Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.

Alterations in cardiac contractile and regulatory proteins contribute to age-related cardiac dysfunction in male rats.

Aging is associated with cardiac contractile abnormalities, but the etiology of these contractile deficits is unclear. We hypothesized that cardiac contractile and regulatory protein expression is altered during aging. To investigate this possibility, left ventricular (LV) lysates were prepared from young (6 months) and old (24 months) Fischer344 rats. There are no age-related changes in SERCA2 expression or phospholamban phosphorylation. Additionally, neither titin isoform expression nor phosphorylation differed. However, there is a significant increase in ß-isoform of the myosin heavy chain (MyHC) expression and phosphorylation of TnI and MyBP-C during aging. In permeabilized strips of papillary muscle, force and Ca2+ sensitivity are reduced during aging, consistent with the increase in ß-MyHC expression and TnI phosphorylation. However, the increase in MyBP-C phosphorylation during aging may represent a mechanism to compensate for age-related contractile deficits. In isolated cardiomyocytes loaded with Fura-2, the peak of the Ca2+ transient is reduced, but the kinetics of the Ca2+ transient are not altered. Furthermore, the extent of shortening and the rates of both sarcomere shortening and re-lengthening are reduced. These results demonstrate that aging is associated with changes in contractile and regulatory protein expression and phosphorylation, which affect the mechanical properties of cardiac muscle.

DNA damage-induced proteasome phosphorylation controls substrate recognition and facilitates DNA repair.

Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation, which is an integral part of the DNA repair program. Although details of the ubiquitination processes have been intensively studied, little is known about whether and how the 26S proteasome is regulated in the DNA damage response (DDR). Here, we show that human Rpn10/PSMD4, one of the three ubiquitin receptors of the 26S proteasome, is rapidly phosphorylated in response to different types of DNA damage. The phosphorylation occurs at Rpn10-Ser266 within a conserved SQ motif recognized by ATM/ATR/DNA-PK. Blockade of S266 phosphorylation attenuates homologous recombination-mediated DNA repair and sensitizes cells to genotoxic insults. In vitro and in cellulo experiments indicate that phosphorylation of S266, located in the flexible linker between the two ubiquitin-interacting motifs (UIMs) of Rpn10, alters the configuration of UIMs, and actually reduces ubiquitin chain (substrate) binding. As a result, essential DDR proteins such as BRCA1 are spared from premature degradation and allowed sufficient time to engage in DNA repair, a scenario supported by proximity labeling and quantitative proteomic studies. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.

UPF3B modulates endoplasmic reticulum stress through interaction with inositol-requiring enzyme-1α.

The unfolded protein response (UPR) is a conserved and adaptive intracellular pathway that relieves the endoplasmic reticulum (ER) stress by activating ER transmembrane stress sensors. As a consequence of ER stress, the inhibition of nonsense-mediated mRNA decay (NMD) is due to an increase in the phosphorylation of eIF2α, which has the effect of inhibiting translation. However, the role of NMD in maintaining ER homeostasis remains unclear. In this study, we found that the three NMD factors, up-frameshift (UPF)1, UPF2, or UPF3B, were required to negate the UPR. Among these three NMD factors, only UPF3B interacted with inositol-requiring enzyme-1α (IRE1α). This interaction inhibited the kinase activity of IRE1α, abolished autophosphorylation, and reduced IRE1α clustering for ER stress. BiP and UPF3B jointly control the activation of IRE1α on both sides of the ER membrane. Under stress conditions, the phosphorylation of UPF3B was increased and the phosphorylated sites were identified. Both the UPF3BY160D genetic mutation and phosphorylation at Thr169 of UPF3B abolished its interaction with IRE1α and UPF2, respectively, leading to activation of ER stress and NMD dysfunction. Our study reveals a key physiological role for UPF3B in the reciprocal regulatory relationship between NMD and ER stress.

Bisecting N-Acetylglucosamine Correlates with Phospho-Tau181 in Subjective Cognitive Decline but not in Control Cases.

Background: The N-glycan structure bisecting N-acetylglucosamine (bisecting GlcNAc) is present on several N-glycans that are elevated in Alzheimer's disease (AD), and previous studies have shown that bisecting GlcNAc levels correlate with total tau and phospho-tau181 in cerebrospinal fluid at early stages of AD. A recent population-based study showed that bisecting GlcNAc correlates with total tau also in blood and that this correlation could predict conversion to dementia. Objective: In this study, we have further investigated how bisecting GlcNAc relates to total tau and phospho-tau 181 in cerebrospinal fluid samples from controls and cases with early cognitive deficits, stratified by amyloid/tau status and gender. Methods: Relative levels of bisecting GlcNAc in cerebrospinal fluid were measured by an enzyme-linked lectin assay in individuals with subjective cognitive decline, mild cognitive impairment and controls from the Norwegian Dementia Disease Initiation cohort. Results: As in our previous study, the correlation between bisecting GlcNAc and total tau or phospho-tau181 was particularly strong in the subjective cognitive decline group. The correlation was observed in amyloid negative and tau negative as well as amyloid positive and tau positive individuals, both in females and in males. Interestingly, among the amyloid negative and tau negative individuals, the correlation was observed in individuals with subjective cognitive decline but not in the controls. Conclusions: Thus, bisecting GlcNAc could be a biomarker for early cognitive decline.

Fine-tuning AMPK in physiology and disease using point-mutant mouse models.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that monitors the cellular energy status to adapt it to the fluctuating nutritional and environmental conditions in an organism. AMPK plays an integral part in a wide array of physiological processes, such as cell growth, autophagy and mitochondrial function, and is implicated in diverse diseases, including cancer, metabolic disorders, cardiovascular diseases and neurodegenerative diseases. AMPK orchestrates many different physiological outcomes by phosphorylating a broad range of downstream substrates. However, the importance of AMPK-mediated regulation of these substrates in vivo remains an ongoing area of investigation to better understand its precise role in cellular and metabolic homeostasis. Here, we provide a comprehensive overview of our understanding of the kinase function of AMPK in vivo, as uncovered from mouse models that harbor phosphorylation mutations in AMPK substrates. We discuss some of the inherent limitations of these mouse models, highlight the broader implications of these studies for understanding human health and disease, and explore the valuable insights gained that could inform future therapeutic strategies for the treatment of metabolic and non-metabolic disorders.

TRIM44 enhances autophagy via SQSTM1 oligomerization in response to oxidative stress.

The deubiquitinase tripartite motif containing 44 (TRIM44) plays a critical role in linking the proteotoxic stress response with autophagic degradation, which is significant in the context of cancer and neurological diseases. Although TRIM44 is recognized as a prognostic marker in various cancers, the complex molecular mechanisms through which it facilitates autophagic degradation, particularly under oxidative stress conditions, have not been fully explored. In this study, we demonstrate that TRIM44 significantly enhances autophagy in response to oxidative stress, reducing cytotoxicity in cancer cells treated with arsenic trioxide. Our research emphasizes the critical role of the posttranslational modification of sequestosome-1 (SQSTM1) and its importance in improving sequestration during autophagic degradation under oxidative stress. We found that TRIM44 notably promotes SQSTM1 oligomerization in both PB1 domain-dependent and oxidation-dependent manners. Furthermore, TRIM44 amplifies the interaction between protein kinase A and oligomerized SQSTM1, leading to enhanced phosphorylation of SQSTM1 at S349. This phosphorylation event activates NFE2L2, a key transcription factor in the oxidative stress response, highlighting the importance of TRIM44 in modulating SQSTM1-mediated autophagy. Our findings support that TRIM44 plays pivotal roles in regulating autophagic sensitivity to oxidative stress, with implications for cancer, aging, aging-associated diseases, and neurodegenerative disorders.

RXR nuclear receptor signaling modulates lipid metabolism and triggers lysosomal clearance of alpha-synuclein in neuronal models of synucleinopathy.

Disease-modifying strategies for Parkinson disease (PD), the most common synucleinopathy, represent a critical unmet medical need. Accumulation of the neuronal protein alpha-synuclein (αS) and abnormal lipid metabolism have each been implicated in PD pathogenesis. Here, we elucidate how retinoid-X-receptor (RXR) nuclear receptor signaling impacts these two aspects of PD pathogenesis. We find that activated RXR differentially regulates fatty acid desaturases, significantly reducing the transcript levels of the largely brain-specific desaturase SCD5 in human cultured neural cells and PD patient-derived neurons. This was associated with reduced perilipin-2 protein levels in patient neurons, reversal of αS-induced increases in lipid droplet (LD) size, and a reduction of triglyceride levels in human cultured cells. With regard to αS proteostasis, our study reveals that RXR agonism stimulates lysosomal clearance of αS. Our data support the involvement of Polo-like kinase 2 activity and αS S129 phosphorylation in mediating this benefit. The lowering of cellular αS levels was associated with reduced cytotoxicity. Compared to RXR activation, the RXR antagonist HX531 had the opposite effects on LD size, SCD, αS turnover, and cytotoxicity, all supporting pathway specificity. Together, our findings show that RXR-activating ligands can modulate fatty acid metabolism and αS turnover to confer benefit in cellular models of PD, including patient neurons. We offer a new paradigm to investigate nuclear receptor ligands as a promising strategy for PD and related synucleinopathies.

Far-infrared radiation alleviates steatohepatitis and fibrosis in metabolic dysfunction-associated fatty liver disease.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a disease that causes an abnormal accumulation of fat in the liver, triggering inflammation and fibrosis, the mechanism of which is not fully understood and for which there is a lack of specific drug therapy. Far-infrared radiation (FIR) has demonstrated evident therapeutic efficacy across various diseases, and novel nanomaterial graphene patches can emit it through electric heating. This study aimed to investigate the potential protective effects of FIR against MAFLD. Mice were fed with a MCD diet to mimic MAFLD progression, and histopathology analysis, biochemical analysis, RT-qPCR, and Western blotting analysis were performed to assess the effect of FIR on MAFLD in vivo. The effect of FIR treatment on MAFLD in vitro was investigated by biochemical analysis and gene expression profiling of hepatocytes. Mice subjected to the MCD diet and treated with FIR exhibited reduced hepatic lipid deposition, inflammation, fibrosis and liver damage. The therapeutic effect exerted by FIR in mice may be caused by the enhancement of AMPK phosphorylation and inhibition of the TGFß1-SMAD2/3 pathway. Besides, FIR intervention alleviated MAFLD in hepatocytes in vitro and the results were verified by gene expression profiling. Our results revealed a promising potential of FIR as a novel therapeutic approach for MAFLD.

LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis.

CD44 is associated with a high risk of metastasis, recurrence, and drug resistance in various cancers. Here we report that platelet endothelial aggregation receptor 1 (PEAR1) is a CD44 chaperone protein that protected CD44 from endocytosis-mediated degradation and enhances cleavage of the CD44 intracellular domain (CD44-ICD). Furthermore, we found that lysyl oxidase-like protein 2 (LOXL2), an endogenous ligand of PEAR1, bound to the PEAR1-EMI domain and facilitated the interaction between PEAR1 and CD44 by inducing PEAR1 Ser891 phosphorylation in a manner that was independent of its enzyme activity. Levels of PEAR1 protein and PEAR1 phosphorylation at Ser891 were increased in patients with triple-negative breast cancer (TNBC), were positively correlated with expression of LOXL2 and CD44, and were negatively correlated with overall survival. The level of PEAR1 Ser891 phosphorylation was identified as the best independent prognostic factor in TNBC patients. The prognostic efficacy of the combination of PEAR1 phosphorylation at Ser891 and CD44 expression was superior to that of PEAR1 phosphorylation at Ser891 alone. Blocking the interaction between LOXL2 and PEAR1 with monoclonal antibodies significantly inhibited TNBC metastasis, representing a promising therapeutic strategy for TNBC.

MNK-driven eIF4E phosphorylation regulates the fibrogenic transformation of mesenchymal cells and chronic lung allograft dysfunction.

Tissue fibrosis remains unamenable to meaningful therapeutic interventions and is the primary cause of chronic graft failure after organ transplantation. Eukaryotic translation initiation factor (eIF4E), a key translational regulator, serves as convergent target of multiple upstream profibrotic signaling pathways that contribute to mesenchymal cell (MC) activation. Here, we investigate the role of MAP kinase-interacting serine/threonine kinase-induced (MNK-induced) direct phosphorylation of eIF4E at serine 209 (Ser209) in maintaining fibrotic transformation of MCs and determine the contribution of the MNK/eIF4E pathway to the pathogenesis of chronic lung allograft dysfunction (CLAD). MCs from patients with CLAD demonstrated constitutively higher eIF4E phosphorylation at Ser209, and eIF4E phospho-Ser209 was found to be critical in regulating key fibrogenic protein autotaxin, leading to sustained ß-catenin activation and profibrotic functions of CLAD MCs. MNK1 signaling was upregulated in CLAD MCs, and genetic or pharmacologic targeting of MNK1 activity inhibited eIF4E phospho-Ser209 and profibrotic functions of CLAD MCs in vitro. Treatment with an MNK1/2 inhibitor (eFT-508) abrogated allograft fibrosis in an orthotopic murine lung-transplant model. Together these studies identify what we believe is a previously unrecognized MNK/eIF4E/ATX/ß-catenin signaling pathway of fibrotic transformation of MCs and present the first evidence, to our knowledge, for the utility of MNK inhibitors in fibrosis.

VEGFD signaling balances stability and activity-dependent structural plasticity of dendrites.

Mature neurons have stable dendritic architecture, which is essential for the nervous system to operate correctly. The ability to undergo structural plasticity, required to support adaptive processes like memory formation, is still present in mature neurons. It is unclear what molecular and cellular processes control this delicate balance between dendritic structural plasticity and stabilization. Failures in the preservation of optimal dendrite structure due to atrophy or maladaptive plasticity result in abnormal connectivity and are associated with various neurological diseases. Vascular endothelial growth factor D (VEGFD) is critical for the maintenance of mature dendritic trees. Here, we describe how VEGFD affects the neuronal cytoskeleton and demonstrate that VEGFD exerts its effects on dendrite stabilization by influencing the actin cortex and reducing microtubule dynamics. Further, we found that during synaptic activity-induced structural plasticity VEGFD is downregulated. Our findings revealed that VEGFD, acting on its cognate receptor VEGFR3, opposes structural changes by negatively regulating dendrite growth in cultured hippocampal neurons and in vivo in the adult mouse hippocampus with consequences on memory formation. A phosphoproteomic screening identified several regulatory proteins of the cytoskeleton modulated by VEGFD. Among the actin cortex-associated proteins, we found that VEGFD induces dephosphorylation of ezrin at tyrosine 478 via activation of the striatal-enriched protein tyrosine phosphatase (STEP). Activity-triggered structural plasticity of dendrites was impaired by expression of a phospho-deficient mutant ezrin in vitro and in vivo. Thus, VEGFD governs the equilibrium between stabilization and plasticity of dendrites by acting as a molecular brake of structural remodeling.

Pyrocurzerenone suppresses human oral cancer cell metastasis by inhibiting the expression of ERK1/2 and cathepsin S proteins.

Pyrocurzerenone is a natural compound found in Curcuma zedoaria and Chloranthus serratus. However, the anticancer effect of pyrocurzerenone in oral cancer remains unclear. Using the MTT assay, wound healing assay, transwell assay and western blot analysis, we investigated the impact of pyrocurzerenone on antimetastatic activity, as well as the critical signalling pathways that underlie the processes of oral cancer cell lines SCC-9, SCC-1 and SAS in this work. Our findings suggested that pyrocurzerenone inhibits cell migration and invasion ability in oral cancer cell lines. Furthermore, phosphorylation of ERK1/2 had significant inhibitory effects in SCC-9 and SCC-1 cell lines. Combining ERK1/2 inhibitors with pyrocurzerenone decreased the migration and invasion activity of SCC-9 and SCC-1 cell lines. We also found that the expressed level of cathepsin S decreased under pyrocurzerenone treatment. This study showed that pyrocurzerenone reduced ERK1/2 expression of the proteins and cathepsin S, suggesting that it could be a valuable treatment to inhibit human oral cancer cell metastasis.

Remote liver ischemic preconditioning protects against renal ischemia/reperfusion injury via phosphorylation of extracellular signal-regulated kinases 1 and 2 in mice.

Perioperative acute kidney injury (AKI), which is mainly mediated by renal ischemia‒reperfusion (I/R) injury, is commonly observed in clinical practice. However, effective measures for preventing and treating this perioperative complication are still lacking in the clinic. Thus, we designed this study to examine whether remote liver ischemic preconditioning (RLIPC) has a protective effect on damage caused by renal I/R injury. In a rodent model, 30 mice were divided into five groups to assess the effects of RLIPC and ERK1/2 inhibition on AKI. The groups included the sham-operated (sham), kidney ischemia and reperfusion (CON), remote liver ischemic preconditioning (RLIPC), CON with the ERK1/2 inhibitor U0126 (CON+U0126), and RLIPC with U0126 (RLIPC+U0126). RLIPC consisted of 4 liver ischemia cycles before renal ischemia. Renal function and injury were assessed through biochemical assays, histology, cell apoptosis and protein phosphorylation analysis. RLIPC significantly mitigated renal dysfunction, tissue damage, inflammation, and apoptosis caused by I/R, which was associated with ERK1/2 phosphorylation. Furthermore, ERK1/2 inhibition with U0126 negated the protective effects of RLIPC and exacerbated renal injury. To summarize, we demonstrated that RLIPC has a strong renoprotective effect on kidneys post I/R injury and that this effect may be mediated by phosphorylation of ERK1/2.

The involvement of the Stat1/Nrf2 pathway in exacerbating Crizotinib-induced liver injury: implications for ferroptosis.

Crizotinib carries an FDA hepatotoxicity warning, yet analysis of the FAERS database suggests that the severity of its hepatotoxicity risks, including progression to hepatitis and liver failure, might be underreported. However, the underlying mechanism remains poorly understood, and effective intervention strategies are lacking. Here, mRNA-sequencing analysis, along with KEGG and GO analyses, revealed that DEGs linked to Crizotinib-induced hepatotoxicity predominantly associate with the ferroptosis pathway which was identified as the principal mechanism behind Crizotinib-induced hepatocyte death. Furthermore, we found that ferroptosis inhibitors, namely Ferrostatin-1 and Deferoxamine mesylate, significantly reduced Crizotinib-induced hepatotoxicity and ferroptosis in both in vivo and in vitro settings. We have also discovered that overexpression of AAV8-mediated Nrf2 could mitigate Crizotinib-induced hepatotoxicity and ferroptosis in vivo by restoring the imbalance in glutathione metabolism, iron homeostasis, and lipid peroxidation. Additionally, both Stat1 deficiency and the Stat1 inhibitor NSC118218 were found to reduce Crizotinib-induced ferroptosis. Mechanistically, Crizotinib induces the phosphorylation of Stat1 at Ser727 but not Tyr701, promoting the transcriptional inhibition of Nrf2 expression after its entry into the nucleus to promote ferroptosis. Meanwhile, we found that MgIG and GA protected against hepatotoxicity to counteract ferroptosis without affecting or compromising the anti-cancer activity of Crizotinib, with a mechanism potentially related to the Stat1/Nrf2 pathway. Overall, our findings identify that the phosphorylation activation of Stat1 Ser727, rather than Tyr701, promotes ferroptosis through transcriptional inhibition of Nrf2, and highlight MgIG and GA as potential therapeutic approaches to enhance the safety of Crizotinib-based cancer therapy.

Rictor, an mTORC2 Protein, Regulates Murine Lymphatic Valve Formation Through the AKT-FOXO1 Signaling.

BACKGROUND: Lymphatic valves are specialized structures in collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves. Conventionally, in many cell types, AKT is phosphorylated at Ser473 by the mTORC2 (mammalian target of rapamycin complex 2). However, mTORC2 has not yet been implicated in lymphatic valve formation. METHODS: In vivo and in vitro techniques were used to investigate the role of Rictor, a critical component of mTORC2, in lymphatic endothelium. RESULTS: Here, we showed that embryonic and postnatal lymphatic deletion of Rictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human dermal lymphatic endothelial cells not only reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions but also abolished the upregulation of AKT activity and valve-forming genes in response to oscillatory shear stress. We further showed that the AKT target, FOXO1 (forkhead box protein O1), a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric lymphatic endothelial cells in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in lymphatic vessels of the ear and mesentery. CONCLUSIONS: Our work identifies a novel role for RICTOR in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately enables the formation and maintenance of lymphatic valves.

Sestrin2 drives ER-phagy in response to protein misfolding.

Protein biogenesis within the endoplasmic reticulum (ER) is crucial for organismal function. Errors during protein folding necessitate the removal of faulty products. ER-associated protein degradation and ER-phagy target misfolded proteins for proteasomal and lysosomal degradation. The mechanisms initiating ER-phagy in response to ER proteostasis defects are not well understood. By studying mouse primary cells and patient samples as a model of ER storage disorders (ERSDs), we show that accumulation of faulty products within the ER triggers a response involving SESTRIN2, a nutrient sensor controlling mTORC1 signaling. SESTRIN2 induction by XBP1 inhibits mTORC1's phosphorylation of TFEB/TFE3, allowing these transcription factors to enter the nucleus and upregulate the ER-phagy receptor FAM134B along with lysosomal genes. This response promotes ER-phagy of misfolded proteins via FAM134B-Calnexin complex. Pharmacological induction of FAM134B improves clearance of misfolded proteins in ERSDs. Our study identifies the interplay between nutrient signaling and ER quality control, suggesting therapeutic strategies for ERSDs.

Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses.

Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase ∼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is ∼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an ∼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.

Unveiling novel insights into human IL-6 - IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis.

The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.

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, an essential first step of HR. The resection-promoting function of SART1 requires phosphorylation at threonine 430 and 695 by ATM/ATR. SART1 is recruited to DSB sites in a manner dependent on transcription and its RS domain. SART1 is epistatic with BRCA1, a major HR factor, in the promotion of resection, especially transcription-associated resection in the G2 phase. SART1 and BRCA1 accumulate at DSB sites in an interdependent manner, and epistatically counteract the resection blockade posed by 53BP1 and RIF1. 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 in the G2 phase, thereby promoting faithful repair by HR, and suppressing genome instability.