Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

LncRNA CamK-A Regulates Ca2+-Signaling-Mediated Tumor Microenvironment Remodeling.

Cancer cells entail metabolic adaptation and microenvironmental remodeling to survive and progress. Both calcium (Ca2+) flux and Ca2+-dependent signaling play a crucial role in this process, although the underlying mechanism has yet to be elucidated. Through RNA screening, we identified one long noncoding RNA (lncRNA) named CamK-A (lncRNA for calcium-dependent kinase activation) in tumorigenesis. CamK-A is highly expressed in multiple human cancers and involved in cancer microenvironment remodeling via activation of Ca2+-triggered signaling. Mechanistically, CamK-A activates Ca2+/calmodulin-dependent kinase PNCK, which in turn phosphorylates IκBα and triggers calcium-dependent nuclear factor κB (NF-κB) activation. This regulation results in the tumor microenvironment remodeling, including macrophage recruitment, angiogenesis, and tumor progression. Notably, our human-patient-derived xenograft (PDX) model studies demonstrate that targeting CamK-A robustly impaired cancer development. Clinically, CamK-A expression coordinates with the activation of CaMK-NF-κB axis, and its high expression indicates poor patient survival rate, suggesting its role as a potential biomarker and therapeutic target.

Lysine-372-dependent SUMOylation inhibits the enzymatic activity of glutamine synthases.

Glutamine synthetase (GS) is a crucial enzyme involved in de novo synthesis of glutamine and participates in several biological processes, including nitrogen metabolism, nucleotide synthesis, and amino acid synthesis. Post-translational modification makes GS more adaptable to the needs of cells, and acetylation modification of GS at double sites has attracted considerable attention. Despite very intensive research, how SUMOylation affects GS activity at a molecular level remains unclear. Here, we report that previously undiscovered GS SUMOylation which is deficient mutant K372R of GS exhibits more bluntness under glutamine starvation. Mechanistically, glutamine deprivation triggers the GS SUMOylation, and this SUMOylation impaired the protein stability of GS, within a concomitant decrease in enzymatic activity. In addition, we identified SAE1, Ubc9, and PIAS1 as the assembly enzymes of GS SUMOylation respectively. Furthermore, Senp1/2 functions as a SUMO-specific protease to reverse the SUMOylation of GS. This study provides the first evidence that SUMOylation serves as a regulatory mechanism for determining the GS enzymatic activity, contributing to understanding the GS regulation roles in various cellular and pathophysiological processes.

Methionine deficiency facilitates antitumour immunity by altering m6A methylation of immune checkpoint transcripts.

OBJECTIVE: Methionine metabolism is involved in a myriad of cellular functions, including methylation reactions and redox maintenance. Nevertheless, it remains unclear whether methionine metabolism, RNA methylation and antitumour immunity are molecularly intertwined. DESIGN: The antitumour immunity effect of methionine-restricted diet (MRD) feeding was assessed in murine models. The mechanisms of methionine and YTH domain-containing family protein 1 (YTHDF1) in tumour immune escape were determined in vitro and in vivo. The synergistic effects of MRD or YTHDF1 depletion with PD-1 blockade were also investigated. RESULTS: We found that dietary methionine restriction reduced tumour growth and enhanced antitumour immunity by increasing the number and cytotoxicity of tumour-infiltrating CD8+ T cells in different mouse models. Mechanistically, the S-adenosylmethionine derived from methionine metabolism promoted the N6-methyladenosine (m6A) methylation and translation of immune checkpoints, including PD-L1 and V-domain Ig suppressor of T cell activation (VISTA), in tumour cells. Furthermore, MRD or m6A-specific binding protein YTHDF1 depletion inhibited tumour growth by restoring the infiltration of CD8+ T cells, and synergised with PD-1 blockade for better tumour control. Clinically, YTHDF1 expression correlated with poor prognosis and immunotherapy outcomes for cancer patients. CONCLUSIONS: Methionine and YTHDF1 play a critical role in anticancer immunity through regulating the functions of T cells. Targeting methionine metabolism or YTHDF1 could be a potential new strategy for cancer immunotherapy.

Integrin signaling in cancer: bidirectional mechanisms and therapeutic opportunities.

Integrins are transmembrane receptors that possess distinct ligand-binding specificities in the extracellular domain and signaling properties in the cytoplasmic domain. While most integrins have a short cytoplasmic tail, integrin ß4 has a long cytoplasmic tail that can indirectly interact with the actin cytoskeleton. Additionally, 'inside-out' signals can induce integrins to adopt a high-affinity extended conformation for their appropriate ligands. These properties enable integrins to transmit bidirectional cellular signals, making it a critical regulator of various biological processes.Integrin expression and function are tightly linked to various aspects of tumor progression, including initiation, angiogenesis, cell motility, invasion, and metastasis. Certain integrins have been shown to drive tumorigenesis or amplify oncogenic signals by interacting with corresponding receptors, while others have marginal or even suppressive effects. Additionally, different α/ß subtypes of integrins can exhibit opposite effects. Integrin-mediated signaling pathways including Ras- and Rho-GTPase, TGFß, Hippo, Wnt, Notch, and sonic hedgehog (Shh) are involved in various stages of tumorigenesis. Therefore, understanding the complex regulatory mechanisms and molecular specificities of integrins are crucial to delaying cancer progression and suppressing tumorigenesis. Furthermore, the development of integrin-based therapeutics for cancer are of great importance.This review provides an overview of integrin-dependent bidirectional signaling mechanisms in cancer that can either support or oppose tumorigenesis by interacting with various signaling pathways. Finally, we focus on the future opportunities for emergent therapeutics based on integrin agonists. Video Abstract.

PLIN2 promotes HCC cells proliferation by inhibiting the degradation of HIF1α.

PLIN2 has been found to be dysregulated in several human malignancies, which influences cancer progression. However, the roles of PLIN2 in regulating hepatocellular carcinoma (HCC) progression are still unclear. Here, we revealed that PLIN2 was frequently upregulated in HCC cells and tissues, and increased PLIN2 expression was associated with poor prognosis outcomes in HCC. In HCC cells, overexpressing PLIN2 promoted cell proliferation, PLIN2-deficiency inhibited cell vitality. Mechanistically, silencing of PLIN2 expression downregulated hypoxia inducible factor 1-α (HIF1α) expression and this downregulation in turn inhibited the targeting genes of HIF1α. Furthermore, we found that PLIN2 stabilized and retarded the degradation of the HIF1α through autophagy-lysosomal pathway by inhibiting AMPK/ULK1. Collectively, we clarified the carcinogenic role of PLIN2 in HCC and suggested a prognostic biomarker for diagnosis and clinical therapy in the future.

Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK.

The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.

Structural Basis of the Inhibition of L-Methionine γ-Lyase from Fusobacterium nucleatum.

Fusobacterium nucleatum is a lesion-associated obligate anaerobic pathogen of destructive periodontal disease; it is also implicated in the progression and severity of colorectal cancer. Four genes (FN0625, FN1055, FN1220, and FN1419) of F. nucleatum are involved in producing hydrogen sulfide (H2S), which plays an essential role against oxidative stress. The molecular functions of Fn1419 are known, but their mechanisms remain unclear. We determined the crystal structure of Fn1419 at 2.5 Å, showing the unique conformation of the PLP-binding site when compared with L-methionine γ-lyase (MGL) proteins. Inhibitor screening for Fn1419 with L-cysteine showed that two natural compounds, gallic acid and dihydromyricetin, selectively inhibit the H2S production of Fn1419. The chemicals of gallic acid, dihydromyricetin, and its analogs containing trihydroxybenzene, were potentially responsible for the enzyme-inhibiting activity on Fn1419. Molecular docking and mutational analyses suggested that Gly112, Pro159, Val337, and Arg373 are involved in gallic acid binding and positioned close to the substrate and pyridoxal-5'-phosphate-binding site. Gallic acid has little effect on the other H2S-producing enzymes (Fn1220 and Fn1055). Overall, we proposed a molecular mechanism underlying the action of Fn1419 from F. nucleatum and found a new lead compound for inhibitor development.

Structural and Biochemical Analyses of the Butanol Dehydrogenase from Fusobacterium nucleatum.

Butanol dehydrogenase (BDH) plays a significant role in the biosynthesis of butanol in bacteria by catalyzing butanal conversion to butanol at the expense of the NAD(P)H cofactor. BDH is an attractive enzyme for industrial application in butanol production; however, its molecular function remains largely uncharacterized. In this study, we found that Fusobacterium nucleatum YqdH (FnYqdH) converts aldehyde into alcohol by utilizing NAD(P)H, with broad substrate specificity toward aldehydes but not alcohols. An in vitro metal ion substitution experiment showed that FnYqdH has higher enzyme activity in the presence of Co2+. Crystal structures of FnYqdH, in its apo and complexed forms (with NAD and Co2+), were determined at 1.98 and 2.72 Å resolution, respectively. The crystal structure of apo- and cofactor-binding states of FnYqdH showed an open conformation between the nucleotide binding and catalytic domain. Key residues involved in the catalytic and cofactor-binding sites of FnYqdH were identified by mutagenesis and microscale thermophoresis assays. The structural conformation and preferred optimal metal ion of FnYqdH differed from that of TmBDH (homolog protein of FnYqdH). Overall, we proposed an alternative model for putative proton relay in FnYqdH, thereby providing better insight into the molecular function of BDH.

AQP3-mediated H2 O2 uptake inhibits LUAD autophagy by inactivating PTEN.

It is widely accepted that redox reprogramming participates in malignant transformation of lung adenocarcinoma (LUAD). However, the source of excessive reactive oxygen species (ROS) and the downstream signaling regulatory mechanism are complicated and unintelligible. In the current study, we newly identified the aquaporin 3 (AQP3) as a LUAD oncogenic factor with capacity to transport exogenous hydrogen peroxide (H2 O2 ) and increase intracellular ROS levels. Subsequently, we demonstrated that AQP3 was necessary for the facilitated diffusion of exogenous H2 O2 in LUAD cells and that the AQP3-dependent transport of H2 O2 accelerated cell growth and inhibited rapamycin-induced autophagy. Mechanistically, AQP3-mediated H2 O2 uptake increased intracellular ROS levels to inactivate PTEN and activate the AKT/mTOR pathway to subsequently inhibit autophagy and promote proliferation in LUAD cells. Finally, we suggested that AQP3 depletion retarded subcutaneous tumorigenesis in vivo and simultaneously decreased ROS levels and promoted autophagy. These findings underscore the importance of AQP3-induced oxidative stress in malignant transformation and suggest a therapeutic target for LUAD.

Rational Design of Crystallization-Induced-Emission Probes To Detect Amorphous Protein Aggregation in Live Cells.

Unlike amyloid aggregates, amorphous protein aggregates with no defined structures have been challenging to target and detect in a complex cellular milieu. In this study, we rationally designed sensors of amorphous protein aggregation from aggregation-induced-emission probes (AIEgens). Utilizing dicyanoisophorone as a model AIEgen scaffold, we first sensitized the fluorescence of AIEgens to a nonpolar and viscous environment mimicking the interior of amorphous aggregated proteins. We identified a generally applicable moiety (dimethylaminophenylene) for selective binding and fluorescence enhancement. Regulation of the electron-withdrawing groups tuned the emission wavelength while retaining selective detection. Finally, we utilized the optimized probe to systematically image aggregated proteome upon proteostasis network regulation. Overall, we present a rational approach to develop amorphous protein aggregation sensors from AIEgens with controllable sensitivity, spectral coverage, and cellular performance.

Stable Super-Resolution Imaging of Lipid Droplet Dynamics through a Buffer Strategy with a Hydrogen-Bond Sensitive Fluorogenic Probe.

Although super-resolution imaging offers an opportunity to visualize cellular structures and organelles at the nanoscale level, cellular heterogeneity and unpredictability still pose a significant challenge in the dynamic imaging of live cells. It is thus vital to develop better-performing and more photostable probes for long-term super-resolution imaging. Herein, we report a probe, LD-FG, for imaging lipid droplet (LD) dynamics using structured illumination microscopy (SIM). LD-FG allows wash-free imaging of LDs, owing to a hydrogen-bond sensitive fluorogenic response. The replacement of photobleached LD-FG by intact probe molecules outside the LDs ensures the long-time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Using this probe, two LD coalescence modes were discovered. The dynamic imaging also allowed us to propose a new model of LD maturation during adipocyte differentiation, i.e., a fast LD coalescence followed by a slow ripening step. The excellent performance of LD-FG makes the buffer strategy an effective method for designing fluorescent probes for cell dynamic imaging.

Integrated Metabolomics and Lipidomics Analyses Reveal Metabolic Reprogramming in Human Glioma with IDH1 Mutation.

Mutations in isocitrate dehydrogenase ( IDH) 1 are high-frequency events in low-grade glioma and secondary glioblastoma, and IDH1 mutant gliomas are vulnerable to interventions. Metabolic reprogramming is a hallmark of cancer. In this study, comprehensive metabolism investigation of clinical IDH1 mutant glioma specimens was performed to explore its specific metabolic reprogramming in real microenvironment. Massive metabolic alterations from glycolysis to lipid metabolism were identified in the IDH1 mutant glioma tissue when compared to IDH1 wild-type glioma. Of note, tricarboxylic acid (TCA) cycle intermediates were in similar levels in both groups, with more pyruvate found entering the TCA cycle in IDH1 mutant glioma. The pool of fatty acyl chains was also reduced, displayed as decreased triglycerides and sphingolipids, although membrane phosphatidyl lipids were not changed. The lower fatty acyl pool may be mediated by the lower protein expression levels of long-chain acyl-CoA synthetase 1 (ACSL1), ACSL4, and very long-chain acyl-CoA synthetase 3 (ACSVL3) in IDH1 mutant glioma. Lower ACSL1 was further found to contribute to the better survival of IDH1 mutant glioma patients based on the The Cancer Genome Atlas (TCGA) RNA sequencing data. Our research provides valuable insights into the tissue metabolism of human IDH1 mutant glioma and unravels new lipid-related targets.

Comprehensive Profiling by Non-targeted Stable Isotope Tracing Capillary Electrophoresis-Mass Spectrometry: A New Tool Complementing Metabolomic Analyses of Polar Metabolites.

Mass spectrometry (MS) driven metabolomics is a frequently used tool in various areas of life sciences; however, the analysis of polar metabolites is less commonly included. In general, metabolomic analyses lead to the detection of the total amount of all covered metabolites. This is currently a major limitation with respect to metabolites showing high turnover rates, but no changes in their concentration. Such metabolites and pathways could be crucial metabolic nodes (e.g., potential drug targets in cancer metabolism). A stable-isotope tracing capillary electrophoresis-mass spectrometry (CE-MS) metabolomic approach was developed to cover both polar metabolites and isotopologues in a non-targeted way. An in-house developed software enables high throughput processing of complex multidimensional data. The practicability is demonstrated analyzing [U-13 C]-glucose exposed prostate cancer and non-cancer cells. This CE-MS-driven analytical strategy complements polar metabolite profiles through isotopologue labeling patterns, thereby improving not only the metabolomic coverage, but also the understanding of metabolism.

Metabolomics and transcriptomics profiles reveal the dysregulation of the tricarboxylic acid cycle and related mechanisms in prostate cancer.

Genetic alterations drive metabolic reprograming to meet increased biosynthetic precursor and energy demands for cancer cell proliferation and survival in unfavorable environments. A systematic study of gene-metabolite regulatory networks and metabolic dysregulation should reveal the molecular mechanisms underlying prostate cancer (PCa) pathogenesis. Herein, we performed gas chromatography-mass spectrometry (GC-MS)-based metabolomics and RNA-seq analyses in prostate tumors and matched adjacent normal tissues (ANTs) to elucidate the molecular alterations and potential underlying regulatory mechanisms in PCa. Significant accumulation of metabolic intermediates and enrichment of genes in the tricarboxylic acid (TCA) cycle were observed in tumor tissues, indicating TCA cycle hyperactivation in PCa tissues. In addition, the levels of fumarate and malate were highly correlated with the Gleason score, tumor stage and expression of genes encoding related enzymes and were significantly related to the expression of genes involved in branched chain amino acid degradation. Using an integrated omics approach, we further revealed the potential anaplerotic routes from pyruvate, glutamine catabolism and branched chain amino acid (BCAA) degradation contributing to replenishing metabolites for TCA cycle. Integrated omics techniques enable the performance of network-based analyses to gain a comprehensive and in-depth understanding of PCa pathophysiology and may facilitate the development of new and effective therapeutic strategies.

High-throughput metabolic profiling based on small amount of hepatic cells.

Common metabolomics platforms require about 106 cells, which has a limited throughput due to the time-consuming steps of cell culture and preparation. There is a demand for metabolic profiling methods to improve analytical throughput and detection sensitivity based on small amount of cells. In this study, we proposed a high-throughput scheme, integrating 96-well plate cell cultivation, in-situ cell pretreatment, and sensitive dansylation labeling coupled with LC-MS analysis of metabolites inside HepG2 cells (of the order of magnitude of 103 cells in each well). A simple and rapid cell pretreatment was performed showing good extraction efficiency and good precision (the RSDs smaller than 5%) for polar metabolites. The recovery in metabolite extraction evaluated with three isotope-labeled amino acids was from 89.7 to 106.3% at low, medium, and high concentrations. The suitability of the method was illustrated by exploring influences of different fatty acids on HepG2 cells.

Proteomic analysis of the human cyclin-dependent kinase family reveals a novel CDK5 complex involved in cell growth and migration.

Cyclin-dependent kinases (CDKs) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in the control of cell-cycle progression, transcription, and neuronal functions. However, the functions, substrates, and regulation of many CDKs are poorly understood. To systematically investigate these features of CDKs, we conducted a proteomic analysis of the CDK family and identified their associated protein complexes in two different cell lines using a modified SAINT (Significance Analysis of INTeractome) method. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000593 and DOI 10.6019/PXD000593. We identified 753 high-confidence candidate interaction proteins (HCIPs) in HEK293T cells and 352 HCIPs in MCF10A cells. We subsequently focused on a neuron-specific CDK, CDK5, and uncovered two novel CDK5-binding partners, KIAA0528 and fibroblast growth factor (acidic) intracellular binding protein (FIBP), in non-neuronal cells. We showed that these three proteins form a stable complex, with KIAA0528 and FIBP being required for the assembly and stability of the complex. Furthermore, CDK5-, KIAA0528-, or FIBP-depleted breast cancer cells displayed impaired proliferation and decreased migration, suggesting that this complex is required for cell growth and migration in non-neural cells. Our study uncovers new aspects of CDK functions, which provide direction for further investigation of these critical protein kinases.

Long non-coding RNA HOTAIR in carcinogenesis and metastasis.

Long non-coding RNAs (lncRNAs) have gained massive attention in recent years as a potentially new and crucial layer of gene regulation. LncRNAs are prevalently transcribed in the genome, but their roles in gene regulation and disease development are largely unknown. HOX antisense intergenic RNA (HOTAIR), a lncRNA located in the HOXC locus, has been shown to repress HOXD gene expression and promote breast cancer metastasis. Mechanistically, HOTAIR interacts with and recruits polycomb repressive complex 2 (PRC2) and regulates chromosome occupancy by EZH2 (a subunit of PRC2), which leads to histone H3 lysine 27 trimethylation of the HOXD locus. Moreover, HOTAIR is pervasively overexpressed in most human cancers compared with noncancerous adjacent tissues. This review summarizes the studies on the HOTAIR lncRNA over the past 6 years.

Exploring Protein S-Palmitoylation: Mechanisms, Detection, and Strategies for Inhibitor Discovery.

S-palmitoylation is a reversible and dynamic process that involves the addition of long-chain fatty acids to proteins. This protein modification regulates various aspects of protein function, including subcellular localization, stability, conformation, and biomolecular interactions. The zinc finger DHHC (ZDHHC) domain-containing protein family is the main group of enzymes responsible for catalyzing protein S-palmitoylation, and 23 members have been identified in mammalian cells. Many proteins that undergo S-palmitoylation have been linked to disease pathogenesis and progression, suggesting that the development of effective inhibitors is a promising therapeutic strategy. Reducing the protein S-palmitoylation level can target either the PATs directly or their substrates. However, there are rare clinically effective S-palmitoylation inhibitors. This review aims to provide an overview of the S-palmitoylation field, including the catalytic mechanism of ZDHHC, S-palmitoylation detection methods, and the functional impact of protein S-palmitoylation. Additionally, this review focuses on current strategies for expanding the chemical toolbox to develop novel and effective inhibitors that can reduce the level of S-palmitoylation of the target protein.