A sensitive ERK fluorescent probe reveals the significance of minimal EGF-induced transcription.

Extracellular signal-regulated kinase (ERK) regulates multiple cellular functions through distinct activation patterns. Genetically encoded fluorescent probes are instrumental in dissecting the ERK activity dynamics in living cells. Here we modified a previously reported Förster resonance energy transfer (FRET) probe for ERK, EKAREN5 by replacing its mTurquoise2 and YPet sequences with mTurquoise-GL and a synonymous codon variant of YPet, respectively.

A chemical approach to extend flower longevity of Japanese morning glory via inhibition of master senescence regulator EPHEMERAL1.

Petal senescence in flowering plants is a type of programmed cell death with highly regulated onset and progression. A NAM/ATAF1,2/CUC2 transcription factor, EPHEMERAL1 (EPH1), has been identified as a key regulator of petal senescence in Japanese morning glory (Ipomoea nil). Here we used a novel chemical approach to delay petal senescence in Japanese morning glory by inhibiting the DNA-binding activity of EPH1.

Schlafen 11 further sensitizes BRCA-deficient cells to PARP inhibitors through single-strand DNA gap accumulation behind replication forks.

The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency.

Crystal structure of L-2-keto-3-deoxyfuconate 4-dehydrogenase reveals a unique binding mode as a α-furanosyl hemiketal of substrates.

L-2-Keto-3-deoxyfuconate 4-dehydrogenase (L-KDFDH) catalyzes the NAD-dependent oxidization of L-2-keto-3-deoxyfuconate (L-KDF) to L-2,4-diketo-3-deoxyfuconate (L-2,4-DKDF) in the non-phosphorylating L-fucose pathway from bacteria, and its substrate was previously considered to be the acyclic α-keto form of L-KDF. On the other hand, BDH2, a mammalian homolog with L-KDFDH, functions as a dehydrogenase for cis-4-hydroxy-L-proline (C4LHyp) with the cyclic structure. We found that L-KDFDH and BDH2 utilize C4LHyp and L-KDF, respectively.

HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR.

Bub1 suppresses inflammatory arthritis-associated bone loss in mice through inhibition of TNFα-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease-modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets.

Amelioration of oxygen-induced retinopathy in neonatal mice with fetal growth restriction.

With the aim of optimizing the balance of maintaining a safe oxygen saturation and reducing the risk of retinopathy of prematurity in human neonates with fetal growth restriction (FGR), the present study investigated the distinct effects of oxygen supplementation on the retinal neovasculature using a murine premature neonatal oxygen-induced retinopathy (OIR) model with or without fetal growth restriction. For comparison with normal birth-weight neonates, maternal low-protein diet-induced FGR neonates were subjected to fluctuating oxygen levels to generate oxygen-induced retinopathy. The retinal neovasculature was histologically evaluated, and comprehensive transcriptome analysis was conducted.

The E3 ligase DTX2 inhibits RUNX1 function by binding its C terminus and prevents the growth of RUNX1-dependent leukemia cells.

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. RUNX1 function is tightly controlled through posttranslational modifications, including ubiquitination and acetylation. However, its regulation via ubiquitination, especially proteasome-independent ubiquitination, is poorly understood.

LOX-1 deficiency increases ruptured abdominal aortic aneurysm via thinning of adventitial collagen.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Conversely, LOX-1 deficiency has been shown to decrease inflammation and atherosclerosis, both of which have been proposed to contribute to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown.

Functional Blockage of S100A8/A9 Ameliorates Ischemia-Reperfusion Injury in the Lung.

(1) Background: Lung ischemia-reperfusion (IR) injury increases the mortality and morbidity of patients undergoing lung transplantation. The objective of this study was to identify the key initiator of lung IR injury and to evaluate pharmacological therapeutic approaches using a functional inhibitor against the identified molecule. (2) Methods: Using a mouse hilar clamp model, the combination of RNA sequencing and histological investigations revealed that neutrophil-derived S100A8/A9 plays a central role in inflammatory reactions during lung IR injury.

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways.

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC.

Protein-protein interactions between jasmonate-related master regulator MYC and transcriptional mediator MED25 depend on a short binding domain.

A network of protein-protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression.

Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness.

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition.

Active aneurysm thrombosis after Kawasaki disease in an adult: Insight into anticoagulation therapy.

The management of systemic artery aneurysms secondary to Kawasaki disease (KD) in adults remains a therapeutic challenge. KD guidelines recommend the use of anticoagulation therapy with warfarin in addition to antiplatelet therapy when a giant coronary aneurysm or a history of thrombosis is documented. However, long-term use of warfarin presents several concerns.

Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4.

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT).

The vacuolar amino acid transport system is a novel, direct target of GATA transcription factors.

In Saccharomyces cerevisiae, Avt4 exports neutral and basic amino acids from vacuoles. Previous studies have suggested that the GATA transcription factors, Gln3 and Gat1, which are key regulators that adapt cells in response to changes in amino acid status, are involved in the AVT4 transcription. Here, we show that mutations in the putative GATA-binding sites of the AVT4 promoter reduced AVT4 expression.

MIND bomb 2 prevents RIPK1 kinase activity-dependent and -independent apoptosis through ubiquitylation of cFLIP.

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIP), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II.

Teriparatide relieves ovariectomy-induced hyperalgesia in rats, suggesting the involvement of functional regulation in primary sensory neurons by PTH-mediated signaling.

Clinical studies have reported that teriparatide (TPTD), a human parathyroid hormone analog, reduces back pain in osteoporotic patients. However, the mechanistic insights of this pharmacological action remain elusive. This study investigated the antinociceptive effect of TPTD mainly on primary sensory neurons in ovariectomized (OVX) rats.

Raf-like kinases CBC1 and CBC2 negatively regulate stomatal opening by negatively regulating plasma membrane H-ATPase phosphorylation in Arabidopsis.

Stomatal pores, which are surrounded by pairs of guard cells in the plant epidermis, regulate gas exchange between plants and the atmosphere, thereby controlling photosynthesis and transpiration. Blue light works as a signal to guard cells, to induce intracellular signaling and open stomata. Blue light receptor phototropins (phots) are activated by blue light; phot-mediated signals promote plasma membrane (PM) H+-ATPase activity via C-terminal Thr phosphorylation, serving as the driving force for stomatal opening in guard cells.

Subquinocin, a small molecule inhibitor of CYLD and USP-family deubiquitinating enzymes, promotes NF-κB signaling.

The tumor suppressor CYLD negatively regulates polyubiquitination-dependent cellular signaling such as nuclear factor (NF)-κB signaling. In addition to CYLD, multiple deubiquitinating enzymes (DUBs) are also involved in the regulation of this signaling pathway, and distinct role of CYLD is yet to be clarified. Here, we identified a small chemical named Subquinocin that inhibited the DUB activity of recombinant CYLD using a wheat cell-free protein synthesis and an AlphaScreen technology.

Pyrrothiogatain acts as an inhibitor of GATA family proteins and inhibits Th2 cell differentiation in vitro.

The transcription factor GATA3 is a master regulator that modulates T helper 2 (Th2) cell differentiation and induces expression of Th2 cytokines, such as IL-4, IL-5, and IL-13. Th2 cytokines are involved in the protective immune response against foreign pathogens, such as parasites. However, excessive production of Th2 cytokines results in type-2 allergic inflammation.

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling.