Regulation Of Cellular Metabolism Research Articles

A Nano-Aggregatable Acedan Derivative for Clathrin-Mediated Cellular Uptake and Two-Photon Imaging of Diabetes-Associated Lipid Droplets.

Lipid droplets (LDs), the essential cytosolic fat storage organelles, have emerged as pivotal regulators of cellular metabolism and are implicated in various diseases. The noninvasive monitoring of LDs necessitates fluorescent probes with precise organelle selectivity and biocompatibility. Addressing this need, we have engineered a probe by strategically modifying the structure of a conventional two-photon-absorbing dipolar dye, acedan. This innovative approach induces nanoaggregate formation in aqueous environments, leading to aggregation-induced fluorescence quenching. Upon cellular uptake via clathrin-mediated endocytosis, the probe selectively illuminates within LDs through a disassembly process, effectively distinguishing LDs from the cytosol with exceptional specificity. This breakthrough enables the high-fidelity imaging of LDs in both cellular and tissue environments. In a pioneering investigation, we probed LDs in a diabetes model induced by streptozotocin, unveiling significantly heightened LD accumulation in cardiac tissues compared to other organs, as evidenced by TP imaging. Furthermore, our exploration of a lipopolysaccharide-mediated cardiomyopathy model revealed an LD accumulation during heart injury. Thus, our developed probe holds immense potential for elucidating LD-associated diseases and advancing related research endeavors.

Sirt5 regulates chondrocyte metabolism and osteoarthritis development through protein lysine malonylation.

Chondrocyte metabolic dysfunction plays an important role in osteoarthritis (OA) development during aging and obesity. Protein post-translational modifications (PTMs) have recently emerged as an important regulator of cellular metabolism. We aim to study one type of PTM, lysine malonylation (MaK) and its regulator Sirt5 in OA development. Human and mouse cartilage tissues were used to measure SIRT5 and MaK levels. Both systemic and cartilage-specific conditional knockout mouse models were subject to high-fat diet (HFD) treatment to induce obesity and OA. Proteomics analysis was performed in Sirt5 -/- and WT chondrocytes. SIRT5 mutation was identified in the Utah Population Database (UPDB). We found that SIRT5 decreases while MAK increases in the cartilage during aging. A combination of Sirt5 deficiency and obesity exacerbates joint degeneration in a sex dependent manner in mice. We further delineate the malonylome in chondrocytes, pinpointing MaK's predominant impact on various metabolic pathways such as carbon metabolism and glycolysis. Lastly, we identified a rare coding mutation in SIRT5 that dominantly segregates in a family with OA. The mutation results in substitution of an evolutionally invariant phenylalanine (Phe-F) to leucine (Leu-L) (F101L) in the catalytic domain. The mutant protein results in higher MaK level and decreased expression of cartilage ECM genes and upregulation of inflammation associated genes. We found that Sirt5 mediated MaK is an important regulator of chondrocyte cellular metabolism and dysregulation of Sirt5-MaK could be an important mechanism underlying aging and obesity associated OA development.

Theory of Lipid Metabolism Disorders in Rhinitis and Asthma (Lipid Droplets).

Lipid droplets are important for the storage of neutral lipids in cells; moreover, they participate in a variety of activities in cells and are multifunctional organelles. In the past few decades, lipid droplets have been extensively studied and found to play important roles in cellular energy balance, signal regulation and metabolic regulation. In particular, the formation and function of lipid droplets in adipocytes and mast cells have received much attention. This article reviews the formation, structure and function of lipid droplets in mast cells and elaborates on the relationship between lipid droplets and both adipocyte metabolism and mast cell-mediated allergic inflammation, to provide ideas for the treatment of allergic inflammation by targeting lipid droplets. This study provides important evidence for the role of lipid metabolism disorders in rhinitis and asthma.

Unraveling Protein-Metabolite Interactions in Precision Nutrition: A Case Study of Blueberry-Derived Metabolites Using Advanced Computational Methods

Metabolomics, the study of small-molecule metabolites within biological systems, has become a potent instrument for understanding cellular processes. Despite its profound insights into health, disease, and drug development, identifying the protein partners for metabolites, especially dietary phytochemicals, remains challenging. In the present study, we introduced an innovative in silico, structure-based target prediction approach to efficiently predict protein targets for metabolites. We analyzed 27 blood serum metabolites from nutrition intervention studies’ blueberry-rich diets, known for their health benefits, yet with elusive mechanisms of action. Our findings reveal that blueberry-derived metabolites predominantly interact with Carbonic Anhydrase (CA) family proteins, which are crucial in acid-base regulation, respiration, fluid balance, bone metabolism, neurotransmission, and specific aspects of cellular metabolism. Molecular docking showed that these metabolites bind to a common pocket on CA proteins, with binding energies ranging from −5.0 kcal/mol to −9.0 kcal/mol. Further molecular dynamics (MD) simulations confirmed the stable binding of metabolites near the Zn binding site, consistent with known compound interactions. These results highlight the potential health benefits of blueberry metabolites through interaction with CA proteins.

Impact of the SIK3 Pathway Inhibition on Osteoclast Differentiation via Oxidative Phosphorylation.

Maintenance of bone homeostasis and the balance between bone resorption and formation are crucial for maintaining skeletal integrity. This study sought to investigate the role of salt-inducible kinase 3 (SIK3), a key regulator in cellular energy metabolism, during the differentiation of osteoclasts. Despite osteoclasts being high energy-consuming cells essential for breaking down mineralized bone tissue, the specific function of SIK3 in this process remains unclear. To address this issue, we generated osteoclast-specific SIK3 conditional knockout mice and assessed the impact of SIK3 deletion on bone homeostasis. Our findings revealed that SIK3 conditional knockout mice exhibited increased bone mass and an osteopetrosis phenotype, suggesting a pivotal role for SIK3 in bone resorption. Moreover, we assessed the impact of pterosin B, a SIK3 inhibitor, on osteoclast differentiation. The treatment with pterosin B inhibited osteoclast differentiation, reduced the numbers of multinucleated osteoclasts, and suppressed resorption activity in vitro. Gene expression analysis demonstrated that SIK3 deletion and pterosin B treatment influence a common set of genes involved in osteoclast differentiation and bone resorption. Furthermore, pterosin B treatment altered intracellular metabolism, particularly affecting key metabolic pathways, such as the tricarboxylic acid cycle and oxidative phosphorylation. These results provide valuable insights into the involvement of SIK3 in osteoclast differentiation and the molecular mechanisms underlying osteoclast function and bone diseases.

The key roles of thyroid hormone in mitochondrial regulation, at interface of human health and disease.

Mitochondria are highly plastic and dynamic organelles long known as the powerhouse of cellular bioenergetics, but also endowed with a critical role in stress responses and homeostasis maintenance, supporting and integrating activities across multifaced cellular processes. As a such, mitochondria dysfunctions are leading causes of a wide range of diseases and pathologies. Thyroid hormones (THs) are endocrine regulators of cellular metabolism, regulating intracellular nutrients fueling of sugars, amino acids and fatty acids. For instance, THs regulate the balance between the anabolism and catabolism of all the macro-molecules, influencing energy homeostasis during different nutritional conditions. Noteworthy, not only most of the TH-dependent metabolic modulations act via the mitochondria, but also THs have been proved to regulate the mitochondrial biosynthesis, dynamics and function. The significance of such an interplay is different in the context of specific tissues and strongly impacts on cellular homeostasis. Thus, a comprehensive understanding of THs-dependent mitochondrial functions and dynamics is required to develop more precise strategies for targeting mitochondrial function. Herein, we describe the mechanisms of TH-dependent metabolic regulation with a focus on mitochondrial action, in different tissue contexts, thus providing new insights for targeted modulation of mitochondrial dynamics.

Circadian disruption, clock genes, and metabolic health.

A growing body of research has identified circadian-rhythm disruption as a risk factor for metabolic health. However, the underlying biological basis remains complex, and complete molecular mechanisms are unknown. There is emerging evidence from animal and human research to suggest that the expression of core circadian genes, such as circadian locomotor output cycles kaput gene (CLOCK), brain and muscle ARNT-Like 1 gene (BMAL1), period (PER), and cyptochrome (CRY), and the consequent expression of hundreds of circadian output genes are integral to the regulation of cellular metabolism. These circadian mechanisms represent potential pathophysiological pathways linking circadian disruption to adverse metabolic health outcomes, including obesity, metabolic syndrome, and type 2 diabetes. Here, we aim to summarize select evidence from in vivo animal models and compare these results with epidemiologic research findings to advance understanding of existing foundational evidence and potential mechanistic links between circadian disruption and altered clock gene expression contributions to metabolic health-related pathologies. Findings have important implications for the treatment, prevention, and control of metabolic pathologies underlying leading causes of death and disability, including diabetes, cardiovascular disease, and cancer.

Mitochondrial regulation of adult hippocampal neurogenesis: Insights into neurological function and neurodevelopmental disorders

Mitochondria are essential regulators of cellular energy metabolism and play a crucial role in the maintenance and function of neuronal cells. Studies in the last decade have highlighted the importance of mitochondrial dynamics and bioenergetics in adult neurogenesis, a process that significantly influences cognitive function and brain plasticity. In this review, we examine the mechanisms by which mitochondria regulate adult neurogenesis, focusing on the impact of mitochondrial function on the behavior of neural stem/progenitor cells and the maturation and plasticity of newborn neurons in the adult mouse hippocampus. In addition, we explore the link between mitochondrial dysfunction, adult hippocampal neurogenesis and genes associated with cognitive deficits in neurodevelopmental disorders. In particular, we provide insights into how alterations in the transcriptional regulator NR2F1 affect mitochondrial dynamics and may contribute to the pathophysiology of the emerging neurodevelopmental disorder Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS). Understanding how genes involved in embryonic and adult neurogenesis affect mitochondrial function in neurological diseases might open new directions for therapeutic interventions aimed at boosting mitochondrial function during postnatal life.

KLF13 restrains Dll4-muscular Notch2 axis to improve the muscle atrophy.

Muscle atrophy can cause muscle dysfunction and weakness. Krüppel-like factor 13 (KLF13), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscles and implicated in the pathogenesis of several diseases. This study investigated the role of KLF13 in muscle atrophy, which could be a novel therapeutic target. The effects of gene knockdown and pharmacological targeting of KLF13 on skeletal muscle atrophy were investigated using cell-based and animal models. Clofoctol, an antibiotic and KLF13 agonist, was also investigated as a candidate for repurposing. The mechanisms related to skeletal muscle atrophy were assessed by measuring the expression levels and activation statuses of key regulatory pathways and validated using gene knockdown and RNA sequencing. In a dexamethasone-induced muscle atrophy mouse model, the KLF13 knockout group had decreased muscle strength (N) (1.77±0.10 vs. 1.48±0.16, P<0.01), muscle weight (%) [gastrocnemius (Gas): 76.0±5.69 vs. 60.7±7.23, P<0.001; tibialis anterior (TA): 75.8±6.21 vs. 67.5±5.01, P<0.05], and exhaustive running distance (m) (495.5±64.8 vs. 315.5±60.9, P<0.05) compared with the control group. KLF13 overexpression preserved muscle mass (Gas: 100±6.38 vs. 120±14.4, P<0.01) and the exhaustive running distance (423.8±59.04 vs. 530.2±77.45, P<0.05) in an in vivo diabetes-induced skeletal muscle atrophy model. Clofoctol treatment protected against dexamethasone-induced muscle atrophy. Myotubes treated with dexamethasone, an atrophy-inducing glucocorticoid, were aggravated by KLF13 knockout, but anti-atrophic effects were achieved by inducing KLF13 overexpression. We performed a transcriptome analysis and luciferase reporter assays to further explore this mechanism, finding that delta-like 4 (Dll4) was a novel target gene of KLF13. The KLF13 transcript repressed Dll4, inhibiting the Dll4-Notch2 axis and preventing muscle atrophy. Dexamethasone inhibited KLF13 expression by inhibiting myogenic differentiation 1 (i.e., MYOD1)-mediated KLF13 transcriptional activation and promoting F-Box and WD repeat domain containing 7 (i.e., FBXW7)-mediated KLF13 ubiquitination. This study sheds new light on the mechanisms underlying skeletal muscle atrophy and potential drug targets. KLF13 regulates muscle atrophy and is a potential therapeutic target. Clofoctol is an attractive compound for repurposing studies to treat skeletal muscle atrophy.

Mitochondria- and ER-associated actin are required for mitochondrial fusion.

Mitochondria play a crucial role in the regulation of cellular metabolism and signalling. Mitochondrial activity is modulated by the processes of mitochondrial fission and fusion, which are required to properly balance respiratory and metabolic functions, transfer material between mitochondria, and remove defective mitochondria. Mitochondrial fission occurs at sites of contact between the endoplasmic reticulum (ER) and mitochondria, and is dependent on the formation of actin filaments that drive mitochondrial constriction and the recruitment and activation of the dynamin-related GTPase fission protein DRP1. The requirement for mitochondria- and ER-associated actin filaments in mitochondrial fission remains unclear, and the role of actin in mitochondrial fusion remains entirely unexplored. Here we show that preventing the formation of actin filaments on either mitochondria or the ER disrupts both mitochondrial fission and fusion. We show that fusion but not fission is dependent on Arp2/3, whereas both fission and fusion are dependent on INF2 formin-dependent actin polymerization. We also show that mitochondria-associated actin marks fusion sites prior to the dynamin family GTPase fusion protein MFN2. Together, our work introduces a novel method for perturbing organelle-associated actin filaments, and demonstrates a previously unknown role for actin in mitochondrial fusion.

Key gene modules and hub genes associated with pyrethroid and organophosphate resistance in Anopheles mosquitoes: a systems biology approach

Indoor residual spraying (IRS) and insecticide-treated nets (ITNs) are the main methods used to control mosquito populations for malaria prevention. The efficacy of these strategies is threatened by the spread of insecticide resistance (IR), limiting the success of malaria control. Studies of the genetic evolution leading to insecticide resistance could enable the identification of molecular markers that can be used for IR surveillance and an improved understanding of the molecular mechanisms associated with IR. This study used a weighted gene co-expression network analysis (WGCNA) algorithm, a systems biology approach, to identify genes with similar co-expression patterns (modules) and hub genes that are potential molecular markers for insecticide resistance surveillance in Kenya and Benin. A total of 20 and 26 gene co-expression modules were identified via average linkage hierarchical clustering from Anopheles arabiensis and An. gambiae, respectively, and hub genes (highly connected genes) were identified within each module. Three specific genes stood out: serine protease, E3 ubiquitin-protein ligase, and cuticular proteins, which were top hub genes in both species and could serve as potential markers and targets for monitoring IR in these malaria vectors. In addition to the identified markers, we explored molecular mechanisms using enrichment maps that revealed a complex process involving multiple steps, from odorant binding and neuronal signaling to cellular responses, immune modulation, cellular metabolism, and gene regulation. Incorporation of these dynamics into the development of new insecticides and the tracking of insecticide resistance could improve the sustainable and cost-effective deployment of interventions.

NIR-II light-powered core-shell prodrug nanomotors enhance cancer therapy through synergistic oxidative stress-photothermo modulation

Near-infrared-II (NIR-II) photothermal therapy is emerging as a cutting-edge modality for tumor ablation due to its good biosafety, high penetration ability and spatiotemporal controllability. Despite efforts, establishing a link between cellular metabolic regulation and photothermal performance is still promising in synergistic cancer therapy. Herein, we developed a core-shell semiconducting polymer@metal-phenolic network (SP@GFP) nanomotor by assembling diphenol-terminated cisplatin prodrug ligand (cPt-DA) and iron (III) (Fe3+) through metal coordination on SP particles in the presence of GOx and DSPE-PEG-cRGD, for NIR-II-propelled self-propulsion and synergistic cancer therapy. Remotely driving the SP@GFP nanomotor with an NIR-II laser through a thermophoresis mechanism would allow for in-depth penetration and accumulation. The synergistic photothermal effect and continuous Fe2+-mediated ROS generation of SP@GFP nanomotor could activate photothermal, chemotherapeutic effects and ferroptosis pathway for cancer cells through reshaping cellular metabolic pathways (HSP and GPX4). By combining the concepts of chemotherapeutic prodrugs, catalytic ROS generation, photothermal response and cellular metabolic regulation, the NIR-II laser-controlled core-shell SP@GFP nanomotor displayed improved outcomes for enhanced cancer therapy through synergistic oxidative stress-photothermo modulation. Statement of significance•A NIR-II-powered core-shell SP@GFP prodrug nanomotor was constructed through metal coordination assembly for enhanced cancer therapy;•NIR-II laser propelled the propulsion of SP@GFP nanomotor through thermophoresis mechanism for in depth penetration and accumulation.•Therapeutic outcomes were improved through synergistic modulating oxidative stress and photothermal performance.

ZIF-8-based Nanoparticles for Inflammation Treatment and Oxidative Stress Reduction in Periodontitis.

Periodontitis, an inflammatory bone resorption disease associated with dental plaque, poses significant challenges for effective treatment. In this study, we developed Mino@ZIF-8 nanoparticles inspired by the periodontal microenvironment and the unique properties of zeolitic imidazolate framework 8, aiming to address the complex pathogenesis of periodontitis. Transcriptome analysis revealed the active engagement of Mino@ZIF-8 nanoparticles in innate and adaptive inflammatory host defense and cellular metabolic remodeling. Through sustained release of the anti-inflammatory and antibacterial agent minocycline hydrochloride (Mino) and the generation of Zn2+ with pro-antioxidant effects during degradation, Mino@ZIF-8 nanoparticles synergistically alleviate inflammation and oxidative damage. Notably, our study focuses on the pivotal role of zinc ions in mitochondrial oxidation protection. Under lipopolysaccharide (LPS) stimulation, periodontal ligament cells undergo a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis, leading to reduced ATP production and increased reactive oxygen species levels. However, Zn2+ effectively rebalances the glycolysis-OXPHOS imbalance, restoring cellular bioenergetics, mitigating oxidative damage, rescuing impaired mitochondria, and suppressing inflammatory cytokine production through modulation of the AKT/GSK3β/NRF2 pathway. This research not only presents a promising approach for periodontitis treatment but also offers novel therapeutic opportunities for zinc-containing materials, providing valuable insights into the design of biomaterials targeting cellular energy metabolism regulation.

Oleic acid enhances proliferation and calcium mobilization of CD3/CD28 activated CD4+ T cells through incorporation into membrane lipids.

Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function.

Integrative RNA-seq and ChIP-seq analysis unveils metabolic regulation as a conserved antiviral mechanism of chicken p53.

The tumor suppressor p53, primarily functioning as a transcription factor, has exhibited antiviral capabilities against various viruses in chickens, including infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). Nevertheless, the existence of a universal antiviral mechanism employed by chicken p53 (chp53) against these viruses remains uncertain. This study conducted a comprehensive comparison of molecular networks involved in chp53's antiviral function against IBDV, ALV-J, and ILTV. This was achieved through an integrated analysis of ChIP-seq data, examining chp53's genome-wide chromatin occupancy, and RNA-seq data from chicken cells infected with these viruses. The consistent observation of chp53 target gene enrichment in metabolic pathways, confirmed via ChIP-qPCR, suggests a ubiquitous regulation of host cellular metabolism by chp53 across different viruses. Further genome binding motif conservation analysis and transcriptional co-factor prediction suggest conserved transcriptional regulation mechanism by which chp53 regulates host cellular metabolism during viral infection. These findings offer novel insights into the antiviral role of chp53 and propose that targeting the virus-host metabolic interaction through regulating p53 could serve as a universal strategy for antiviral therapies in chickens.IMPORTANCEThe current study conducted a comprehensive analysis, comparing molecular networks underlying chp53's antiviral role against infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). This was achieved through a combined assessment of ChIP-seq and RNA-seq data obtained from infected chicken cells. Notably, enrichment of chp53 target genes in metabolic pathways was consistently observed across viral infections, indicating a universal role of chp53 in regulating cellular metabolism during diverse viral infections. These findings offer novel insights into the antiviral capabilities of chicken p53, laying a foundation for the potential development of broad-spectrum antiviral therapies in chickens.

Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells

The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted a SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared to the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.

Targeting pyruvate kinase M2 for the treatment of kidney disease.

Pyruvate kinase M2 (PKM2), a rate limiting enzyme in glycolysis, is a cellular regulator that has received extensive attention and regards as a metabolic regulator of cellular metabolism and energy. Kidney is a highly metabolically active organ, and glycolysis is the important energy resource for kidney. The accumulated evidences indicates that the enzymatic activity of PKM2 is disturbed in kidney disease progression and treatment, especially diabetic kidney disease and acute kidney injury. Modulating PKM2 post-translational modification determines its enzymatic activity and nuclear translocation that serves as an important interventional approach to regulate PKM2. Emerging evidences show that PKM2 and its post-translational modification participate in kidney disease progression and treatment through modulating metabolism regulation, podocyte injury, fibroblast activation and proliferation, macrophage polarization, and T cell regulation. Interestingly, PKM2 activators (TEPP-46, DASA-58, mitapivat, and TP-1454) and PKM2 inhibitors (shikonin, alkannin, compound 3k and compound 3h) have exhibited potential therapeutic property in kidney disease, which indicates the pleiotropic effects of PKM2 in kidney. In the future, the deep investigation of PKM2 pleiotropic effects in kidney is urgently needed to determine the therapeutic effect of PKM2 activator/inhibitor to benefit patients. The information in this review highlights that PKM2 functions as a potential biomarker and therapeutic target for kidney diseases.

Intramolecular Disulfide Bridges in Voltage-Dependent Anion Channel 2 (VDAC2) Protein from Rattus norvegicus Revealed by High-Resolution Mass Spectrometry.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

D-SPIN constructs gene regulatory network models from multiplexed scRNA-seq data revealing organizing principles of cellular perturbation response.

Gene regulatory networks within cells modulate the expression of the genome in response to signals and changing environmental conditions. Reconstructions of gene regulatory networks can reveal the information processing and control principles used by cells to maintain homeostasis and execute cell-state transitions. Here, we introduce a computational framework, D-SPIN, that generates quantitative models of gene regulatory networks from single-cell mRNA-seq datasets collected across thousands of distinct perturbation conditions. D-SPIN models the cell as a collection of interacting gene-expression programs, and constructs a probabilistic model to infer regulatory interactions between gene-expression programs and external perturbations. Using large Perturb-seq and drug-response datasets, we demonstrate that D-SPIN models reveal the organization of cellular pathways, sub-functions of macromolecular complexes, and the logic of cellular regulation of transcription, translation, metabolism, and protein degradation in response to gene knockdown perturbations. D-SPIN can also be applied to dissect drug response mechanisms in heterogeneous cell populations, elucidating how combinations of immunomodulatory drugs can induce novel cell states through additive recruitment of gene expression programs. D-SPIN provides a computational framework for constructing interpretable models of gene-regulatory networks to reveal principles of cellular information processing and physiological control.

ANGPTL3 regulates the peroxisomal translocation of SmarcAL1 in response to cell growth states.

Angiopoietin-like 3 (ANGPTL3) is a key regulator of lipoprotein metabolism, known for its potent inhibition on intravascular lipoprotein and endothelial lipase activities. Recent studies have shed light on the cellular functions of ANGPTL3. However, the precise mechanism underlying its regulation of cellular lipid metabolism remains elusive. We recently reported that ANGPTL3 interacts with the chromatin regulator SMARCAL1, which plays a pivotal role in maintaining cellular lipid homeostasis. Here, through a combination of in vitro and in vivo functional analyses, we provide evidence that ANGPTL3 indeed influences cellular lipid metabolism. Increased expression of Angptl3 prompted the formation of lipid droplets (LDs) in response to slow growth conditions. Notably, under the conditions, Angptl3 accumulated within cytoplasmic peroxisomes, where it interacts with SmarcAL1, which translocated from nucleus as observed previously. This translocation induced changes in gene expression favoring triglyceride (TG) accumulation. Indeed, ANGPTL3 gene knockout (KO) in human cells increased the expression of key lipid genes, which could be linked to elevated nuclear localization of SMARCAL1, whereas the expression of these genes decreased in SMARCAL1 KO cells. Consistent with these findings, the injection of Angptl3 protein to mice led to hepatic fat accumulation derived from circulating blood, a phenotype likely indicative of its long-term effect on blood TG, linked to SmarcAL1 activities. Thus, our results suggest that the Angptl3-SmarcAL1 pathway may confer the capacity for TG storage in cells in response to varying growth states, which may have broad implications for this pathway in regulating energy storage and trafficking.