously that HOIL1 is essential for survival following Citrobacter rodentium gastrointestinal infection of mice, but the mechanism of protection by HOIL1 was not examined. C. rodentium is a murine model for human attaching and effacing (A/E) pathogens, enteropathogenic and enterohemorrhagic Escherichia coli, that cause diarrhea and food-borne illnesses, and lead to severe disease in children and immunocompromised individuals. In this study, we found that C. rodentium infection caused severe colitis and dissemination of C. rodentium to systemic organs in Hoil1-/-mice. HOIL1 was important in radiation-resistant cells and in the innate immune response to limit early replication of C. rodentium in the intestine, and to modulate induction of inflammatory cytokines. Using cell type-specific knock-out mice, we found that HOIL1 was dispensable in intestinal epithelial cells (IEC), but was required in CD11c- and lysozyme 2-expressing myeloid cells to prevent weight loss and systemic dissemination of C. rodentium. While HOIL1-deficiency did not affect populations of neutrophils or macrophages, dendritic cells and group 3 innate lymphoid cell (ILC3) numbers were reduced, resulting in a defect in IL-22 induction during C. rodentium infection. Understanding the role HOIL1 plays in limiting the pathogenesis of A/E lesion-forming bacteria will provide further insights into the innate immune response to gastrointestinal pathogens and inflammatory disorders.

immunology↗

bioRxiv2022

HOIL1 regulates group 2 innate lymphoid cell numbers and type 2 inflammation in the small intestine

Patients with mutations in HOIL1 experience a complex immune disorder including intestinal inflammation. To investigate the role of HOIL1 in regulating intestinal inflammation, we employed a mouse model of HOIL1 deficiency. The ileum of Hoil1-/- mice displayed features of type 2 inflammation including tuft cell and goblet cell hyperplasia, and elevated expression of Il13, Il5 and Il25 mRNA. Inflammation persisted in the absence of T and B cells, and bone marrow chimeric mice revealed a requirement for HOIL1 expression in radiation-resistant cells to regulate inflammation. Although disruption of IL-4 receptor alpha (IL4R) signaling on intestinal epithelial cells ameliorated tuft and goblet cell hyperplasia, expression of Il5 and Il13 mRNA remained elevated. KLRG1hi CD90lo group 2 innate lymphoid cell (ILC2) were increased independent of IL4R signaling, tuft cell hyperplasia and IL-25 induction. Antibiotic treatment dampened intestinal inflammation indicating commensal microbes as a contributing factor. We have identified a key role for HOIL1, a component of the Linear Ubiquitin Chain Assembly Complex, in regulating type 2 inflammation in the small intestine. Understanding the mechanism by which HOIL1 regulates type 2 inflammation will advance our understanding of intestinal homeostasis and inflammatory disorders and may lead to the identification of new targets for treatment.

immunology↗

bioRxiv2021

Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on chip. The microfluidic device contains a 10 x 10 element array, each element containing sets of electrodes for two electric field actuated operations-electrowetting for merging droplets to mix reagents and electroporation for transformation. It can perform up to 100 genetic modifications in parallel, providing a scalable platform for generating the large number of engineered strains required for combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the systems capabilities through CRISPR-based engineering of two test cases-1) disruption of the function of enzyme galactokinase (galK) in E. coli and 2) targeted engineering of glutamine synthetase gene (glnA) and blue-pigment synthetase (bpsA) enzyme to improve indigoidine production in E. coli.

synthetic biology↗

bioRxiv2020

Comprehensive analyses of the cysteine thiol oxidation of PKM2 reveals the effects of multiple oxidation on cellular oxidative stress response

Redox regulation of proteins via cysteine residue oxidation is known to be involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer acts as pyruvate kinase (PK), whereas the PKM2 dimer, which is induced by Cys358 oxidation, has reduced PK activity. Here, we identified four oxidation-sensitive cysteine residues (Cys152, Cys358, Cys423, and Cys424) responsible for three different oxidation forms. Possibly due to obstruction of the dimer-dimer interface, sulfenylation (-SOH) at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulfide bonds with heterologous proteins. In addition, intramolecular polysulfide linkage (-Sn-, n[greater double equals]3) possible between Cys152 and Cys358 also is induced. We found that cells expressing the oxidation-resistant, constitutive-tetramer PKM2 (PKM2C358,424A) show a higher intracellular reactive oxygen species (ROS) and greater sensitivity to ROS-generating reagents and ROS-inducible anti-cancer drugs. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to the elimination of excess ROS and oxidative stress.

molecular biology↗