bims-cesirm 2025-11-30 papers

bims-cesirm

Biomed News

on Cell Signaling mediated regulation of metabolism

Issue of 2025–11–30
thirty-two papers selected by
Tigist Tamir, University of North Carolina

Novel high-resolution ion mobility mass spectrometry for site-specific quantification of the sirtuin-5 regulated kidney succinylome.
Reprogramming SREBP1-dependent lipogenesis and inflammation in high-risk breast with licochalcone A: A novel path to cancer prevention.
Molecular Architecture of the human Citrate Synthase-Malate Dehydrogenase 2 metabolon.
High-Throughput De Novo Protein Design Yields Novel Immunomodulatory Agonists.
The non-kinase function of CDK6 is a key driver of acquired resistance to CDK4/6 inhibitors in Estrogen Receptor-positive breast cancer.
Dietary sulfur amino acid restriction improves glucose homeostasis through hepatic de novo serine synthesis.
Unique response of polysulfide in serum albumin to oxidative stress.
PANK2-mediated de novo CoA synthesis is required for metabolic switching to fatty acid oxidation.
Direct cell reprogramming by a designed agonist inducing HER2-FGFR proximity.
Multiomics analyses of human colorectal cancer reveal changes in mitochondrial metabolism associated with chemotherapy resistance.
Multiplex design and discovery of proximity handles for programmable proteome editing.
Targeting glutaryl-CoA dehydrogenase-driven acetyl coenzyme A acetyltransferase 2 crotonylation dysregulates cholesterol metabolism in pancreatic cancer cells.
Catalytic activity of FAHD1 correlates with nuclear morphology and proteomic states in human osteosarcoma cells.
Persistent Oxidation of Mitochondrial and Transmembrane Proteins in Rat Cerebrum and Heart Regardless of Age or Nutrition.
Macropinocytosis enables metabolic recycling of extracellular DNA in cancer cells.
Isoform-specific regulation of PKM by acetylation.
Acetyl-CoA carboxylase maintains energetic balance for functional oogenesis.
Mitochondria redistribution organizes the immunosuppressive tumor ecosystem.
Many circulating indole and phenol metabolites are host derived.
Mapping the diverse topologies of protein-protein interaction fitness landscapes.
Oxidative stress reactivates androgen receptor signaling via USP36 to drive castration resistance in prostate cancer.
Metabolic Reprogramming Intermediates of Glucose Regulate Macrophage Polarization: An Important Direction for Ameliorating Pulmonary Vascular Remodeling.
Ferroptotic resistance involved in PTEN-loss prostate cancer progression.
Distal Mutations Rewire Allosteric Networks to Control Substrate Specificity in PTP1B.
Adaptive nuclear GTP synthesis promotes glioblastoma treatment resistance and is a targetable vulnerability in patients.
Stabilization of FASN by USP5-mediated deubiquitination promotes hepatocellular carcinoma progression.
Elevated tumor-associated androgen receptor activity correlates with poor immune infiltration and immunotherapy response across cancer types.
Uncovering the molecular basis of kinase activity and substrate recognition with phospho-PCA.
The functional landscape of the human ubiquitinome.
Optogenetic control of PLC-γ1 activity polarizes cell motility.
Uncovering the glutamate carboxypeptidase II microenvironment using a multi-labeling proteomic approach.
Nutrient Availability Dictates Cancer Metabolism-Based Therapeutic Responses to Non-oncology Drugs.

bioRxiv. 2025 Nov 03. pii: 2025.11.01.686011. [Epub ahead of print]

Novel high-resolution ion mobility mass spectrometry for site-specific quantification of the sirtuin-5 regulated kidney succinylome.

Birgit Schilling, Leonard Rorrer, Lauren Royer, Anne Silva-Barbosa, Katherine Pfister, Eric Goetzman, Sunder Sims-Lucas, Daniel DeBord, Joanna Bons.

Protein post-translational modifications (PTMs) dynamically regulate essential biological and cellular processes. Lysine succinylation changes the amino acid charge, potentially affecting protein structures and functions, and dysregulation of protein succinylation may lead to metabolic disorders. Proteome-wide succinylation quantification using proteomic tools remains challenging, especially due to the low abundance of succinylated peptides and the frequent presence of isomeric PTM forms. Ion mobility spectrometry workflows that can differentiate peptidoforms with different PTM distributions represent a powerful strategy to alleviate these challenges. Recently, a new Parallel Accumulation with Mobility Aligned Fragmentation (PAMAF™) operating mode for high-resolution ion mobility-mass spectrometry (HRIM-MS) analysis based on the structures for lossless ion manipulation (SLIM) technology was introduced. Here, we first assessed the performance of PAMAF mode for protein succinylation analysis using synthetic succinylated peptides, demonstrating residue-level differentiation of co-eluting isomers and isobars and precise PTM site localization. We leveraged this novel approach to investigate succinylome remodeling in kidney tissues from wild-type and Sirtuin-5 (Sirt5) knock-out mice, a NAD + -dependent lysine de-succinylase. PAMAF acquisitions yielded ∼1,000 confidently identified and accurately quantified succinylated peptides and sites from mouse kidney. Sirt5 regulated succinylation of mitochondrial proteins involved in metabolic processes, including fatty acid oxidation, the tricarboxylic acid cycle, and propionate metabolism.

DOI: https://doi.org/10.1101/2025.11.01.686011
Int J Cancer. 2025 Nov 27.

Reprogramming SREBP1-dependent lipogenesis and inflammation in high-risk breast with licochalcone A: A novel path to cancer prevention.

Atieh Hajirahimkhan, Elizabeth T Bartom, Carolina H Chung, Xingyu Guo, Kyli Berkley, Seyyedmohsen Hosseinibarkooie, Zahra Assadi, Shao Huan Samuel Weng, Raymond Moellering, Oukseub Lee, Ruohui Chen, Wonhwa Cho, Sriram Chandrasekaran, Susan E Clare, Seema A Khan.

  Anti-estrogens have had a limited impact on breast cancer (BC) prevention. Novel agents with better tolerability, and efficacy beyond estrogen receptor (ER) positive BC are needed. We studied licochalcone A (LicA) for ER-agnostic BC prevention. We demonstrated that LicA significantly reduced proliferation in seven human breast cell lines and suppressed ER+ and ER- xenograft tumors in mice. We confirmed these observations ex vivo in the contralateral unaffected breast (CUB) of women with unilateral sporadic BC, and BC cell lines using RNA sequencing, metabolism flux modeling, confirmatory NanoString nCounter metabolic pathway panel analysis in independent sets of specimens, proteomics, and western blots. We found that LicA targets sterol regulatory element binding protein 1 (SREBP1) with subsequent metabolic-inflammatory changes, lowering spatiotemporally resolved cholesterol levels inside malignant cells to the levels in normal mammary cells. Mechanistically, in CUBs we observed that LicA downregulated PI3K-AKT-SREBP1-dependent lipogenesis, NF-kB-dependent inflammation, and de novo nucleotide biosynthesis, stalling proliferation. Studies in cell lines showed suppression of PI3K and AKT phosphorylation, SREBP1 protein expression, and the SREBP1-dependent enzymes such as ACAT2, ACLY, FASN, SCD, consistent with reduced NEDD8 required for SREBP1 stabilization. We found a significant reduction in NF-kB expression, its nuclear translocation mediator karyopherin β1, and prostaglandin E2 synthesis. We demonstrated a reduction in PRPS1-catalyzed de novo nucleotide biosynthesis, and downregulation of proliferative markers MKI67, RRM2, and the survival marker BCL2. LicA reduces pro-tumorigenic aberrations in lipid homeostasis and inflammation through SREBP1. It is a promising non-endocrine candidate for BC prevention. Future studies in immunocompetent BC prevention models are warranted.

Keywords:  SREBP; antiproliferation; anti‐inflammation; breast cancer prevention; licochalcone a

DOI:  https://doi.org/10.1002/ijc.70226
bioRxiv. 2025 Oct 14. pii: 2025.10.13.681887. [Epub ahead of print]

Molecular Architecture of the human Citrate Synthase-Malate Dehydrogenase 2 metabolon.

Angela J Kayll, Umanga Rupakheti, Renee St John, Joseph J Provost, Christopher E Berndsen.

Metabolons are transient biomolecular complexes that enhance the efficiency of metabolic pathways through substrate channeling. These complexes are difficult to study because of the transient nature, thus limiting our understanding of how they are formed and regulated. The citric acid cycle is proposed to contain many such complexes although few have been characterized structurally. Here, we provide direct structural evidence for the complex of human Citrate Synthase and human mitochondrial Malate Dehydrogenase 2, which is part of the larger proposed citric acid cycle metabolon. Our structural model supports previous crosslinking studies and suggests that hMDH2 can interact with each subunit of the hCS dimer, forming up to a hexameric complex. However, this complex appears transient as titration of hMDH2 into hCS in activity assays does not saturate. We further show that the interaction site with hCS is non-specific, as hCS could also stimulate oxaloacetate formation by cytosolic and plant MDH enzymes. This structural model will provide a base for understanding the structure and regulation of the broader citric acid cycle metabolon.

DOI: https://doi.org/10.1101/2025.10.13.681887
bioRxiv. 2025 Oct 13. pii: 2025.10.12.681920. [Epub ahead of print]

High-Throughput De Novo Protein Design Yields Novel Immunomodulatory Agonists.

Mohamad Abedi, Marc Expòsit, Brian Coventry, Divij Mathew, Shruti Jain, Aditya Krishnakumar, Inna Goreshnik, Sophie L Gray-Gaillard, Margaret Lunn-Halbert, Ta-Yi Yu, Matthias Glögl, Uma Mitchell, Riya Keshri, Jung Ho Chun, Hannele Ruohola-Baker, E John Wherry, David Baker.

  Cytokines regulate cell behavior by bringing together specific receptor subunits to trigger downstream signaling. Designed molecules that bring together non-natural receptor pairs could have novel signaling responses and cell specificities. We present a high-throughput de-novo design approach to create novel cytokines by generating and fusing pairs of computationally designed binders. By combining 33 designed receptor-binding domains, we generated over a thousand potential de novo designed "Novokines", of which 75 activated pSTAT signaling in peripheral blood mononuclear cells. We characterized 18 of these, including new pairings of established common receptors, cross-family pairings such as TrkA-γcommon, and a series of pairings with interferon receptor-1 (IFNAR1), revealing that IFNAR1 can function as a versatile common receptor similar to γcommon or βcommon. We identify novokines that drive monocyte proliferation, T cell survival and CD4+ T cell-specific proliferation. Our framework provides a blueprint for expanding the understanding of cytokine signaling and generating novel therapeutic proteins.

Keywords:  Cell Signaling; Computational Protein Design; Cytokine; Synthetic Biology

DOI:  https://doi.org/10.1101/2025.10.12.681920
bioRxiv. 2025 Nov 13. pii: 2025.11.11.687883. [Epub ahead of print]

The non-kinase function of CDK6 is a key driver of acquired resistance to CDK4/6 inhibitors in Estrogen Receptor-positive breast cancer.

S M Nashir Udden, Baishan Jiang, Kamal Pandey, Cheryl M Lewis, Sunati Sahoo, Noelle S Williams, Prasanna G Alluri.

The therapeutic efficacy of CDK4/6 inhibitors in ER+ breast cancer (BC) patients is presumed to arise from inhibition of the kinase function of CDK4 and 6 proteins. Despite their initial efficacy, development of acquired resistance to CDK4/6 inhibitors in metastatic ER+ BC patients is nearly universal, commonly through compensatory overexpression of CDK6. Here, we show that primary ER+ tumors show high CDK4 but very low to undetectable expression of CDK6, which suggests that the therapeutic efficacy of CDK4/6 inhibitors in ER+ BC patients is largely driven by inhibition of CDK4, rather than CDK6. Furthermore, we show that overexpression of CDK6 confers acquired resistance to CDK4/6 inhibitors, largely, through a kinase-independent function. Our findings challenge the dogma that CDK4/6-mediated phosphorylation of retinoblastoma protein (pRB) is necessary to ER+ BC cells to progress from G1 to S phase of cell cycle. Consequently, by taking advantage of the non-kinase function of CDK6, ER+ BC cells acquire independence from the kinase function of CDK4 and 6 for cell cycle progression. These findings highlight the limitations of the current standard-of-care treatments, which focus on merely inhibiting the kinase function of CDK4 and 6, and uncover the non-kinase function of CDK6 as a new targetable vulnerability to overcome acquired resistance to CDK4/6 inhibitors in ER+ BC.

DOI: https://doi.org/10.1101/2025.11.11.687883
bioRxiv. 2025 Oct 17. pii: 2025.10.16.682938. [Epub ahead of print]

Dietary sulfur amino acid restriction improves glucose homeostasis through hepatic de novo serine synthesis.

Andres F Ortega, Cha Mee Vang, Ferrol I Rome, Kaitlyn M Andreoni, Aiden M Phoebe, Alisa B Nelson, Peter A Crawford, James J Galligan, Stanley Ching-Cheng Huang, Curtis C Hughey.

Dietary sulfur amino acid restriction (SAAR) improves whole-body glucose homeostasis, elevates liver insulin action, and lowers liver triglycerides. These adaptations are associated with an increased expression of hepatic de novo serine synthesis enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). This study tested the hypothesis that enhanced hepatic serine synthesis is necessary for glucose and lipid adaptations to SAAR. Hepatocyte-specific PSAT1 knockout (KO) mice and wild type (WT) littermates were fed a high-fat control or SAAR diet. In WT mice, SAAR increased liver PSAT1 protein (~70-fold), serine concentration (~2-fold), and 13C-serine (~20-fold) following an intravenous infusion of [U-13C]glucose. The elevated liver serine and partitioning of circulating glucose to liver serine by SAAR were attenuated in KO mice. This was accompanied by a blunted improvement in glucose tolerance in KO mice fed a SAAR diet. Interestingly, SAAR decreased liver lysine lactoylation, a SAA-supported post-translational modification known to inhibit PHGDH enzymatic activity. This suggests dietary SAAR may increase serine synthesis, in part, by lowering lysine lactoylation. Beyond glucose metabolism, dietary SAAR reduced body weight, adiposity, and liver triglycerides similarly in WT and KO mice. Collectively, these results demonstrate that hepatic PSAT1 is necessary for glucose, but not lipid, adaptations to SAAR.

DOI: https://doi.org/10.1101/2025.10.16.682938
Sci Rep. 2025 Nov 27. 15(1): 42689

Unique response of polysulfide in serum albumin to oxidative stress.

Mayumi Ikeda-Imafuku, Maki Sakai, Keitaro Umezawa, Hidehiko Nakagawa, Toru Maruyama, Masaki Otagiri, Takaaki Akaike, Tomohiro Sawa, Ming Xian, Yuri Miura, Tatsuhiro Ishida, Yu Ishima.

  Supersulfide species have been identified as molecules that have catenated sulfur atoms as persulfides (R-SSH) and polysulfides (R-SSnH). The physiological activities of intracellular supersulfides have been widely studied, but little is known about the role of supersulfides in extracellular biological fluids. We therefore analyzed the pathological changes and oxidative stress responses of the reduced and oxidized forms of polysulfides in the plasma of patients with diabetic nephropathy. We measured the reduced and oxidized forms of polysulfide with elimination of sulfides using dithiothreitol and ascorbic acid plus alkali conditions, respectively. Oxidation decreased oxidized forms of polysulfide and further polysulfide in human serum albumin, increasing its antioxidative activity. We identified seven cysteine residues that have polysulfides in a reduced form after oxidation in albumin. Further oxidation decreased the levels of the reduced forms of polysulfide and the antioxidative effect of serum albumin. Similar changes were also observed in a mouse model of rhabdomyolysis-induced acute kidney injury; the levels of reduced polysulfide in plasma transiently increased 1 h after glycerol administration, which was accompanied by increased antioxidative activity. In the sera of patients with diabetic nephropathy and chronic renal failure, both forms of polysulfides were decreased compared with those of healthy subjects. Analysis of the stages of renal damage revealed that dithiothreitol-liberated polysulfide increased from stages 1-3 and decreased in stage 5. These results suggest that the antioxidative activity of serum, including that of serum albumin, is regulated by the switch from the oxidized form of polysulfide to its reduced counterparts in response to oxidative stress.

Keywords:  Kidney disease; Oxidative stress; Polysulfides; Serum albumin; Supersulfides

DOI:  https://doi.org/10.1038/s41598-025-26702-w
bioRxiv. 2025 Nov 18. pii: 2025.10.07.680981. [Epub ahead of print]

PANK2-mediated de novo CoA synthesis is required for metabolic switching to fatty acid oxidation.

Sandra M Nordlie, Robert Schafer, Adam Rauckhorst, Ana M Santos-Exposito, Andres Prieto-Rodriguez, Bryce A Wilson, Idil A Evans, Una Hadziamehtovic, Frances Nowlen, Carlos Santos-Ocaña, Eric B Taylor, Michael C Kruer, Sergio Padilla-Lopez.

Humans have 3 different PANK enzymes (PANK1-3) that catalyze the first step in the de novo synthesis of Coenzyme A (CoA). All PANKs are feedback inhibited by acyl-CoAs but only PANK2 can overcome this inhibition by binding palmitoyl-carnitine. Previous studies, conducted under glucose-replete conditions, have failed to detect a PANK2-mediated contribution to CoA synthesis. We found that exposure to BSA-conjugated palmitate (PAL-BSA) led to activation of fatty acid oxidation (FAO) and the accumulation of both palmitoyl-carnitine and palmitoyl-CoA in HEK293T cells, suggesting that PANK2 is active under these conditions. Isotope tracing experiments with 13 C 15 N-pantothenate showed that PANK2 uniquely sustains de novo CoA synthesis and high production of Acetyl-CoA in the presence of long-chain fatty acids, indicating that FAO is limited by CoA availability in these conditions. Consistent with this mechanism, fibroblasts from PKAN patients exhibited impaired oxidation of palmitoyl-carnitine, confirming the functional relevance of our results in a disease context.

DOI: https://doi.org/10.1101/2025.10.07.680981
bioRxiv. 2025 Oct 13. pii: 2025.10.12.681903. [Epub ahead of print]

Direct cell reprogramming by a designed agonist inducing HER2-FGFR proximity.

Riya Keshri, Marc Exposit, Mohamad Abedi, Derrick R Hicks, Zachary Foreman, Ashish Phal, Yen Chian Lim, Philip Barrett, Catherine Sniezek, Jinlong Lin, Thomas Schlichthaerle, Alexander J Robinson, Damien Detraux, Tung Chan Ching, Keija Wu, Brian Coventry, Lemuel Chang, Alec S T Smith, David L Mack, Devin K Schweppe, Beatriz Estrada Martin, Kalina Hristova, Julie Mathieu, David Baker, Hannele Ruohola-Baker.

Growth factor induced receptor dimerization and activation of downstream pathways can modulate cell fate decisions. Here, we investigate the potential of de novo designed synthetic ligands, termed Novokines, to reprogram cell identity by inducing proximity of novel pairs of receptor subunits. We find that a design, H2F, that brings together HER2 (which has no known natural ligand) and the FGF receptor has potent signaling activity. H2F induces robust signaling and reprograms fibroblasts into myogenic cells. Unlike native FGF ligands, H2F selectively activates the MAPK pathway without engaging PLCγ-mediated Ca²⁺ signaling. FRET assays confirm H2F-mediated HER2-FGFR proximity, and phosphoproteomic analysis reveals activation of MAPK effectors. H2F-induced ERK phosphorylation is abolished in cells expressing a kinase-dead FGFR1 (K514M) mutant, confirming the requirement for FGFR catalytic activity. H2F treatment significantly increases myofiber formation from adult patient-derived primary myoblasts, demonstrating its capacity to promote myogenic regeneration. Our findings demonstrate that synthetic receptor pairings can rewire signaling outputs to drive regeneration, providing a programmable platform for cell fate engineering.

DOI: https://doi.org/10.1101/2025.10.12.681903
Front Oncol. 2025 ;15 1625797

Multiomics analyses of human colorectal cancer reveal changes in mitochondrial metabolism associated with chemotherapy resistance.

Shiyi Chen, Qian Li, Wei Zheng.

   Background: Mitochondria are essential organelles involved in energy production, cellular metabolism, and signal transduction. They have important impacts on tumorigenesis and cancer progression. Nevertheless, the associations between mitochondrial metabolic processes and chemotherapy resistance in colorectal cancer (CRC) are not well understood.
Methods: We generated a chemotherapy-resistant colorectal cancer cell line, HCT-15/DOX, via doxorubicin (DOX) induction. We then performed proteomic and metabolomic analyses via LC-MS/MS technology on both the parental and the DOX-resistant cell lines. Additionally, transmission electron microscopy was used to examine changes in mitochondrial morphology between the two cell lines.
Results: The results revealed significant dysregulation of 185 proteins and 1099 metabolites in HCT-15/DOX cells relative to parental cells, highlighting the impact of chemotherapy resistance on cellular processes. The key functional proteins that were identified included upregulated SDHA, BCKDHB, CRYZ, NUDT6, CPT1A, and POLG, and downregulated CRAT, FDPS, SFXN1, and ATAD3B. Additionally, through combined multiomics pathway enrichment analysis, pyrimidine metabolism, purine metabolism, ascorbate and aldarate metabolism, propanoate metabolism, and the citrate cycle (TCA cycle) were identified as important metabolic processes associated with CRC chemotherapy resistance. Transmission electron microscopy analysis revealed that HCT-15/DOX cells had increased mitochondrial number, length, and area.
Conclusions: This research highlights notable differences in mitochondrial morphology and diverse mitochondrial metabolic functions between parental and DOX-resistant HCT-15 CRC cells. The findings of the present study provide insights into the mitochondrial metabolic changes associated with CRC chemotherapy resistance, offering valuable insights into the mechanisms underlying these changes and identifying potential therapeutic targets for addressing CRC chemotherapy resistance.

Keywords:  chemotherapy resistance; colorectal cancer; metabolomics; mitochondrial metabolism; proteomics

DOI:  https://doi.org/10.3389/fonc.2025.1625797
bioRxiv. 2025 Oct 13. pii: 2025.10.13.681693. [Epub ahead of print]

Multiplex design and discovery of proximity handles for programmable proteome editing.

Chase C Suiter, Green Ahn, Melodie Chiu, Yi Fu, Shayan Sadre, Jessica J Simon, David S Lee, Douglas M Fowler, Dustin J Maly, David Baker, Jay Shendure.

Although we now have a rich toolset for genome editing, an equivalent framework for manipulating the proteome with a comparable flexibility and specificity remains elusive. A promising strategy for "proteome editing" is to use bifunctional molecules (e.g. PROteolysis-Targeting Chimeras or PROTACs1) that bring a target protein into proximity with a degradation or stabilization effector, but their broader application is constrained by a limited repertoire of well-characterized target or effector "handles". We asked whether coupling de novo protein design to a multiplex screening framework could address this gap by accelerating the discovery of effector handles for intracellular protein degradation, stabilization, or relocalization. Using LABEL-seq2, a sequencing-based assay that enables multiplex, quantitative measurement of protein abundance, we screened 9,715 de novo designed candidate effector handles for their ability to recruit a target protein to components of the ubiquitin-proteasome system3 (UPS) (FBXL12, TRAF2, UCHL1, USP38) or the autophagy pathway4 (GABARAP, GABARAPL2, MAP1LC3A). In a single experiment, we discovered hundreds of de novo designed effector handles that reproducibly drove either intracellular degradation (n = 277) or stabilization (n = 204) of a reporter protein. Validation of a subset of these hits in an orthogonal assay confirmed that sequencing-based measurements from the primary screen reliably reflected changes in intracellular abundance of the target protein. Successful effector handles were discovered for both the UPS (n = 194) and autophagy (n = 287) pathways, which provide complementary routes for programmable proteome editing. Autophagy-recruiting effector handles generalized to endogenous targets, as substituting the reporter-specific target handle with a high-affinity MCL1 binder5 reduced endogenous levels of this intracellular oncoprotein6. Moreover, directing autophagy-recruiting effector handles to the outer mitochondrial membrane dramatically perturbed mitochondrial networks in a manner consistent with synthetic tethering and sequestration7,8. Beyond generating a diverse repertoire of protein abundance or localization effector handles, our results establish a scalable, low-cost platform that links deep learning-guided protein design to functional cellular readouts, and chart a course toward a general framework for programmable proteome editing.

DOI: https://doi.org/10.1101/2025.10.13.681693
Int J Biol Macromol. 2025 Nov 22. pii: S0141-8130(25)09739-9. [Epub ahead of print] 149182

Targeting glutaryl-CoA dehydrogenase-driven acetyl coenzyme A acetyltransferase 2 crotonylation dysregulates cholesterol metabolism in pancreatic cancer cells.

Feng Han, Hao Zhang, Qing Ye, Qu Zhang, Wandi Shen, Ka Bian, Fanghong Chen, Jiayou Guo, Mingchen Mu, Jianxin Ma.

  Cancer cells rely on the metabolic reprogramming of amino acids to regulate the production of specific metabolites in the tumor microenvironment and support tumor growth. We demonstrated that the upregulation of glutaryl-CoA dehydrogenase (GCDH), a key gene encoding a metabolic enzyme involved in the conversion of lysine and tryptophan to crotonyl-CoA, was correlated with worse prognosis in patients with pancreatic ductal adenocarcinoma (PDAC), as GCDH depletion inhibits PDAC growth. Mechanistically, GCDH promotes cholesterol biosynthesis by enhancing the CREB Binding Protein (CBP)-mediated crotonylation (Kcr) modification of acetyl coenzyme A acetyltransferase 2(ACAT2). Importantly, ACAT2-Kcr induces the dissociation of the Insig1-SCAP complex, thereby facilitating the transport and activation of the SCAP-SREBP2 complex during its translocation from the endoplasmic reticulum (ER) to the Golgi apparatus. Conversely, ACAT2 decrotonylation triggers ER stress and promotes the apoptosis of PDAC cells. This study reveals the roles of GCDH in controlling cholesterol metabolism and PDAC progression and suggests that GCDH is a new target for therapeutic intervention in patients with PDAC.

Keywords:  Acetyl coenzyme A acetyltransferase 2; Cholesterol metabolism; Crotonylation; Glutaryl-CoA dehydrogenase; Pancreatic ductal adenocarcinoma

DOI:  https://doi.org/10.1016/j.ijbiomac.2025.149182
Sci Rep. 2025 Nov 25.

Catalytic activity of FAHD1 correlates with nuclear morphology and proteomic states in human osteosarcoma cells.

Andreas S Pfarl, Anne Heberle, Alexander K H Weiss.

FAHD1 is a mitochondrial enzyme involved in oxaloacetate metabolism, with emerging links to cellular redox balance, Ca2+-metabolism, and structural features. Building on previous work (Heberle et al. Sci Rep 14:9231, 2024), we investigated how overexpression of human FAHD1 (hFAHD1) variants, including wild-type, the catalytically inactive hFAHD1-K123A, and the hyperactive hFAHD1-T192S, affects nuclear morphology in U2OS osteosarcoma cells. Using high-content microscopy and automated classification, we observed variant-specific shifts in nuclear shape distributions. Notably, expression of K123A was associated with a higher frequency of large, rounded nuclei and a reduction in elongated forms, while the T192S variant produced subtler changes. By aligning morphological clusters with available proteomic profiles, we identified suggestive correlations with differences in biosynthetic activity and chromatin organization. These findings indicate that altered FAHD1 activity is correlated with changes in nuclear morphology and may be associated with broader cellular organization. Our results are descriptive and hypothesis-generating, highlighting possible links between mitochondrial metabolic states and nuclear architecture that warrant further validation.

DOI: https://doi.org/10.1038/s41598-025-29460-x
Int J Mol Sci. 2025 Nov 18. pii: 11155. [Epub ahead of print]26(22):

Persistent Oxidation of Mitochondrial and Transmembrane Proteins in Rat Cerebrum and Heart Regardless of Age or Nutrition.

Wangya Yang, Shipan Fan, Carina Ramallo-Guevara, Manuela Kratochwil, Sandra Thilmany, Michiru D Sugawa, Norbert A Dencher, Ansgar Poetsch.

  Reactive oxygen species (ROS), inevitable by-products of aerobic metabolism, act both as regulators of signaling pathways and as mediators of oxidative stress and aging-related damage. Protein oxidative post-translational modifications (Ox-PTMs) are recognized hallmarks of aging and metabolic decline, yet the persistence of protein oxidation under different physiological conditions, such as age and diet, remains unclear. Here, we applied proteomics to mitochondrial and membrane-enriched fractions of male Fischer 344 rat cerebrum and heart, comparing Ox-PTMs across young and aged animals subjected to ad libitum nutrition (AL) or calorie restriction (CR). We identified 139 mitochondrial and membrane-associated proteins consistently exhibiting high levels of oxidation, including tricarboxylic acid (TCA) cycle enzymes, respiratory chain subunits, ATP synthase components, cytoskeletal proteins, and synaptic vesicle regulators. Functional enrichment and network analyses revealed that oxidized proteins clustered in modules related to mitochondrial energy metabolism, membrane transport, and excitation-contraction coupling. Notably, many proteins remained persistently oxidized, predominantly as mono-oxidation, without significant changes during aging or CR. Moreover, the enzymatic activity of mitochondrial complexes was not only preserved but significantly enhanced in specific contexts, and the structural integrity of the respiratory chain was maintained. These findings indicate a dual strategy for coping with oxidative stress: CR reduces ROS production to limit oxidative burden, while protein and network robustness enable functional adaptation to persistent oxidation, collectively shaping mitochondrial function and cellular homeostasis under differing physiological conditions.

Keywords:  aging; calorie restriction; mitochondria; oxidative post-translational modifications; rat cerebrum; rat heart; reactive oxygen species

DOI:  https://doi.org/10.3390/ijms262211155
bioRxiv. 2025 Oct 23. pii: 2025.10.22.684010. [Epub ahead of print]

Macropinocytosis enables metabolic recycling of extracellular DNA in cancer cells.

Wen-Hsuan Yang, Olivia T Stamatatos, Evdokia Michalopoulou, Ava Sann, Paolo Cifani, Linda Van Aelst, Justin R Cross, Tse-Luen Wee, Michael J Lukey.

Avid nutrient consumption is a metabolic hallmark of cancer and leads to regional depletion of key metabolites within the tumor microenvironment (TME). Cancer cells consequently employ diverse strategies to acquire the fuels needed for growth, including bulk uptake of the extracellular medium by macropinocytosis. Here, we show that breast and pancreatic cancer cells macropinocytically internalize extracellular DNA (exDNA), an abundant component of the TME, and deliver it to lysosomes for degradation. This provides a supply of nucleotides that sustains growth when de novo biosynthesis is impaired by glutamine restriction or pharmacological blockade. Mechanistically, this process is dependent on the non-redundant lysosomal equilibrative nucleoside transporter SLC29A3 (ENT3), which mediates the export of nucleosides from the lysosomal lumen into the cytosol. Accordingly, genetic ablation of SLC29A3 or pharmacological disruption of lysosomal function prevents exDNA scavenging and potently sensitizes breast tumors to antimetabolite chemotherapy in vivo . These findings reveal a previously unrecognized nutrient acquisition pathway through which cancer cells recycle exDNA into metabolic building blocks and highlight SLC29A3 as a mediator of metabolic flexibility and a potential target to improve chemotherapy response.

DOI: https://doi.org/10.1101/2025.10.22.684010
Proc Natl Acad Sci U S A. 2025 Dec 02. 122(48): e2527086122

Isoform-specific regulation of PKM by acetylation.

Dariia Pavlenko, Joaquin Tamargo-Azpilicueta, Hila Nudelman, Yuval Ankri, Anat Shahar, Irene Díaz-Moreno, Eyal Arbely.

  Pyruvate kinase (PK) is a crucial glycolytic protein involved in vital cellular processes ranging from cell proliferation to immune responses. The activity and functions of PK are tightly regulated by diverse mechanisms, including posttranslational Nϵ-lysine acetylation. Although previous studies have explored the impact of acetylation on selected lysine residues within the M2 isoform of PK (PKM2), a more comprehensive selection of acetylation sites and their respective effects on both PKM2 and the highly homologous PKM1 isoform is lacking. Here, we describe the structural, functional, and regulatory effects of site-specific acetylation on an expanded set of conserved lysines in PKM2 and selected lysines in PKM1. To study homogeneously acetylated proteins, we genetically encoded the incorporation of acetylated lysine into PKM variants expressed in bacteria and cultured mammalian cells. Our integrated biochemical, structural, and computational approach revealed K115 acetylation as an inhibitory modification in both PKM1 and PKM2 that stabilizes a closed active site conformation of the proteins. We also show that, in contrast to K115 acetylation, previously reported acetylation of K305 inhibits PKM2 but has no effect on the activity and oligomerization of PKM1. These findings propose the existence of both uniform and isoform-specific regulatory mechanisms of PKM, mediated by acetylation.

Keywords:  deacetylation; genetic code expansion; glycolysis; metabolic regulation; posttranslational modifications

DOI:  https://doi.org/10.1073/pnas.2527086122
Nat Commun. 2025 Nov 27. 16(1): 10677

Acetyl-CoA carboxylase maintains energetic balance for functional oogenesis.

Oyundari Amartuvshin, Chi-Hung Lin, Yi-Ting Ke, Han-Jung Lee, Kreeti Kajal, Tsai-Ling Huang, Wen-Der Wang, Tsai-Ming Lu, Ling-Huei Yih, Chen-Yuan Tseng, Hwei-Jan Hsu.

Reproduction is tightly linked to nutrient availability and metabolic homeostasis, yet how specific metabolic pathways coordinate with cellular signaling to control oogenesis remains unclear. Through a targeted RNAi screen in the Drosophila germline, we identify Acetyl-CoA Carboxylase (Acc), the rate-limiting enzyme in fatty acid synthesis (FAS), as an essential regulator of germline stem cell (GSC) maintenance and oocyte development. Acc loss shifts cellular metabolism toward fatty acid oxidation (FAO), fueling the TCA cycle and electron transport chain, which elevates ATP levels and hyperactivates TOR signaling. This metabolic reprogramming induces excessive protein synthesis, disrupting endosomal trafficking and fusome branching, a germline-specific organelle essential for synchronized cell divisions and oocyte selection. These defects are rescued by inhibiting FAO, suppressing TOR activity, reducing protein synthesis, or restricting dietary protein intake. Our study establishes a direct metabolic-signaling-structural axis in the female germline and highlights Acc as a key metabolic checkpoint that safeguards energy balance, intracellular trafficking, and oocyte fate.

DOI: https://doi.org/10.1038/s41467-025-65708-w
bioRxiv. 2025 Nov 13. pii: 2025.11.11.687895. [Epub ahead of print]

Mitochondria redistribution organizes the immunosuppressive tumor ecosystem.

Azusa Terasaki, Alexis T Weiner, Yuhao Tan, Viktoria Szeifert, Keshav Bhatnagar, Cara C Rada, Vishnu Shankar, Courtney Kernick, Mahnoor Mahmood, Lukas Wiggers, Viviana R Rodrigues, Payam A Gammage, Theodore Roth, Jeffrey D Axelrod, Edgar Engleman, Bo Li, Derick Okwan-Duodu.

Hostile conditions in the tumor microenvironment restrict cellular respiration, yet mitochondrial metabolism remains indispensable for tumor growth and the activity of immunosuppressive cells. How tumor ecosystems sustain mitochondrial output has been unclear. Here, we show that cancer cells resolve this paradox by acting as hubs of intercellular mitochondrial redistribution. Using mitochondrial reporter systems, we demonstrate that cancer cells import host-derived mitochondria, integrate them into their endogenous network, and subsequently relay these hybrid organelles to neighboring immune cells. Mitochondria redistribution reprograms recipient neutrophils, macrophages, and CD4+ T cells into highly suppressive states but drives CD8+ T cell exhaustion. Within cancer cells, fusion of incoming mitochondria induces filamentous P5CS assembly, enhances biosynthetic output, and enables the refurbishment of damaged organelles into fully functional units. Disrupting mitochondrial redistribution collapses the immunosuppressive ecosystem and impairs tumor growth. Thus, cancer cells do not hoard resources but orchestrate a redistribution program that fortifies their own metabolic resilience, derails anti-tumor immunity, and sustains immunosuppressive partners.
HIGHLIGHTS: Tumor cells regulate their ecosystem by redistributing mitochondriaRedistributed mitochondria expand immunosuppressive cells but exhausts CD8+ T cellsMitochondria fusion within cancer cells, which precedes redistribution, optimizes metabolic output by triggering conformational changes in P5CSMitochondria fusion allows cancer cells to incorporate and refurbish seemingly incompetent host-derived mitochondria, improving efficiency in the tumor ecosystem.

DOI: https://doi.org/10.1101/2025.11.11.687895
bioRxiv. 2025 Oct 06. pii: 2025.10.05.680522. [Epub ahead of print]

Many circulating indole and phenol metabolites are host derived.

Jenna E AbuSalim, Kellen Olszewski, Salma Youssef, Craig J Hunter, Michael R MacArthur, Alessa Henneberg, Rolf-Peter Ryseck, Christiane A Opitz, Mohamed S Donia, Joshua D Rabinowitz.

Indole and phenol metabolites are typically thought to be products of bacterial digestion of tryptophan (indoles) and phenylalanine or tyrosine (phenols). Interest in controlling gut microbial production of these metabolites has continually grown as they have important physiological impacts, with indoles agonizing AhR signaling, and phenols being associated with healthy body weight. While there is a growing wealth of research into which bacteria produce these metabolites, host contribution to their circulating pools has not been adequately characterized. Here, through stable isotope tracing in cell culture and mice, we show that mammalian cells can make aryl-pyruvates, -lactates, -acetates, and -carboxylic acids. Levels of these metabolites in mice and human patients are insensitive to perturbations of the microbiome. In contrast, bacterial metabolism is required to synthesize aryl-propionates and free indole, phenol, and cresol. Overall, we show that host metabolism is a primary contributor to circulating indole and phenol metabolite pools.

DOI: https://doi.org/10.1101/2025.10.05.680522
bioRxiv. 2025 Oct 15. pii: 2025.10.14.682342. [Epub ahead of print]

Mapping the diverse topologies of protein-protein interaction fitness landscapes.

Shannon S Lu, Matthew J Styles, Cheng Frank Gao, Aditya Nandy, Christopher Basile, Joshua A Pixley, Siyuan Tao, Aaron R Dinner, Suriyanarayanan Vaikuntanathan, Bryan C Dickinson.

De novo binder discovery is unpredictable and inefficient due to a lack of quantitative understanding of protein-protein interaction (PPI) sequence-function landscapes. Here, we use our PANCS-Binder technology to perform >1,300 independent selections of various library sizes and compositions of a randomized small protein to identify binders to a panel of 96 distinct target proteins. For successful selections, we discovered reproducible fitness landscapes that group into a few, target-specific, clusters. Each cluster defines a minimal binding motif whose frequency is inversely proportional to the number of specified amino acids (∼2-8) and determines selection success, which is quantifiable by the density of binders to the target within a theoretical sequence space. We leverage these data to develop a supervised contrastive learning approach that discriminates binders from non-binders and demonstrates generalization beyond a threshold amount of data. Together, this framework renders PPI landscapes measurable and predictive, accelerating de novo binder discovery and optimization.

DOI: https://doi.org/10.1101/2025.10.14.682342
Sci Rep. 2025 Nov 26. 15(1): 42185

Oxidative stress reactivates androgen receptor signaling via USP36 to drive castration resistance in prostate cancer.

Changhui Fan, Zhiheng Huang, Junfeng Gao, Yulong Gu, Ning Wang, Bo Zhou.

  Castration-resistant prostate cancer (CRPC) often emerges within a few years following androgen deprivation therapy, and therapeutic options remain limited. Androgen deprivation induces oxidative stress in prostate cancer (PCa) cells, leading to aberrant activation of androgen receptor (AR) signaling. This study aims to clarify the molecular mechanism underlying oxidative stress-induced AR activation in CRPC. Transcriptional activity of the prostate-specific antigen (PSA) promoter was evaluated using a dual-luciferase reporter assay under various treatments. To identify AR-interacting proteins under oxidative stress, TurboID-mediated proximity biotin labeling coupled with mass spectrometry was employed following H2O2 exposure. Protein-protein interactions between AR and ubiquitin-specific peptidase 36 (USP36) were validated by co-immunoprecipitation (Co-IP). Subcellular AR expression was assessed via Immunofluorescence in PCa cells. Low doses H2O2 (10 and 20 μM) enhanced viability and induced oxidative stress in PCa cells, and these concentrations were therefore selected for subsequent experiments. H2O2 treatment activated the AR-PSA signaling axis. The deubiquitinating enzyme USP36 was identified among the proteins that interact with AR upon H2O2 stimulation. Co-IP confirmed the specific binding between AR and USP36. Functional studies revealed that USP36 deubiquitinates and stabilizes AR. Notably, knockdown of USP36 abolished H2O2-induced activation of the AR-PSA pathway. H2O2 promotes the interaction between USP36 and AR, resulting in AR stabilization, transcriptional activation of PSA, and conferring androgen resistance. These findings provide mechanistic insights into how oxidative stress reactivates AR signaling in PCa and highlight potential therapeutic strategies for different stages of PCa.

Keywords:  Androgen receptor; Oxidative stress; Prostate cancer; Prostate-specific antigen; Ubiquitin specific peptidase 36

DOI:  https://doi.org/10.1038/s41598-025-25964-8
J Inflamm Res. 2025 ;18 16045-16062

Metabolic Reprogramming Intermediates of Glucose Regulate Macrophage Polarization: An Important Direction for Ameliorating Pulmonary Vascular Remodeling.

Junqi Wang, Rong Yuan, Shengkang Zhang, Zhaojun Xu, Lan Song.

  Pulmonary vascular remodeling (PVR) is a key pathological basis for various lung diseases and is centered on macrophage-driven pathological vascular remodeling. Macrophage functional polarization is closely related to metabolic reprogramming, a process that not only encompasses energy supply but also dictates cellular function through metabolic intermediates. To bridge the knowledge gap between metabolic regulation and clinical translation in PVR, this review focuses on key metabolites produced during glucose metabolism: pyruvate, citrate, succinate, and itaconate. These intermediates are not merely metabolic byproducts; rather, they directly influence the pathological processes of vascular endothelial cells, smooth muscle cells, and the extracellular matrix by modulating the polarization of macrophages. This review systematically elucidates the precise regulatory mechanisms of these metabolic signals, with the aim of providing new diagnostic and therapeutic targets for PVR. It emphasizes the immense potential of targeting metabolic intermediates for future precision medicine, ultimately promoting a paradigm shift in PVR therapy from traditional anti-proliferative interventions to an innovative model based on metabolic reprogramming.

Keywords:  citrate; glycolytic reprogramming; itaconate; macrophage polarization; pulmonary vascular remodeling; pyruvate; succinate

DOI:  https://doi.org/10.2147/JIR.S541649
Discov Oncol. 2025 Nov 25. 16(1): 2166

Ferroptotic resistance involved in PTEN-loss prostate cancer progression.

Yaoyao Jing, Donghui Xing, Zhigang Zhao, Xiaofang Wang.

  Ferroptosis, a newly recognized form of regulated cell death characterized by iron accumulation and lipid peroxidation, plays a pivotal role in cancer development. Herein, we investigated the mechanisms of ferroptotic resistance mediated by Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) loss in prostate cancer cells. The ferroptosis inducer Erastin was used to evaluate the effects on cell viability, colony formation, and Reactive Oxygen Species (ROS) level in three prostate cancer cell lines: PTEN wild-type (DU145 cells) and two of the PTEN null cells (PC3 and LNCaP cells). DU145 cells were prone to ferroptosis, whereas PC3 and LNCaP cells exhibited reduced sensitivity to Erastin-induced ferroptosis. Mechanistically, PTEN loss increased Glutathione peroxidase 4 (GPX4) expression and subsequently decreased intracellular ROS level, which was associated with elevated GPX4 mRNA levels. Knockdown of GPX4 by RNAi reversed the resistance of PTEN-deficient PC3 and LNCaP cells to Erastin. Collectively, our findings suggest that ferroptosis can serve as a potential therapeutic strategy for prostate cancer, and PTEN status may influence cellular sensitivity to ferroptosis.

Keywords:  Ferroptosis; GPX4; PTEN; Prostate cancer

DOI:  https://doi.org/10.1007/s12672-025-03990-2
Biochemistry. 2025 Nov 25.

Distal Mutations Rewire Allosteric Networks to Control Substrate Specificity in PTP1B.

Xiaoyuan Wang, Ryan M Anderson, Jinchan Liu, Victor Batista, J Patrick Loria.

Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of cellular signaling pathways, and its dysregulation is linked to diabetes, obesity, cancer, and immune dysfunction. While the catalytic mechanism of PTP1B is conserved across protein tyrosine phosphatases, its regulation by distal allosteric sites remains less understood. Here, we investigate how mutations at four allosteric sites (Y153, I275, M282, and E297) alter the PTP1B substrate specificity and enzymatic dynamics. Kinetic analyses with phosphotyrosine peptides and p-nitrophenylphosphate reveal that allosteric mutants display distinct changes in catalytic efficiency (kcat/Km), in some cases reversing substrate preference relative to the wild-type enzyme. Solution NMR spectroscopy and microsecond molecular dynamics simulations demonstrate that these mutations perturb long-range communication networks, disrupting coupling between helices α3 and α7 and altering acid-loop flexibility and active-site dynamics. Notably, the E297A mutation has the most pronounced effects, rigidifying the acid loop and weakening allosteric communication to the catalytic center. Community network analysis highlights the acid loop and helix α7 as central hubs linking distal sites to the active site. Together, these results establish that distal mutations can reshape PTP1B's dynamic landscape, thereby modulating substrate specificity. This work expands our understanding of allosteric regulation in PTP1B and provides a framework for targeting dynamic networks to control phosphatase activity.

DOI: https://doi.org/10.1021/acs.biochem.5c00539
medRxiv. 2025 Nov 13. pii: 2025.11.11.25340023. [Epub ahead of print]

Adaptive nuclear GTP synthesis promotes glioblastoma treatment resistance and is a targetable vulnerability in patients.

Andrew J Scott, Ningning Liang, Wajd N Al-Holou, Jie Xu, Alexandra O'Brien, Angelica Lin, Vidhi Pareek, Zhou Sha, Zitong Zhao, John Z Yang, Annabel Yang, Ayesha U Kothari, Kari Wilder-Romans, Zhe Wu, Jiane Feng, Navyateja Korimerla, Ameer Elaimy, Sravya Palavalasa, Jiali Chen, Lu Wang, Li Zhang, Anthony C Andren, Peter Sajjakulnukit, Bernard Marini, Jason A Heth, Meredith A Morgan, Theodore S Lawrence, Stephen J Benkovic, Deepak Nagrath, Sriram Chandrasekaran, Nathan R Qi, Duxin Sun, Bo Wen, Manjunath P Pai, Nathan Clarke, Krithika Suresh, Yoshie Umemura, Costas A Lyssiotis, Weihua Zhou, Daniel R Wahl.

Glioblastoma (GBM), the most lethal of all brain cancers, resists therapy by rewiring metabolism and relying on GTP signaling to promote DNA repair and radiation therapy (RT) resistance. How GBM modulates GTP levels for this signaling in response to RT-induced DNA damage, and the therapeutic tractability of this metabolic activity in the context of standard-of-care chemoradiation therapy, remain unaddressed. Here, we identify acute changes in glioma metabolism within hours of RT, including an acute post-RT rewiring of guanylate synthesis driven by nuclear translocation of the rate-limiting de novo guanylate synthesis enzyme IMPDH1. This subcellular IMPDH1 re-localization and nuclear GTP accumulation are dependent on the DNA damage signal kinase DNA-PK. Targeting intracranial GTP synthesis with the FDA-approved inhibitor mycophenolate mofetil (MMF) slows repair of DNA damage and extends survival of orthotopic murine models treated with combined RT and temozolomide. Extending our findings to humans, we performed a phase 0 clinical trial revealing that oral MMF administration leads to active intracranial drug concentrations, with target engagement indicated by reversal of IMPDH upstream and downstream metabolites in recurrent GBM tumors. Together, these findings implicate IMPDH as a potential metabolic target in GBM whose pharmacological inhibition is feasible and could complement standard-of-care chemoradiation therapy.

DOI: https://doi.org/10.1101/2025.11.11.25340023
Oncogenesis. 2025 Nov 28. 14(1): 46

Stabilization of FASN by USP5-mediated deubiquitination promotes hepatocellular carcinoma progression.

Qinliang Fang, Changhong Luo, Yuyan Lu, Xijun Chen, Ping Zhan, Qin Yao, Huita Wu, Fuqiang Wang, Zhenyu Yin, Chengrong Xie.

The deubiquitinating enzyme Ubiquitin specific peptidase 5 (USP5) has attracted substantial notice for its vital role in cancer progression. However, the USP5-mediated deubiquitination of corresponding protein substrates and its functional role in hepatocellular carcinoma (HCC) have not been fully investigated. Here, we demonstrated that USP5 expression was significantly elevated in HCC tissues. The overexpression of USP5 was closely associated with larger tumor sizes, more satellite nodules and tumor emboli, and predicted unfavorable clinical outcome in HCC patients as well. Functionally, USP5 facilitated cell proliferation, migration, and invasion, and induced lipid accumulation in vitro, along with enhanced tumor growth in vivo. Moreover, knockdown of USP5 expression showed a profound effect on lipidomic profiling, specially reduced the content of palmitic acid (PA). Treatment of PA could partially rescue the suppression of HCC mediated by USP5 knockdown. Further mechanistic investigation uncovered that Fatty acid synthase (FASN), the crucial enzyme catalyzing PA synthesis, was a downstream target of USP5. USP5 interacted with FASN, repressing the ubiquitination modification of FASN and preventing its degradation. Notably, the positive correlation between USP5 and FASN expression in HCC tissues was observed, and USP5 exerted oncogenic effects partly via FASN. Our findings revealed that USP5 promotes HCC progression through deubiquitinating FASN, and targeting the USP5-FASN-PA axis could potentially serve as a strategic approach for the therapy of HCC.

DOI: https://doi.org/10.1038/s41389-025-00589-8
Cancer Res Commun. 2025 Nov 26.

Elevated tumor-associated androgen receptor activity correlates with poor immune infiltration and immunotherapy response across cancer types.

Ya-Mei Hu, Faming Zhao, Julie N Graff, Canping Chen, Yi Zhang, Jayne M Stommel, Jinho Lee, Gabriel M Zangirolani, Joshua Rose, George V Thomas, Hui Wu, Adel Kardosh, Gordon B Mills, Joshi J Alumkal, Amy E Moran, Zheng Xia.

The role of androgen receptor (AR) signaling in modulating antitumor immune responses has received increasing attention in recent years; however, its broader impact across diverse cancer types and between sexes remains largely unexplored. Here, we investigated how AR activity correlates with tumor-infiltrating leukocytes, patient prognosis, and immunotherapy response across cancers and sexes. We inferred AR activity using a network-based approach across bulk RNA-seq (TCGA), single-cell RNA-seq (prostate cancer meta-atlas), and immunotherapy cohorts. Pathway analysis and Cox regression assessed mechanisms and survival. Immune infiltration and signatures were evaluated via TIMER and ssGSEA. Key findings were validated using Digital Spatial Profiling and immunohistochemistry. Our pan-cancer analysis of 33 TCGA cancer types revealed broad variability in AR activity, highest in prostate adenocarcinoma. Genes significantly correlated with AR activity show negative associations and are enriched in immune activation pathways. Notably, AR activity inversely correlated with leukocyte abundance and IFN-γ pathway activity across tumors and sexes-unlike estrogen or progesterone receptors. Longitudinal biopsy analysis in metastatic prostate cancer showed AR inhibition enhanced immune cell and IFN-γ signatures. Single-cell analysis confirmed that tumor-intrinsic AR activity inversely correlates with immune infiltration in prostate cancer. Furthermore, low AR activity is significantly associated with favorable immunotherapy responses in hormone-independent cohorts. Spatial proteomics showed a negative correlation between AR and CD45 protein in sarcoma and ovarian cancers. These findings suggest AR activity as a pan-cancer predictive biomarker of immunotherapy response and support that AR blockade in immunotherapy-refractory tumors represents a promising treatment strategy, regardless of tumor type or patient sex.

DOI: https://doi.org/10.1158/2767-9764.CRC-25-0409
bioRxiv. 2025 Nov 13. pii: 2025.11.07.687244. [Epub ahead of print]

Uncovering the molecular basis of kinase activity and substrate recognition with phospho-PCA.

Changhua Yu, Edward Pao, David Van Valen.

Protein kinases relay information to various cellular processes, and their dysregulation underlies numerous human diseases. Despite their importance, our understanding of how kinase domains and their variants impact protein stability, catalytic activity, and substrate recognition is incomplete. In this work, we develop the phosphorylation protein complementation assay (phospho-PCA), which enables quantitative measurements of kinase-substrate interactions by coupling them to the growth of bud-ding yeast, thereby enabling deep mutational scanning of kinase domains. When combined with deep mutational scans targeting folding stability and Bayesian modeling, phospho-PCA can disentangle the relative impact of mutations on kinase domain stability, catalytic activity, and substrate specificity. We demonstrate the accuracy and breadth of phospho-PCA, showing its applicability to both tyrosine and serine/threonine kinase domains. We then apply our method to three closely related protein kinases with distinct substrate preferences, evaluating over 15,000 kinase variants against a panel of three substrates for both catalytic activity and substrate specificity. The resulting dataset constitutes the largest and most detailed variant-to-function map assembled for this enzyme family to date, revealing numerous mutations that alter kinase activity and substrate specificity. Physics-based modeling reveals how these mutations operate through diverse mechanisms, including long-range allosteric communication, to alter both activity and substrate specificity. Given its scalability, we believe phospho-PCA can measure the functional impact of variants across the entire human kinome.

DOI: https://doi.org/10.1101/2025.11.07.687244
bioRxiv. 2025 Oct 08. pii: 2025.10.08.681129. [Epub ahead of print]

The functional landscape of the human ubiquitinome.

Julian van Gerwen, Maximilian Fottner, Shengbo Wang, Bede Busby, Ellen Boswell, Paul Schnacke, Andrea C Carrano, Malina A Bakowski, Emily R Troemel, Romain Studer, Marta Strumillo, Maria-Jesus Martin, J Wade Harper, Kathrin Lang, Andrew R Jones, Eric J Bennett, Juan Antonio Vizcaíno, Inigo Barrio-Hernandez, Pedro Beltrao.

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. Mass spectrometry-based proteomics has allowed proteome-scale quantification of hundreds of thousands of ubiquitination sites (ubi-sites), however the functional importance and regulatory roles of most ubi-sites remain undefined. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. We identified a core subset of ubi-sites under evolutionary constraint through alignment of ubiquitin proteomics data from six non-human species, and determined ultra-conserved ubi-sites recurring at regulatory hotspots within protein domains. Perturbation proteomics revealed that these highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize functional ubi-sites with roles in cellular signaling, we constructed a functional score for more than 100,000 ubi-sites by integrating evolutionary, proteomic, and structural features using machine learning. Our score identifies ubi-sites regulating diverse protein functions and rationalizes mechanisms of genetic disease. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying protein ubiquitination.

DOI: https://doi.org/10.1101/2025.10.08.681129
bioRxiv. 2025 Oct 11. pii: 2025.10.09.681531. [Epub ahead of print]

Optogenetic control of PLC-γ1 activity polarizes cell motility.

Ravikanth Appalabhotla, Priscila F Siesser, Harrison Truscott, Nicole Hajicek, John Sondek, James E Bear, Jason M Haugh.

Phospholipase C-γ1 (PLC-γ1) signaling is required for mesenchymal chemotaxis, but is it sufficient to bias motility? PLC-γ1 enzyme activity is basally autoinhibited, and light-controlled membrane recruitment of wild-type (WT) PLC-γ1 (OptoPLC-γ1) in Plcg1- null fibroblasts does not trigger lipid hydrolysis, complicating efforts to isolate its contribution. Utilizing cancer-associated mutations to investigate the regulatory logic of PLC-γ1, we demonstrate that the canonical hallmark of enzyme activity, phosphorylated Tyr783 (pTyr783), is not a proxy for activity level, but is rather a marker of dysregulated autoinhibition. Accordingly, OptoPLC-γ1 with a deregulating mutation (P867R, S345F, or D1165H) exhibits elevated phosphorylation, and membrane localization of such is sufficient to activate substrate hydrolysis and concomitant motility responses. In particular, local recruitment of OptoPLC-γ1 S345F polarizes cell motility on demand. This response is spatially dose-sensitive and only partially reduced by blocking canonical PLC-γ1 signaling yet is lipase-dependent. Our findings reframe the interpretation of PLC-γ1 regulation and demonstrate that local activation of PLC-γ1 is sufficient to direct cell motility.

DOI: https://doi.org/10.1101/2025.10.09.681531
Sci Rep. 2025 Nov 27.

Uncovering the glutamate carboxypeptidase II microenvironment using a multi-labeling proteomic approach.

Jana Pokorná, Martin Hadzima, Alena Křenková, Joshua D Smith, Vladimír Šubr, Robin Kryštůfek, Martin Hubálek, Jana Starková, Karolína Šrámková, Tomáš Etrych, Libor Kostka, František Sedlák, Jan Konvalinka, Pavel Šácha.

  Glutamate carboxypeptidase II (GCPII) is a membrane-bound metallopeptidase predominantly expressed in neural and prostatic tissues, with significantly elevated levels in prostate carcinoma that increase with tumor grade. Despite its significance as a target for imaging and therapy in prostate cancer, its physiological function in this tissue remains poorly understood. To help fill this knowledge gap, we developed an integrated approach combining proximity labeling technologies for proteomic profiling (horseradish peroxidase, µMap, and riboflavin tetraacetate labeling) with our previously established iBody platform, which targets GCPII with a small-molecule specific inhibitor with proven efficacy as a chemical probe. Proximity labeling proteomic experiments on U251 MG-GCPII cells were followed by mass spectrometry and statistical analysis of protein abundances obtained by label-free quantification. Additionally, selected identified proteins were further validated through Western blot analyses and GCPII pulldown assays using cell lysates. This work identifies a network of GCPII-associated proteins that are potentially involved in cancer metabolism, migration, invasiveness, progression, and immune evasion. Our novel proximity proteomics labeling strategy provides a low-background framework, efficient biotinylation, and enhanced target binding via the avidity effect. Among the approaches tested, riboflavin tetraacetate-based iBody labeling exhibited the highest precision, underscoring its potential for membrane protein interactome mapping.

Keywords:  HRP; PSMA; Protein proximity labeling; Riboflavin; iBody; µMap

DOI:  https://doi.org/10.1038/s41598-025-28826-5
Cancer Res. 2025 Nov 25.

Nutrient Availability Dictates Cancer Metabolism-Based Therapeutic Responses to Non-oncology Drugs.

Woo Yang Pyun, Jae Hyung Park, Jae Won Roh, Dongkyu Jeon, Jongwan Kim, Ji Eun Paik, Seok Chan Cho, So Yeon Park, Hocheol Lim, Hyungwoo Kim, Young Jin Jang, Jaehoon Lee, Jong Hwa Byun, Dong Woo Son, Soo-Youl Kim, Kun-Liang Guan, Won Dong Lee, Heon Yung Gee, Han-Woong Lee, Kyoung Tai No, Yu Suk Choi, Tadayoshi Hashimoto, Takayuki Yoshino, Han-Sol Jeong, Wan Namkung, Joo Hyun Nam, Hyun Woo Park.

Metabolic dysregulation is a major hallmark of cancer, making interventions that modify tumor nutrient availability attractive adjuvants for improving clinical outcomes for cancer patients. Clarifying how the nutritional status of individual patients affects the metabolic vulnerability of tumors to drugs is needed to inform personalized treatment guidelines. Working toward the goal of oncometabolic precision medicine, we developed the cancer metabolism-based synthetic lethality platform (CM-SLP), a high-throughput screening platform that explores the metabolic vulnerability of cancer cells to non-oncology drugs induced by altered nutrient availability and predicts potential synthetic lethal interactions with nutrient conditions. Promising CM-SLP candidates included propafenone and biguanides as representative non-oncology drugs that cooperatively enhanced cytotoxicity via dysregulated metabolic pathways. Furthermore, the mTOR and Hippo pathways mediated the response to combined propafenone/hypoglycemia or biguanides/hypoglycemia treatments, respectively, and mTOR or TEAD inhibitors circumvented the need for dietary interventions to enhance cancer cell death. Together, these results indicate that CM-SLP represents a promising approach for integrating metabolic profiling into precision oncology, offering therapeutic avenues tailored to individual patient needs.

DOI: https://doi.org/10.1158/0008-5472.CAN-25-1123