Explore the vast range of titles available.

Protein phosphorylation is a central post‐translational modification regulating cellular signaling, frequently dysregulated in cancer. Mass spectrometry (MS)‐based phosphoproteomics has emerged as a powerful approach to systematically profile phosphorylation events, thereby revealing aberrant kinase activity and therapeutic vulnerabilities that are not captured by genomic or transcriptomic analyses. Recent advances across the workflow—including optimized sample preparation and phosphopeptide enrichment, isotope‐ or label‐free quantitative strategies, high‐resolution mass spectrometry platforms, specialized algorithms for site identification and quantification, and integrative informatics analyses—have enabled the detection of tens of thousands of phosphorylation sites even from small clinical specimens. These developments have facilitated the characterization of signaling pathways across diverse cancer types, leading to the identification of targetable kinases and informing therapeutic strategies. In this review, we highlight studies that employed phosphoproteomic analyses of clinical specimens or patient‐derived cancer cells to delineate signaling characteristics and to propose and validate therapeutic targets. Collectively, MS‐based phosphoproteomics is poised to become a cornerstone of precision oncology. By enabling comprehensive and quantitative mapping of phosphorylation events, this technology allows mechanistic dissection of cancer signaling pathways and uncovers therapeutic vulnerabilities that may be exploited with targeted agents.

Non-alcoholic steatohepatitis (NASH) has pathological characteristics similar to those of alcoholic hepatitis, despite the absence of a drinking history. The greatest threat associated with NASH is its progression to cirrhosis and hepatocellular carcinoma. The pathophysiology of NASH is not fully understood to date. In this study, we investigated the pathophysiology of NASH from the perspective of glycolysis and the Warburg effect, with a particular focus on microRNA regulation in liver-specific macrophages, also known as Kupffer cells. We established NASH rat and mouse models and evaluated various parameters including the liver-to-body weight ratio, blood indexes, and histopathology. A quantitative phosphoproteomic analysis of the NASH rat model livers revealed the activation of glycolysis. Western blotting and immunohistochemistry results indicated that the expression of pyruvate kinase muscle 2 (PKM2), a rate-limiting enzyme of glycolysis, was upregulated in the liver tissues of both NASH models. Moreover, increases in PKM2 and p-PKM2 were observed in the early phase of NASH. These observations were partially induced by the downregulation of microRNA122-5p (miR-122-5p) and occurred particularly in the Kupffer cells. Our results suggest that the activation of glycolysis in Kupffer cells during NASH was partially induced by the upregulation of PKM2 via miR-122-5p suppression.

Protein phosphorylation plays a pivotal role in cellular signal transduction and plant development. The plant steroid hormone Brassinosteroids (BRs) signal transduction relies primarily on protein kinase‐mediated phosphorylation cascades. However, the specific mechanisms of phosphorylation regulation in BR signalling remain to be fully elucidated. This study focuses on BIN2, an indispensable protein kinase in the BR signalling pathway, utilising single‐celled cotton fibre to investigate the mechanisms by which phosphorylation regulates cell elongation. Firstly, we confirmed the inhibitory role of GhBIN2 in fibre elongation through its overexpression. Subsequently, we employed 4D‐fastDIA quantitative phosphoproteomics and proteomics analysis to map the GhBIN2‐mediated phosphorylation regulatory network. Through a comprehensive analysis of this network, we identified six credible substrates of GhBIN2. Further investigation revealed that GhBIN2 interacts with substrate GhIQD14 and increases its abundance through phosphorylation to negatively regulate fibre elongation. This study deepens the understanding of BR signalling in cotton fibre elongation and provides experimental evidence and new insights for comprehending the regulatory role of protein phosphorylation in plant cell elongation processes.

Recent developments in proteomics have enabled signaling studies where > 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase–substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid‐robotic phosphoproteomics (R2‐P2), an end‐to‐end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry‐ready phosphopeptides. R2‐P2 is rapid, robust, versatile, and high‐throughput. To showcase the method, we applied it, in combination with data‐independent acquisition mass spectrometry, to study signaling dynamics in the mitogen‐activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high‐osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large‐scale signaling studies involving hundreds of perturbations opening the door to systems‐level studies aiming to capture signaling complexity. Synopsis The study presents R2‐P2, an automated end‐to‐end phosphoproteomic sample preparation method. Application of R2‐P2 to study phosphorylation temporal dynamics in yeast stimulated to perturb MAPK signaling reveals treatment‐specific responses and pathway crosstalks. R2‐P2 is a novel workflow for proteomic and phosphoproteomic sample preparation that compares favorably to common methods. R2‐P2 is automated, high‐throughput, rapid, robust, reproducible and yields high phosphopeptide enrichment efficiency. R2‐P2 facilitates large‐scale cell signaling studies. R2‐P2 in combination with DIA‐MS reveals broad and specific signaling events along the different branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Graphical Abstract The study presents R2‐P2, an automated end‐to‐end phosphoproteomic sample preparation method. Application of R2‐P2 to study phosphorylation temporal dynamics in yeast stimulated to perturb MAPK signaling reveals treatment‐specific responses and pathway crosstalks.

Phloretin, a plant‐derived dihydrochalcone bioactive compound, potentially modulates preadipocyte differentiation, although this remains controversial and requires further clarification. To clarify this point, herein, phloretin gavage inhibited mice obesity, reduced adipocyte size, and ameliorated serum lipid parameters, including triglycerides. In vitro, phloretin suppressed the differentiation of 3T3‐L1 and bovine preadipocytes, decreasing lipid droplet content and reducing CEBPα and PPARγ levels. Moreover, during differentiation, glutathione peroxidase (GSH‐Px) ultimately decreased with elevations in malondialdehyde (MDA) and Fe 2+ levels, along with acyl‐CoA synthetase long‐chain family member 4 ( ACSL4 ), P53 , and SLC7A11 expression, indicating differentiation occurring under mild oxidation and slight ferroptosis activation. Similar to (1 S ,3 R )‐RSL3, phloretin further stimulated the ferroptosis pathway by inhibiting glutathione reductase (GR) and GSH‐Px, increasing the content of MDA and Fe 2+ . Simultaneously, the suppression of Fth1 , Slc7a11 , and Sod2 transcription, significantly elevated HMGB1 protein, and markedly downregulated glutathione peroxidase 4 (GPX4) and TFR protein collectively confirmed that phloretin activated the ferroptosis signaling pathway to inhibit differentiation. Further phosphoproteomic analysis identified dm‐phosphorylation sites (p‐eEF2K[S365], p‐AMPKα1[S491], and p‐CaMKK2[S495]) enriched in AMPK/MAPK signaling pathways and transferrin receptor binding. Further molecular docking simulations revealed that phloretin binds to PPARγ or transferrin to activate ferroptosis pathway, revealing the potential crosstalk between ferroptosis signaling pathway and adipogenic differentiation processes.

Protein kinases play crucial roles in tumor progression and modulation of the immunosuppressive tumor microenvironment. However, the specific function of MASTL (microtubule-associated serine/threonine kinase-like) in lung adenocarcinoma (LUAD) remains poorly understood. In this study, we integrated multi-omics bioinformatic analyses with experimental validation to delineate the clinical significance and biological role of MASTL in LUAD. We found that MASTL is markedly overexpressed in LUAD tissues and exhibits substantial diagnostic value. Elevated MASTL expression served as an independent prognostic indicator of poor overall survival, particularly in early-stage patients. Comprehensive immune profiling revealed a strong association between high MASTL expression and an immunosuppressive tumor microenvironment, characterized by impaired dendritic cell function and altered Th1/Th2 balance. Notably, patients with low MASTL expression showed enhanced sensitivity to immune checkpoint blockade therapy. Phosphoproteomic analysis identified serine 370 (S370) as a novel functional phosphorylation site on MASTL, whose activation correlated with higher tumor grade and dysregulation of key oncogenic pathways, including p53, MYC, mTOR, WNT, and HIPPO signaling. Functionally, pharmacological inhibition of MASTL using MKI-1 suppressed LUAD cell proliferation, induced apoptosis and cell cycle arrest, and reduced cancer stem cell-like properties such as self-renewal and metastatic potential. Importantly, MKI-1 administration significantly inhibited tumor growth in a LUAD xenograft model with good tolerability. Collectively, our findings identify MASTL as a pivotal regulator of tumor progression and immune evasion in LUAD and underscore its potential as both a prognostic biomarker and a promising therapeutic target.

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. Parkinson’s disease is a degenerative disorder of the nervous system that affects approximately 1% of the elderly population. Mutations in the gene that encodes an enzyme known as LRRK2 are the most common causes of the inherited form of the disease. Such mutations generally increase the activity of LRRK2 and so drug companies have developed drugs that inhibit LRRK2 to prevent or delay the progression of Parkinson’s disease. However, it was not known what role LRRK2 plays in cells, and why its over-activation is harmful. Steger et al. used a 'proteomics' approach to find other proteins that are regulated by LRRK2. The experiments tested a set of newly developed LRRK2 inhibitors in cells and brain tissue from mice. The mice had mutations in the gene encoding LRRK2 that are often found in human patients with Parkinson’s disease. The experiments show that LRRK2 targets some proteins belonging to the Rab GTPase family, which are involved in transporting molecules and other 'cargoes' around cells. Several Rab GTPases are less active in the mutant mice, which interferes with the ability of these proteins to correctly direct the movement of cargo around the cell. Steger et al.’s findings will help to advance the development of new therapies for Parkinson’s disease. The next challenges are to identify how altering the activity of Rab GTPases leads to degeneration of the nervous system and how LRRK2 inhibitors may slow down these processes.

To investigate the impact of growth hormone (GH) on branched‐chain amino acids (BCAAs) catabolism in males with hypopituitarism, we measured the concentration of amino acids in 133 males with hypopituitarism and 90 age‐matched healthy controls using untargeted metabolome. A rat model of hypopituitarism was established through hypophysectomy, followed by recombinant human GH (rhGH) intervention. Targeted metabolomics and label‐free quantitative phosphoproteomics were utilised to assess amino acid levels in rats and explore the mechanisms of GH's effect on BCAA catabolism. Hypopituitarism exhibited elevated concentrations of BCAAs, which correlated positively with triglyceride, fasting insulin and HOMA‐IR. The BCAAs were significantly elevated following hypophysectomy and were substantially reduced upon rhGH intervention. Phosphorylation proteomics analysis in liver tissues revealed that differentially expressed phosphoproteins (DEPPs) after GH treatment were predominantly involved in ‘RNA metabolic process’, ‘Diseases of signal transduction by growth factor receptors’ and ‘BCAAs degradation’. Notably, 12 proteins in the BCAA degradation pathway showed altered phosphorylation without whole protein changes. Importantly, the expression or phosphorylation modification of BCKDH, BCATs and MuRF1 were restored through rhGH intervention. Hypopituitarism exhibits elevated levels of circulating BCAAs. The increased circulating BCAAs in hypopituitarism may result from enhanced MuRF1‐mediated muscle proteolysis, which greatly exceeds the BCAA degradation capacity. This study provides valuable insights into the effects of GH on BCAA catabolism at the scale of the proteomics level.

Signal transduction governs cellular behavior, and its dysregulation often leads to human disease. To understand this process, we can use network models based on prior knowledge, where nodes represent biomolecules, usually proteins, and edges indicate interactions between them. Several computational methods combine untargeted omics data with prior knowledge to estimate the state of signaling networks in specific biological scenarios. Here, we review, compare, and classify recent network approaches according to their characteristics in terms of input omics data, prior knowledge and underlying methodologies. We highlight existing challenges in the field, such as the general lack of ground truth and the limitations of prior knowledge. We also point out new omics developments that may have a profound impact, such as single‐cell proteomics or large‐scale profiling of protein conformational changes. We provide both an introduction for interested users seeking strategies to study cell signaling on a large scale and an update for seasoned modelers. Graphical Abstract Network models based on prior knowledge are used to understand signal transduction. This Review compares and classifies recent network approaches according to their characteristics in terms of input omics data, prior knowledge, and underlying methodologies.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology upon DUX4 activation, including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered rapid widespread changes in protein phosphorylation following DUX4 induction, indicating that alterations in kinase signaling might play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover that the JNK pathway is involved in DUX4-mediated cell death and provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.