NRASQ61R mutation drives elevated angiopoietin-2 expression in human endothelial cells and a genetic mouse model.

BACKGROUND: Angiopoietin-2 (Ang-2) is increased in the blood of patients with kaposiform lymphangiomatosis (KLA) and kaposiform hemangioendothelioma (KHE). While the genetic causes of KHE are not clear, a somatic activating NRASQ61R mutation has been found in the lesions of KLA patients. PROCEDURE: Our study tested the hypothesis that the NRASQ61R mutation drives elevated Ang-2 expression in endothelial cells. Ang-2 was measured in human endothelial progenitor cells (EPC) expressing NRASQ61R and a genetic mouse model with endothelial targeted NRASQ61R. To determine the signaling pathways driving Ang-2, NRASQ61R EPC were treated with signaling pathway inhibitors. RESULTS: Ang-2 levels were increased in EPC expressing NRASQ61R compared to NRASWT by Western blot analysis of cell lysates and ELISA of the cell culture media. Ang-2 levels were elevated in the blood of NRASQ61R mutant mice. NRASQ61R mutant mice also had reduced platelet counts and splenomegaly with hypervascular lesions, like some KLA patients. mTOR inhibitor rapamycin attenuated Ang-2 expression by NRASQ61R EPC. However, MEK1/2 inhibitor trametinib was more effective blocking increases in Ang-2. CONCLUSIONS: Our studies show that the NRASQ61R mutation in endothelial cells induces Ang-2 expression in vitro and in vivo. In cultured human endothelial cells, NRASQ61R drives elevated Ang-2 through MAP kinase and mTOR-dependent signaling pathways.

Chang'an decoction alleviates endoplasmic reticulum stress by regulating mitofusin 2 to improve colitis.

OBJECTIVE: To evaluate the protective effects of Chang'an decoction (, CAD) on colitis, and investigate the potential mechanisms underlying these effects from the perspectives of endoplasmic reticulum (ER) stress induced by mitofusin 2 (MFN2). METHODS: The composition of CAD was identified by liquid chromatography-mass spectrometry technology. A mice model of dextran sulfate sodium (DSS) induced colitis was established and therapeutic effects of CAD were determined by detecting body weight, disease activity index, colon length and histopathological changes. Then, the expression levels of MFN2, ER stress markers and Nucleotide-binding domain and leucine-rich repeat protein3 (NLRP3) relevant proteins were detected by polymerase chain reaction (PCR), Western blot, immunohistochemistry and immunofluorescence staining. Subsequently, knockdown and overexpression cell model were constructed to further investigate the underlying mechanism of MFN2 mediating ER stress and energy metabolism by PCR, Western blot, electron microscopy and reactive oxygen species (ROS) staining. Finally, inflammatory indicator and tight junction proteins were measured by PCR and immunofluorescence staining to evaluate the protective effects of CAD. RESULTS: Results showed that the indispensable regulatory role of MFN2 in mediating ER stress and mitochondrial damage was involved in the protective effects of CAD on colitis in mice fed with DSS. Network pharmacology analysis also revealed CAD may play a protective effect on colitis by affecting mitochondrial function. In addition, our data also suggested a causative role for MFN2 in the development of inflammatory responses and energy metabolic alterations by constructing a knockdown and overexpression cell model whereby alter proper ER-mitochondria interaction in Caco-2 cells. Furthermore, relative expression analyses of ER stress markers and NLRP3 inflammasome showed the onset of ER stress and activation of NLRP3 inflammasome, which is consistent with the above findings. In contrast, intervention of CAD could improve the mucosal barrier integrity and colonic inflammatory response effectively through inhibiting ER stress response mediated by MFN2. CONCLUSION: CAD could alleviate ER stress by regulating MFN2 to exert therapeutic effects on DSS-induced colitis, which might provide an effective natural therapeutic approach for the treatment of ulcerative colitis.

Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury via PI3K/AKT pathway mediated HIF-1α to regulate the mitochondrial dynamic balance.

BACKGROUND: Myocardial ischemia-reperfusion injury (I/RI) is a major cause of perioperative cardiac-related adverse events and death. Studies have shown that sevoflurane postconditioning (SpostC), which attenuates I/R injury and exerts cardioprotective effects, regulates mitochondrial dynamic balance via HIF-1α, but the exact mechanism is unknown. This study investigates whether the PI3K/AKT pathway in SpostC regulates mitochondrial dynamic balance by mediating HIF-1α, thereby exerting myocardial protective effects. METHODS: The H9C2 cardiomyocytes were cultured to establish the hypoxia-reoxygenation (H/R) model and randomly divided into 4 groups: Control group, H/R group, sevoflurane postconditioning (H/R + SpostC) group and PI3K/AKT blocker (H/R + SpostC + LY) group. Cell survival rate was determined by CCK-8; Apoptosis rate was determined by flow cytometry; mitochondrial membrane potential was evaluated by Mito Tracker™ Red; mRNA expression levels of AKT, HIF-1α, Opa1and Drp1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR); Western Blot assay was used to detect the protein expression levels of AKT, phosphorylated AKT (p-AKT), HIF-1α, Opa1 and Drp1. RESULTS: Compared with the H/R group, the survival rate of cardiomyocytes in the H/R + SpostC group increased, the apoptosis rate decreased and the mitochondrial membrane potential increased. qRT-PCR showed that the mRNA expression of HIF-1α and Opa1 were higher in the H/R + SpostC group compared with the H/R group, whereas the transcription level of Drp1 was lower in the H/R + SpostC group. In the H/R + SpostC + LY group, the mRNA expression of HIF-1α was lower than the H/R + SpostC group. There was no difference in the expression of Opa1 mRNA between the H/R group and the H/R + SpostC + LY group. WB assay results showed that compared with the H/R group, the protein expression levels of HIF-1α, Opa1, P-AKT were increased and Drp1 protein expression levels were decreased in the H/R + SpostC group. HIF-1α, P-AKT protein expression levels were decreased in the H/R + SpostC + LY group compared to the H/R + SpostC group. CONCLUSION: SpostC mediates HIF-1α-regulated mitochondrial fission and fusion-related protein expression to maintain mitochondrial dynamic balance by activating the PI3K/AKT pathway and increasing AKT phosphorylation, thereby attenuating myocardial I/R injury.

Changes in cell morphology and function induced by the NRAS Q61R mutation in lymphatic endothelial cells.

Recently, a low-level somatic mutation in the NRAS gene (c.182 A > G, Q61R) was identified in various specimens from patients with kaposiform lymphangiomatosis. However, it is unknown how these low-frequency mutated cells can affect the characterization and surrounding environment of their lesions. To understand the pathogenesis and association of these gene abnormalities, we established NRASQ61R mutated lymphatic endothelial cells transfected with lentivirus vector and undertook morphological and functional characterization, protein expression profiling, and metabolome analysis. NRASQ61R human dermal lymphatic endothelial cells showed poor tube formation, a low proliferation rate, and high migration ability, with an increase in the ratio of mutated cells. An analysis of signaling pathways showed inactivation of the PIK3/AKT/mTOR pathway and hyperactivation of the RAS/MAPK/ERK pathway, which was improved by MAPK kinase (MEK) inhibitor treatment. This study shows the theoretical circumstances induced in vitro by NRASQ61R-mutated cells in the affected lesions of kaposiform lymphangiomatosis patients.

Charcot-Marie-Tooth type 2A in vivo models: Current updates.

Charcot-Marie-Tooth type 2A (CMT2A) is an inherited sensorimotor neuropathy associated with mutations within the Mitofusin 2 (MFN2) gene. These mutations impair normal mitochondrial functioning via different mechanisms, disturbing the equilibrium between mitochondrial fusion and fission, of mitophagy and mitochondrial axonal transport. Although CMT2A disease causes a significant disability, no resolutive treatment for CMT2A patients to date. In this context, reliable experimental models are essential to precisely dissect the molecular mechanisms of disease and to devise effective therapeutic strategies. The most commonly used models are either in vitro or in vivo, and among the latter murine models are by far the most versatile and popular. Here, we critically revised the most relevant literature focused on the experimental models, providing an update on the mammalian models of CMT2A developed to date. We highlighted the different phenotypic, histopathological and molecular characteristics, and their use in translational studies for bringing potential therapies from the bench to the bedside. In addition, we discussed limitations of these models and perspectives for future improvement.

Molecular Deciphering of Colorectal Cancer: Exploring Molecular Classifications and Analyzing the Interplay among Molecular Biomarkers MMR/MSI, KRAS, NRAS, BRAF and CDX2 - A Comprehensive Literature Review.

Background: Colorectal cancer (CRC) exhibits molecular and morphological diversity, involving genetic, epigenetic alterations, and disruptions in signaling pathways. This necessitates a comprehensive review synthesizing recent advancements in molecular mechanisms, established biomarkers, as well as emerging ones like CDX2 for enhanced CRC assessment. Material and Methods: This review analyzes the last decade's literature and current guidelines to study CRC's molecular intricacies. It extends the analysis beyond traditional biomarkers to include emerging ones like CDX2, examining their interaction with carcinogenic mechanisms and molecular pathways, alongside reviewing current testing methodologies. Results: A multi-biomarker strategy, incorporating both traditional and emerging biomarkers like CDX2, is crucial for optimizing CRC management. This strategy elucidates the complex interaction between biomarkers and the tumor's molecular pathways, significantly influencing prognostic evaluations, therapeutic decision-making, and paving the way for personalized medicine in CRC. Conclusions: This review proposes CDX2 as an emerging prognostic biomarker and emphasizes the necessity of thorough molecular profiling for individualized treatment strategies. By enhancing CRC treatment approaches and prognostic evaluation, this effort marks a step forward in precision oncology, leveraging an enriched understanding of tumor behavior.

Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

Erratum - LncRNA gadd7 promotes mitochondrial membrane potential decrease and apoptosis of alveolar type II epithelial cells by positively regulating MFN1 in an in vitro model of hyperoxia-induced acute lung injury.

This corrects the article published in European Journal of Histochemistry 2023;67:3535. doi: 10.4081/ejh.2023.3535.

Targeted inhibition of branched-chain amino acid metabolism drives apoptosis of glioblastoma by facilitating ubiquitin degradation of Mfn2 and oxidative stress.

Glioblastoma is one of the most challenging malignancies with high aggressiveness and invasiveness and its development and progression of glioblastoma highly depends on branched-chain amino acid (BCAA) metabolism. The study aimed to investigate effects of inhibition of BCAA metabolism with cytosolic branched-chain amino acid transaminase (BCATc) Inhibitor 2 on glioblastoma, elucidate its underlying mechanisms, and explore therapeutic potential of targeting BCAA metabolism. The expression of BCATc was upregulated in glioblastoma and BCATc Inhibitor 2 precipitated apoptosis both in vivo and in vitro with the activation of Bax/Bcl2/Caspase-3/Caspase-9 axis. In addition, BCATc Inhibitor 2 promoted K63-linkage ubiquitination of mitofusin 2 (Mfn2), which subsequently caused lysosomal degradation of Mfn2, and then oxidative stress, mitochondrial fission and loss of mitochondrial membrane potential. Furthermore, BCATc Inhibitor 2 treatment resulted in metabolic reprogramming, and significant inhibition of expression of ATP5A, UQCRC2, SDHB and COX II, indicative of suppressed oxidative phosphorylation. Moreover, Mfn2 overexpression or scavenging mitochondria-originated reactive oxygen species (ROS) with mito-TEMPO ameliorated BCATc Inhibitor 2-induced oxidative stress, mitochondrial membrane potential disruption and mitochondrial fission, and abrogated the inhibitory effect of BCATc Inhibitor 2 on glioblastoma cells through PI3K/AKT/mTOR signaling. All of these findings indicate suppression of BCAA metabolism promotes glioblastoma cell apoptosis via disruption of Mfn2-mediated mitochondrial dynamics and inhibition of PI3K/AKT/mTOR pathway, and suggest that BCAA metabolism can be targeted for developing therapeutic agents to treat glioblastoma.

Cardiac-specific PFKFB3 overexpression prevents diabetic cardiomyopathy via enhancing OPA1 stabilization mediated by K6-linked ubiquitination.

Diabetic cardiomyopathy (DCM) is a prevalent complication of type 2 diabetes (T2D). 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is a glycolysis regulator. However, the potential effects of PFKFB3 in the DCM remain unclear. In comparison to db/m mice, PFKFB3 levels decreased in the hearts of db/db mice. Cardiac-specific PFKFB3 overexpression inhibited myocardial oxidative stress and cardiomyocyte apoptosis, suppressed mitochondrial fragmentation, and partly restored mitochondrial function in db/db mice. Moreover, PFKFB3 overexpression stimulated glycolysis. Interestingly, based on the inhibition of glycolysis, PFKFB3 overexpression still suppressed oxidative stress and apoptosis of cardiomyocytes in vitro, which indicated that PFKFB3 overexpression could alleviate DCM independent of glycolysis. Using mass spectrometry combined with co-immunoprecipitation, we identified optic atrophy 1 (OPA1) interacting with PFKFB3. In db/db mice, the knockdown of OPA1 receded the effects of PFKFB3 overexpression in alleviating cardiac remodeling and dysfunction. Mechanistically, PFKFB3 stabilized OPA1 expression by promoting E3 ligase NEDD4L-mediated atypical K6-linked polyubiquitination and thus prevented the degradation of OPA1 by the proteasomal pathway. Our study indicates that PFKFB3/OPA1 could be potential therapeutic targets for DCM.

Dihydroartemisinin-driven selective anti-lung cancer proliferation by binding to EGFR and inhibition of NRAS signaling pathway-induced DNA damage.

Chemotherapeutic agents can inhibit the proliferation of malignant cells due to their cytotoxicity, which is limited by collateral damage. Dihydroartemisinin (DHA), has a selective anti-cancer effect, whose target and mechanism remain uncovered. The present work aims to examine the selective inhibitory effect of DHA as well as the mechanisms involved. The findings revealed that the Lewis cell line (LLC) and A549 cell line (A549) had an extremely rapid proliferation rate compared with the 16HBE cell line (16HBE). LLC and A549 showed an increased expression of NRAS compared with 16HBE. Interestingly, DHA was found to inhibit the proliferation and facilitate the apoptosis of LLC and A549 with significant anti-cancer efficacy and down-regulation of NRAS. Results from molecular docking and cellular thermal shift assay revealed that DHA could bind to epidermal growth factor receptor (EGFR) molecules, attenuating the EGF binding and thus driving the suppressive effect. LLC and A549 also exhibited obvious DNA damage in response to DHA. Further results demonstrated that over-expression of NRAS abated DHA-induced blockage of NRAS. Moreover, not only the DNA damage was impaired, but the proliferation of lung cancer cells was also revitalized while NRAS was over-expression. Taken together, DHA could induce selective anti-lung cancer efficacy through binding to EGFR and thereby abolishing the NRAS signaling pathway, thus leading to DNA damage, which provides a novel theoretical basis for phytomedicine molecular therapy of malignant tumors.

WTAP-mediated m6A modification of lncRNA Snhg1 improves myocardial ischemia-reperfusion injury via miR-361-5p/OPA1-dependent mitochondrial fusion.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is caused by reperfusion after ischemic heart disease. LncRNA Snhg1 regulates the progression of various diseases. N6-methyladenosine (m6A) is the frequent RNA modification and plays a critical role in MIRI. However, it is unclear whether lncRNA Snhg1 regulates MIRI progression and whether the lncRNA Snhg1 was modified by m6A methylation. METHODS: Mouse cardiomyocytes HL-1 cells were utilized to construct the hypoxia/reoxygenation (H/R) injury model. HL-1 cell viability was evaluated utilizing CCK-8 method. Cell apoptosis, mitochondrial reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were quantitated utilizing flow cytometry. RNA immunoprecipitation and dual-luciferase reporter assays were applied to measure the m6A methylation and the interactions between lncRNA Snhg1 and targeted miRNA or target miRNAs and its target gene. The I/R mouse model was constructed with adenovirus expressing lncRNA Snhg1. HE and TUNEL staining were used to evaluate myocardial tissue damage and apoptosis. RESULTS: LncRNA Snhg1 was down-regulated after H/R injury, and overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization. Besides, lncRNA Snhg1 could target miR-361-5p, and miR-361-5p targeted OPA1. Overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization though the miR-361-5p/OPA1 axis. Furthermore, WTAP induced lncRNA Snhg1 m6A modification in H/R-stimulated HL-1 cells. Moreover, enforced lncRNA Snhg1 repressed I/R-stimulated myocardial tissue damage and apoptosis and regulated the miR-361-5p and OPA1 levels. CONCLUSION: WTAP-mediated m6A modification of lncRNA Snhg1 regulated MIRI progression through modulating myocardial apoptosis, mitochondrial ROS production, and mitochondrial polarization via miR-361-5p/OPA1 axis, providing the evidence for lncRNA as the prospective target for alleviating MIRI progression.

Miro GTPases at the Crossroads of Cytoskeletal Dynamics and Mitochondrial Trafficking.

Miro GTPases are key components in the machinery responsible for transporting mitochondria and peroxisomes along microtubules, and also play important roles in regulating calcium homeostasis and organizing contact sites between mitochondria and the endoplasmic reticulum. Moreover, Miro GTPases have been shown to interact with proteins that actively regulate cytoskeletal organization and dynamics, suggesting that these GTPases participate in organizing cytoskeletal functions and organelle transport. Derailed mitochondrial transport is associated with neuropathological conditions such as Parkinson's and Alzheimer's diseases. This review explores our recent understanding of the diverse roles of Miro GTPases under cytoskeletal control, both under normal conditions and during the course of human diseases such as neuropathological disorders.

Targeting NRAS via miR-1304-5p or farnesyltransferase inhibition confers sensitivity to ALK inhibitors in ALK-mutant neuroblastoma.

Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.

OMA1-Mediated Mitochondrial Dynamics Balance Organellar Homeostasis Upstream of Cellular Stress Responses.

In response to cellular metabolic and signaling cues, the mitochondrial network employs distinct sets of membrane-shaping factors to dynamically modulate organellar structures through a balance of fission and fusion. While these organellar dynamics mediate mitochondrial structure/function homeostasis, they also directly impact critical cell-wide signaling pathways such as apoptosis, autophagy, and the integrated stress response (ISR). Mitochondrial fission is driven by the recruitment of the cytosolic dynamin-related protein-1 (DRP1), while fusion is carried out by mitofusins 1 and 2 (in the outer membrane) and optic atrophy-1 (OPA1) in the inner membrane. This dynamic balance is highly sensitive to cellular stress; when the transmembrane potential across the inner membrane (Δψm) is lost, fusion-active OPA1 is cleaved by the overlapping activity with m-AAA protease-1 (OMA1 metalloprotease, disrupting mitochondrial fusion and leaving dynamin-related protein-1 (DRP1)-mediated fission unopposed, thus causing the collapse of the mitochondrial network to a fragmented state. OMA1 is a unique regulator of stress-sensitive homeostatic mitochondrial balance, acting as a key upstream sensor capable of priming the cell for apoptosis, autophagy, or ISR signaling cascades. Recent evidence indicates that higher-order macromolecular associations within the mitochondrial inner membrane allow these specialized domains to mediate crucial organellar functionalities.

Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Insights into the ATP / GTP selectivity of a GTPase, adenylosuccinate synthetase from Leishmania donovani.

Many GTPases have been shown to utilize ATP too as the phosphoryl donor. Both GTP and ATP are important molecules in the cellular environments and play multiple and discrete functional role within the cells. In our present study, we showed that one of the purine metabolic enzymes Adenylosuccinate synthetase from Leishmania donovani (LdAdSS) which belongs to the BioD-superfamily of GTPases can also carry out the catalysis by hydrolysing ATP instead of its cognate substrate GTP albeit with less efficiency. Biochemical and biophysical studies indicated its ability to bind to ATP too but at a higher concentration of ATP compared to that of GTP. Sequence analysis and molecular dynamic simulations suggested that residues of the switch loop and the G4-G5 (593SAXD596) connected motif of LdAdSS plays a role in determining the nucleotide specificity. Though the crucial interaction between Asp596 and the nucleotide is broken when ATP is bound, interactions between the Ala594 and the adenine ring of ATP could still hold ATP in the GTP binding site. The results of the present study suggested that though LdAdSS is GTP specific, it still shows ATP hydrolysing activity.

Inhibition of MFN1 restores tamoxifen-induced apoptosis in resistant cells by disrupting aberrant mitochondrial fusion dynamics.

Tamoxifen (TAM) resistance presents a major clinical obstacle in the management of estrogen-sensitive breast cancer, highlighting the need to understand the underlying mechanisms and potential therapeutic approaches. We showed that dysregulated mitochondrial dynamics were involved in TAM resistance by protecting against mitochondrial apoptosis. The dysregulated mitochondrial dynamics were associated with increased mitochondrial fusion and decreased fission, thus preventing the release of mitochondrial cytochrome c to the cytoplasm following TAM treatment. Dynamin-related GTPase protein mitofusin 1 (MFN1), which promotes fusion, was upregulated in TAM-resistant cells, and high MFN1 expression indicated a poor prognosis in TAM-treated patients. Mitochondrial translocation of MFN1 and interaction between MFN1 and mitofusin 2 (MFN2) were enhanced to promote mitochondrial outer membrane fusion. The interaction of MFN1 and cristae-shaping protein optic atrophy 1 (OPA1) and OPA1 oligomerization were reduced due to augmented OPA1 proteolytic cleavage, and their apoptosis-promoting function was reduced due to cristae remodeling. Furthermore, the interaction of MFN1 and BAK were increased, which restrained BAK activation following TAM treatment. Knockdown or pharmacological inhibition of MFN1 blocked mitochondrial fusion, restored BAK oligomerization and cytochrome c release, and amplified activation of caspase-3/9, thus sensitizing resistant cells to apoptosis and facilitating the therapeutic effects of TAM both in vivo and in vitro. Conversely, overexpression of MFN1 alleviated TAM-induced mitochondrial apoptosis and promoted TAM resistance in sensitive cells. These results revealed that dysregulated mitochondrial dynamics contributes to the development of TAM resistance, suggesting that targeting MFN1-mediated mitochondrial fusion is a promising strategy to circumvent TAM resistance.

Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Inhibition of adenylyl cyclase by GTPase-deficient Gαi is mechanistically different from that mediated by receptor-activated Gαi.

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/ß5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/ß5 loop, the α4 helix, and the α4/ß6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.