The new study, published in Frontiers in Pharmacology, set out to move beyond rutin’s well-documented antioxidant and anti-inflammatory reputation and identify a concrete molecular target. Using TGF-β1-stimulated MRC-5 human fetal lung fibroblasts and bleomycin-induced pulmonary fibrosis in C57BL/6 mice, the investigators first confirmed that rutin inhibits fibroblast-to-myofibroblast differentiation, suppresses myofibroblast proliferation and migration, and reduces extracellular matrix deposition. Notably, the half-maximal inhibitory concentration of rutin for TGF-β1-activated cells was roughly 60 micrograms per milliliter at 24 hours, dramatically lower than the approximately 250 micrograms per milliliter measured in quiescent cells, indicating a selective sensitivity of the pathological phenotype rather than indiscriminate cytotoxicity. In mice, oral rutin at 10 and 20 milligrams per kilogram improved forced vital capacity, dynamic lung compliance, and total lung capacity, while microCT imaging revealed reduced honeycomb lung, preserved alveolar structure, and thinned alveolar walls. Histological staining with hematoxylin and eosin and Masson’s trichrome confirmed diminished collagen deposition, and Western blot analysis showed downregulation of fibroblast activation protein 1, vimentin, collagen I, and α-SMA—the canonical molecular signatures of myofibroblast activation.

The mechanistic hunt then zeroed in on heterogeneous nuclear ribonucleoprotein L, or hnRNP L, an RNA-binding splicing factor previously implicated in pulmonary fibrosis through its control of circRNA back-splicing. Molecular docking against the AlphaFold-predicted structure suggested potential interactions with residues including ASN-395, ARG-398, ASN-401, GLN-566, LYS-475, ASP-397, ASN-482, and PHE-561, alongside hydrophobic contacts with PHE-477 and PHE-400. Surface plasmon resonance on a CM5 chip confirmed that rutin binds recombinant hnRNP L with an equilibrium dissociation constant of approximately 19.48 micromolar—a moderate but measurable affinity. Critically, the cellular thermal shift assay showed that in rutin-treated cells, hnRNP L exhibited higher thermal stability across a 45 to 70 degree Celsius range, with a significant rightward shift in the denaturation curve, demonstrating that the compound engages its target inside living cells and stabilizes the protein against heat-induced unfolding. Quantitative PCR and Western blotting further revealed that rutin suppressed hnRNP L expression at both the mRNA and protein levels, and rescue experiments showed that overexpressing hnRNP L reversed rutin’s inhibitory effects on α-SMA expression, cell migration, and fibrotic protein markers.

What elevates this work above a routine drug-target pairing is its embrace of liquid–liquid phase separation, the biophysical process by which proteins containing intrinsically disordered regions spontaneously condense into dynamic droplet-like biomolecular condensates. Sequence analysis using the PONDR database identified two intrinsically disordered segments in hnRNP L, spanning amino acids 1–101 and 336–383, marking it as a phase-separation-prone protein. The researchers purified an EGFP-tagged hnRNP L fusion protein and observed that, in the presence of 10 percent PEG6000 as a molecular crowding agent, the protein formed spherical droplets under laser scanning confocal microscopy. Fluorescence recovery after photobleaching showed that when a single condensate was bleached with a high-intensity laser, fluorescence recovered within roughly four minutes, confirming the liquid-like diffusivity and internal mobility characteristic of genuine phase-separated condensates rather than static aggregates. When TGF-β1-stimulated cells were transfected with EGFP-hnRNP L, phase-separated puncta formed in the nucleus; rutin treatment prevented this assembly, and photobleached condensates in the rutin group showed no fluorescence recovery, indicating that the drug had dismantled the condensate network entirely.

The disordered regions proved functionally indispensable. When the researchers constructed a truncation mutant lacking amino acids 1–111 and 274–384, the protein failed to form droplets both in vitro and in cells, and overexpression of this mutant had no significant impact on fibrotic protein expression or on α-SMA mRNA levels in IMR-90 cells. To test whether phase separation was required for hnRNP L’s pro-fibrotic activity in living animals, the team packaged full-length hnRNP L and the truncation mutant into adeno-associated viral vectors (AAV2/5) and delivered them via aerosol directly into the lungs of bleomycin-treated mice. Overexpression of full-length hnRNP L deteriorated lung function, reversed rutin’s beneficial effects on forced vital capacity, dynamic lung compliance, and total lung capacity, worsened honeycomb-like fibrotic structures on microCT, and elevated collagen, vimentin, α-SMA, and FAP1 on Western blot. In striking contrast, overexpression of the phase-separation-deficient mutant did not impair lung function, did not reverse rutin’s therapeutic efficacy, and did not counteract the drug’s inhibitory effects on fibrotic markers. These in vivo rescue data, obtained from a single randomized and blinded cohort of six mice per group, support the model that the anti-fibrotic action of rutin is tied to its interference with hnRNP L condensate assembly.

Having established that hnRNP L undergoes phase separation and that this capacity is required for its pro-fibrotic function, the investigators next asked which downstream splicing events the condensates control. Transcriptome sequencing of TGF-β1-stimulated MRC-5 cells after hnRNP L knockdown, analyzed through Gene Ontology and KEGG enrichment, revealed that glycolysis was among the most significantly altered biological processes. A heatmap of 21 differentially expressed glycolysis-related genes placed pyruvate kinase M, or PKM, among the top-ranking hits. Using the SUPPA2 pipeline on Oxford Nanopore long-read data, the team identified 17,958 significant differential splicing events between control and hnRNP L-depleted groups, with 25 of these events located in genes involved in glycolysis and gluconeogenesis—suggesting that hnRNP L phase separation may orchestrate a broader metabolic reprogramming beyond PKM alone. PKM can be alternatively spliced to produce two isoforms, PKM1 and PKM2, and the PKM2 isoform is a rate-limiting glycolytic enzyme previously shown to promote myofibroblast differentiation and pulmonary fibrosis progression. Quantitative PCR showed that rutin reduced PKM2 expression and increased PKM1 expression at the mRNA level, and Western blotting confirmed the downregulation of PKM2 protein. An RNA immunoprecipitation experiment demonstrated that PKM pre-mRNA physically binds hnRNP L, and that this binding decreases under rutin treatment, consistent with the drug disrupting the splicing factor’s engagement with its target transcript.

The metabolic consequences of this splicing switch proved equally consequential. PKM2 drives the final step of glycolysis and enhances lactate production, and lactate serves as the substrate for histone lactylation—a recently discovered epigenetic modification in which lactyl groups are covalently attached to histone lysine residues, directly influencing gene transcription. The researchers screened three lactylated histones—H3K9la, H4K12la, and H4K16la—and found that rutin significantly reduced H3K9 lactylation. Inhibition of PKM2 with the selective inhibitor PKM2-IN-1 reduced H3K9la, while overexpression of PKM2 reversed this effect, and exogenous lactic acid supplementation attenuated rutin’s inhibitory effects on both H3K9la and α-SMA. Co-immunoprecipitation revealed that P300, the acetyltransferase enzyme responsible for lactylation, increased H3K9la in the TGF-β1 group, whereas rutin inhibited this modification. Chromatin immunoprecipitation followed by PCR demonstrated that TGF-β1 enhanced the occupancy of H3K9la at the PKM promoter, while rutin decreased this interaction—and critically, overexpression of full-length hnRNP L reversed rutin’s effect, whereas the phase-separation-deficient truncation mutant did not. Supplementation with exogenous lactic acid largely restored PKM pre-mRNA and PKM2 transcript abundance in rutin-treated cells, supporting a model in which lactate functionally maintains PKM transcription through H3K9la, forming a self-limiting regulatory circuit in which reduced H3K9la feeds back to suppress PKM2 transcription.

The authors are careful to frame their findings as a mechanistic framework rather than a definitive causal chain, acknowledging that the binding affinity of rutin for hnRNP L is moderate and that the compound’s well-documented polyphenol properties may contribute to its overall anti-fibrotic effects through additional targets. They also note that the intrinsically disordered region truncation mutant lost both phase separation ability and pro-fibrotic activity simultaneously, leaving open the possibility that the truncation impairs other functional domains independent of condensate assembly. Direct visualization of endogenous hnRNP L phase separation in fibrotic lung tissue has not yet been achieved, and the mechanistic studies were conducted in fetal lung fibroblast lines that may not fully recapitulate the behavior of primary adult fibroblasts from patients with idiopathic pulmonary fibrosis. The models also do not incorporate the mechanical microenvironment—increased matrix stiffness is itself a principal regulator of PKM2 in lung fibroblasts—and future work using stiffness-tunable substrates or decellularized fibrotic lung matrices will be needed to determine whether the hnRNP L–PKM2–lactate–H3K9la axis is also modulated by mechanical cues. Lactate may derive from pathways other than PKM2-dependent glycolysis, and H3K9la may be regulated by histone modifiers beyond P300, so locus-specific manipulation of H3K9la at the PKM promoter will be necessary to establish a causal link between this epigenetic mark and PKM transcription.

Perhaps the most significant translational caveat concerns rutin’s pharmacokinetics. The compound suffers from poor oral bioavailability, stemming from low aqueous solubility, limited intestinal absorption, and rapid metabolism—drawbacks that have long hindered its clinical translation despite promising in vitro bioactivity. The researchers therefore positioned rutin as a tool compound to dissect a previously unrecognized molecular mechanism, while recognizing that future chemical optimization, nanocarrier formulation, or structural derivatization will be required to translate this mechanism into therapeutic benefit. No pharmacokinetic profiling of rutin or its metabolites was performed in the present study, and future LC-MS/MS-based analyses of plasma, lung tissue, and urine will be necessary to correlate systemic exposure with target engagement and antifibrotic efficacy. Nevertheless, the study represents a conceptual advance on multiple fronts: it identifies a natural product that pharmacologically targets phase separation, links an RNA splicing factor to metabolic and epigenetic regulation through a single druggable axis, and proposes a self-limiting feedback circuit in which reduced histone lactylation suppresses the very transcript that generates the lactate substrate. Whether this framework survives validation in primary human fibroblasts, independent animal cohorts, and more physiological systems remains to be seen—but it offers a compelling new lens through which to view the molecular logic of fibrotic disease, and a template for how natural products might be repurposed as precision modulators of condensate biology.

Subject of Research: Pulmonary Fibrosis

Article Title: Rutin interferes with hnRNP L phase separation and modulates the PKM2–lactate–H3K9la axis in pulmonary fibrosis

Article References: Luan, J., Wang, Y., Jiang, M., Zhang, H., Lv, C., Zhang, J., & Song, X. (2026). Rutin interferes with hnRNP L phase separation and modulates the PKM2–lactate–H3K9la axis in pulmonary fibrosis. Frontiers in Pharmacology, 17, Article 1787570. https://doi.org/10.3389/fphar.2026.1787570

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1787570

Keywords: Rutin, interferes, hnRNP, phase, separation, modulates, PKM2, lactate, H3K9la, axis, pulmonary, fibrosis

Podocytes are highly specialized epithelial cells wrapped around the glomerular capillaries, and their interlocking foot processes form the slit diaphragm, a structure essential to the glomerular filtration barrier. When podocytes are injured, proteins leak into the urine, producing the massive proteinuria that defines podocytopathies such as minimal change disease, focal segmental glomerulosclerosis and membranous nephropathy. Clinically, infection is the most common trigger of recurrent proteinuria in these conditions: adults with early-onset, frequently relapsing minimal change disease often develop oedema and heavy protein loss shortly after respiratory infections. Yet even when immunosuppressive and supportive therapies work initially, relapses remain frequent, and the mechanisms by which infection-associated inflammatory signals aggravate podocyte injury have remained incompletely defined.

Arachidonic acid offered a plausible suspect. This omega-6 polyunsaturated fatty acid is stored esterified in cell-membrane phospholipids, and circulating free levels are normally very low, roughly 0.1 micromoles per litre, because the molecule is rapidly bound by albumin. During infection or tissue injury, inflammatory cytokines activate membrane phospholipases that liberate free arachidonic acid, raising its circulating concentration. In kidney disease, proteinuria and hypoalbuminaemia may further reduce albumin’s binding capacity, enlarging the biologically available pool of the fatty acid. Previous work had linked arachidonic acid metabolism to renal inflammation and shown that excessive amounts can harm podocytes, but whether the lipid amplifies damage through metabolic stress in minimal change disease-related injury was unknown.

To find out, the researchers used a puromycin aminonucleoside, or PAN, model, a standard experimental mimic of minimal change disease. Male Sprague-Dawley rats received a single intraperitoneal PAN injection, and ten days later, once podocyte injury was established, half of the injured animals received an intraperitoneal dose of arachidonic acid at 3 milligrams per kilogram. The team was careful to frame this PAN-plus-AA condition as an injury-exacerbation model rather than a faithful reproduction of infection-triggered clinical relapse, since no true infectious stimulus was present. Serum measurements confirmed the design worked: arachidonic acid levels rose from about 4.5 nanograms per millilitre in controls to 8.2 in PAN-treated rats and 12.0 in the combined group, with the combined group also exceeding rats given the fatty acid alone.

The structural consequences were striking. Light microscopy showed no prominent glomerular sclerosis or fibrosis in any group, consistent with the subtle appearance of minimal change disease, so the investigators turned to transmission electron microscopy. There, the glomerular basement membrane thickened from 155 nanometres in controls to 227 nanometres after PAN and 274 nanometres after PAN plus arachidonic acid. Foot-process width swelled from 0.42 to 0.73 and then 1.00 micrometres across the same groups, while the density of foot processes per micrometre of basement membrane fell from 2.04 to 1.16 to 0.81, all differences between the PAN and combined groups statistically significant. Nephrin, the slit-diaphragm protein that anchors the filtration barrier, declined after PAN and dropped further with added arachidonic acid, as shown by both immunofluorescence and Western blotting.

The metabolic story proved equally compelling. Electron microscopy revealed severe mitochondrial injury in podocytes from the combined-treatment rats, including swelling, size heterogeneity, matrix rarefaction and fragmented or dissolved cristae, with quantified increases in mean mitochondrial area, cristae-damage rate and overall damage score. The mitochondrial outer-membrane protein TOM20 was reduced in parallel. The team then examined the PKM enzyme family: PKM1 and PKM2 signals within nephrin-positive glomerular regions both fell after PAN and fell further with arachidonic acid, while phosphorylated PKM2 at the Tyr105 residue rose. Because Tyr105 phosphorylation disrupts formation of PKM2’s active tetramer, the increase signals dysfunction rather than activation. Functionally, total pyruvate kinase activity declined, and renal pyruvate, lactate and ATP levels all dropped, reaching their lowest points in the combined group.

Cultured human podocytes reproduced the pattern. Differentiated cells exposed to PAN alone showed disrupted F-actin stress fibres and reduced nephrin; adding arachidonic acid produced the lowest F-actin intensity and the greatest nephrin loss. The same metabolic signature emerged: reduced PKM1 and PKM2 abundance, elevated phospho-PKM2, diminished pyruvate kinase activity, and lower lactate, pyruvate and ATP. Mitochondria fared worse with the combination, showing greater swelling and cristae damage, a reduced JC-1 aggregate-to-monomer ratio indicating loss of membrane potential, decreased TOM20, and elevated mitochondrial reactive oxygen species detected by MitoSOX staining. Downstream, TUNEL assays revealed more apoptotic cells, and DCFH-DA staining plus flow cytometry confirmed heightened intracellular oxidative stress in the combined-treatment group.

The rescue experiment provided the study’s most translational moment. TEPP-46, a selective PKM2 activator that stabilizes the enzyme’s active tetrameric state, was applied to podocytes under PAN-plus-arachidonic-acid conditions. Treatment restored a more organized actin cytoskeleton, reduced apoptosis, increased PKM2 expression while lowering phospho-PKM2, and raised pyruvate kinase activity along with lactate, pyruvate and ATP production. Mitochondrial readouts improved as well: TOM20 abundance rose, electron microscopy showed less swelling and cristae damage, MitoTracker and JC-1 signals indicated better mitochondrial mass and membrane potential, and ROS levels fell. Together these results indicate that PKM2 activation can partially reverse the metabolic and mitochondrial collapse that arachidonic acid imposes on injured podocytes.

The authors frame their findings within a two-axis model of arachidonic acid toxicity. Their earlier work showed that the fatty acid activates a PKA-c-Abl-nephrin pathway driving actin cytoskeletal remodelling; the present study adds a PKM2-associated metabolic axis that impairs energy production and mitochondrial homeostasis. The two pathways may converge, since actin remodelling demands ATP and mitochondrial ROS can destabilize nephrin and the cytoskeleton, though the data do not establish a strict sequence. Limitations are acknowledged: the model lacks a true infectious trigger, lipidomic analysis did not distinguish free from esterified arachidonic acid or eicosanoid products such as those from COX2/PGE2 signalling, glycolytic flux and respiration were not directly measured by ECAR or OCR assays, and the TEPP-46 rescue was performed only in vitro. Notably, omega-3 fatty acids have recently been reported to protect the podocyte and kidney, underscoring the divergent effects of different polyunsaturated fat subclasses. Even so, the study supports further testing of PKM2-targeted metabolic intervention, including in vivo rescue studies and models that better recapitulate infection-associated relapse, offering hope that a metabolic lever might one day help prevent the proteinuric crashes that follow infections in vulnerable patients.

Subject of Research: Arachidonic acid exacerbation of puromycin aminonucleoside-induced podocyte injury through PKM2-linked glycolytic and mitochondrial dysfunction

Article Title: Arachidonic Acid Disrupts PKM2‐Linked Glycolysis‐Related Metabolism and Exacerbates PAN‐Induced Podocyte Injury

Article References: Nie, K., Gao, X., Liu, M., You, D., Weng, M., Zheng, T., Lin, J., Rao, S., Zhuo, Y., Wan, J., & Yang, L. (2026). Arachidonic Acid Disrupts PKM2 ‐Linked Glycolysis‐Related Metabolism and Exacerbates PAN ‐Induced Podocyte Injury. Journal of Cellular and Molecular Medicine, 30(19), Article e71404. https://doi.org/10.1111/jcmm.71404

Image Credits: AI Generated

DOI: 10.1111/jcmm.71404

Keywords: arachidonic acid, podocytes, PKM2, glycolysis, minimal change disease, mitochondrial dysfunction, puromycin aminonucleoside, nephrin, oxidative stress, TEPP-46, kidney disease, proteinuria

Hepatic stellate cells are the quiet custodians of the healthy liver. In their quiescent state, they store vitamin A droplets and maintain the extracellular matrix that provides structural support to the organ. When the liver is injured, however, these cells undergo a dramatic transformation. They lose their lipid droplets, proliferate, migrate to sites of damage, and begin producing massive quantities of collagen and other matrix proteins that accumulate as scar tissue. This activation process is metabolically expensive, and activated stellate cells, much like cancer cells, shift their energy production toward aerobic glycolysis, a phenomenon in which glucose is preferentially converted to lactate even in the presence of oxygen. This metabolic rewiring, sometimes called a Warburg-like shift, supplies the biosynthetic intermediates that rapidly dividing, matrix-producing cells need, but the upstream signals that coordinate this shift during stellate cell activation have remained poorly defined.

The Shanghai-led team set out to find those upstream signals, and their search converged on PRPF19, a ubiquitin ligase best known for its role in pre-mRNA splicing and DNA damage responses. The researchers found that PRPF19 expression was elevated in both human and mouse fibrotic livers, and that within these tissues the protein was enriched specifically in activated hepatic stellate cells. Crucially, the abundance of PRPF19 correlated positively with the severity of fibrosis, suggesting that the protein tracks with disease progression rather than being an incidental byproduct of liver injury. This clinical correlation provided the initial impetus to test whether PRPF19 was merely a marker of scarring or an active participant in the fibrogenic process.

To distinguish between these possibilities, the team turned to conditional genetic mouse models, an approach that allows a gene to be deleted in a specific cell type while leaving it intact elsewhere. Using mice carrying floxed Prpf19 alleles crossed with a tamoxifen-inducible Lrat-CreERT2 driver, they deleted the gene selectively in hepatic stellate cells. The result was striking: loss of Prpf19 in stellate cells attenuated both the activation of these cells and the development of liver fibrosis in vivo. In contrast, deleting Prpf19 in hepatocytes, the metabolic workhorses of the liver, using an Alb-CreERT2 driver produced no such protective effect. This cell-type specificity is a central finding of the study, because it demonstrates that PRPF19 acts within the stellate cells themselves rather than indirectly through neighboring parenchymal cells, and it positions the protein as a genuine driver of the fibrogenic response rather than a passive companion of injury.

With a causal role established, the researchers next asked what PRPF19 was actually doing inside the cells. They deployed an integrated multi-omics strategy, combining transcriptomic profiling of gene expression, proteomic analysis of the cellular protein complement, and metabolomic measurement of small-molecule metabolites. The convergence of these three data streams pointed in a single direction: PRPF19 sustains glycolytic flux, the rate at which glucose is funneled through the glycolytic pathway, and maintains the transcriptional programs that characterize activated stellate cells. When PRPF19 was absent, the cells’ metabolic engine downshifted, and the gene expression signature of activation faded. The metabolomic data in particular indicated that glycolytic intermediates and lactate production depended on PRPF19 levels, tying the protein directly to the energetic state of the cell.

The mechanistic heart of the paper concerns pyruvate kinase M2, or PKM2, the enzyme that catalyzes the final, rate-limiting step of glycolysis by converting phosphoenolpyruvate to pyruvate. PKM2 is famous in cancer biology for its ability to switch between an active tetrameric form in the cytoplasm and a monomeric or dimeric form that can translocate into the nucleus, where it acts as a transcriptional coactivator. The new study shows that PRPF19 promotes a specific chemical modification of PKM2: K63-linked ubiquitination, a form of the ubiquitin tag that, unlike the K48-linked version that condemns proteins to destruction, typically alters a protein’s behavior, localization, or interactions without marking it for degradation. In this case, K63-linked ubiquitination of PKM2 facilitated its movement into the nucleus of the stellate cell.

Once in the nucleus, ubiquitinated PKM2 does something consequential: it partners with hypoxia-inducible factor 1α, or HIF-1α, the master transcription factor that orchestrates cellular responses to low oxygen and drives glycolytic gene expression. The study demonstrates that nuclear PKM2 supports HIF-1α-dependent transcription of both glycolytic genes and fibrogenic genes, creating a feed-forward loop in which the metabolic enzyme and the transcription factor jointly enforce the activated stellate cell phenotype. In other words, PRPF19 sits at the top of a signaling cascade that converts a post-translational modification, the ubiquitin tag on PKM2, into a metabolic shift toward glycolysis and, simultaneously, into the transcriptional activation of collagen production and other fibrogenic outputs. This integration of post-translational signaling with metabolic reprogramming is precisely the kind of upstream regulatory mechanism that the field had been missing.

The significance of this work extends beyond the mechanics of a single protein pair. For decades, antifibrotic drug development has struggled because activated stellate cells are resilient, and broad approaches to suppress scarring have often produced unacceptable side effects. The PRPF19-PKM2 axis offers a more precise target: a pathway that is enriched in activated stellate cells, that is dispensable in hepatocytes at least with respect to fibrosis, and that sits upstream of both the metabolic and transcriptional hallmarks of activation. The cell-type-specific conditional knockout data suggest that interventions aimed at this pathway could, in principle, dampen stellate cell activation without broadly disrupting liver metabolism, although the authors’ findings in mice are early-stage and therapeutic translation would require substantial additional work, including the development of tools to modulate PRPF19 or its ubiquitination of PKM2 in a controlled way.

It is also worth noting what the study does not yet establish. The published work, which was released as a citable accepted manuscript under Springer Nature’s open access policy, reports findings from human tissue correlations and murine genetic models, and the authors, who include Guang-Cong Zhang, Shuang-Feng Zi, Baokui Ye, and Yi-Fan Ma as co-first contributors, declare no competing interests. Whether pharmacological inhibition of the PRPF19-PKM2-HIF-1α circuit can reverse established fibrosis in larger animal models, and whether PRPF19 levels in patient biopsies could serve as a predictive biomarker of disease trajectory, remain open questions for future research. Still, the study adds a compelling new node to the growing map of immunometabolism and fibrosis biology, illustrating how a splicing-associated ubiquitin ligase can moonlight as a metabolic regulator, and how the chemistry of a single ubiquitin linkage can ripple outward to reshape the fate of an entire organ.

Subject of Research: The role of PRPF19-mediated PKM2 ubiquitination in hepatic stellate cell activation and liver fibrosis

Article Title: PRPF19 drives hepatic stellate cell activation and liver fibrosis via PKM2-mediated glycolytic reprogramming

Article References: Zhang, G.-C., Zi, S.-F., Ye, B., Ma, Y.-F., Chen, J., Zhu, J.-M., Liu, T., Yin, J., Shen, X., Yu, X.-N., & Dong, L. (2026). PRPF19 drives hepatic stellate cell activation and liver fibrosis via PKM2-mediated glycolytic reprogramming. Nature Communications. https://doi.org/10.1038/s41467-026-78341-y

Image Credits: AI Generated

DOI: 10.1038/s41467-026-78341-y

Keywords: liver fibrosis, hepatic stellate cells, PRPF19, PKM2, glycolysis, HIF-1α, ubiquitination, metabolic reprogramming, K63-linked ubiquitin, liver disease, fibrogenesis, Nature Communications

The team began by searching for cellular partners of vIRF3 in lymphatic endothelial cells using immunoprecipitation followed by mass spectrometry. Among the proteins that specifically co-purified with the viral factor was a polypeptide of roughly sixty kilodaltons corresponding to pyruvate kinase M. Although vIRF3 could bind both the PKM1 and PKM2 isoforms in pull-down assays, only PKM2 was expressed in lymphatic endothelial cells, so the investigators focused their attention on the vIRF3–PKM2 axis. Detailed binding experiments mapped the interaction to a short region of vIRF3 spanning amino acids 250 to 280. Deleting this segment produced a mutant viral protein, vIRF3-MT, that lost the ability to bind PKM2 while retaining its interactions with other known partners, including IRF7, HIF1α and HDAC5, making it a clean genetic tool for isolating the metabolic consequences of the interaction.

The biological significance of the binding became apparent when the researchers examined the oligomeric state of PKM2. This enzyme exists in two functionally distinct forms: a tetramer that acts as an active pyruvate kinase, channeling carbon from phosphoenolpyruvate into pyruvate and onward into the tricarboxylic acid cycle, and a nuclear dimer that functions as a transcriptional co-factor supporting aerobic glycolysis. When wild-type vIRF3 was expressed in lymphatic endothelial cells, cross-linking experiments showed a striking increase in PKM2 tetramerization, accompanied by elevated pyruvate kinase activity, higher intracellular acetyl coenzyme A levels and reduced lactate concentrations. The mutant viral protein, lacking the PKM2-binding region, produced none of these effects. Seahorse metabolic flux analysis reinforced the picture: cells expressing wild-type vIRF3 showed significantly increased oxygen consumption rates, including higher basal respiration, ATP production and maximal respiratory capacity, while their extracellular acidification rate, a proxy for glycolysis, declined.

To trace where the newly abundant acetyl-CoA came from, the team performed liquid chromatography–mass spectrometry metabolomics and isotope tracing with uniformly labeled carbon-13 glucose. In cells expressing wild-type vIRF3, pyruvate accumulated predominantly in the M+3 isotopologue form, a signature of enhanced PKM2 activity, and the labeled carbon flowed into M+1 and M+2 acetyl-CoA, which then fed the biosynthesis of citrate, α-ketoglutarate, succinate, fumarate and malate. Glutamate labeling patterns indicated that the cells were not relying on glutaminolysis to replenish the cycle, and measurements of fatty acid oxidation intermediates showed that this pathway was actually suppressed: carnitine accumulated while expression of carnitine palmitoyltransferase 1A and downstream β-oxidation intermediates fell. The evidence therefore pointed to enhanced pyruvate catabolism, rather than fatty acid oxidation or glutamine metabolism, as the principal source of the expanded acetyl-CoA pool.

Acetyl-CoA is not merely a fuel; it donates acetyl groups for the lysine acetylation of thousands of proteins, including transcription factors. Consistent with this, wild-type vIRF3 expression raised the global acetylation level of cellular proteins, and this increase depended on citrate exported from mitochondria and processed by ATP citrate lyase, since the ACLY inhibitor BMS-303141 abolished the effect. A human protein acetylation microarray then identified twenty-four proteins whose acetylation rose specifically in the presence of the vIRF3–PKM2 interaction. Among them, the investigators prioritized SMAD5, a transcriptional effector of TGFβ–BMP signaling with established roles in endothelial-to-mesenchymal transition, the process by which endothelial cells lose their identity and acquire invasive, mesenchymal characteristics.

Immunoprecipitation confirmed that SMAD5 acetylation was markedly increased in cells expressing wild-type vIRF3 but not the binding-defective mutant, and co-immunoprecipitation identified the acetyltransferase p300 as the enzyme responsible, since the p300 inhibitor A-485 reduced SMAD5 acetylation. The functional consequences were dramatic. Cells expressing wild-type vIRF3 adopted a spindle-shaped, mesenchymal morphology, downregulated the endothelial markers LYVE-1 and VE-cadherin, and upregulated mesenchymal markers including vimentin, fibronectin, N-cadherin and α-smooth muscle actin. Three-dimensional spheroid invasion assays in Matrigel and Boyden chamber migration assays demonstrated that these behaviors depended on SMAD5: silencing SMAD5 with short hairpin RNAs abolished both the invasive growth and the migration induced by vIRF3.

Critical controls established that this metabolic hijacking is separable from the viral life cycle itself. Using bacterial artificial chromosome-based recombination, the team constructed recombinant KSHV clones carrying either wild-type vIRF3, the Δ250-280 deletion mutant, or a revertant restoring the wild-type sequence. The mutant virus replicated normally, produced comparable infectious virions and expressed latent and lytic genes at levels indistinguishable from wild type. Yet when lymphatic endothelial cells were infected with these viruses, only wild-type and revertant infections raised pyruvate kinase activity and acetyl-CoA levels, drove spheroid invasion, and enabled colony formation in soft agar. The phenotypes were reproduced in primary human lymphatic endothelial cells, confirming that the metabolic reprogramming is a genuine consequence of KSHV infection rather than an artifact of immortalized cell lines.

The in vivo consequences were equally striking. In xenograft experiments with immunodeficient NCr-nu/nu mice, cells infected with wild-type or revertant KSHV formed tumors efficiently, whereas cells infected with the vIRF3-MT virus did not. Moreover, mice bearing wild-type-derived tumors developed secondary lesions in the stomach, spleen and liver that displayed hallmark histopathological features of Kaposi’s sarcoma, including extravasated erythrocytes, hemosiderin deposits and spindle-shaped cells positive for the viral latency antigen LANA, vIRF3, the endothelial marker CD31 and the lymphatic lineage factor PROX1. Human vimentin-positive cells at these sites traced back to the implanted infected endothelial cells. No secondary tumors appeared in mice injected with the PKM2-binding-defective virus, directly implicating the vIRF3–PKM2 interaction in disseminated visceral disease.

The study also delivers a potential therapeutic lead. Because amino acids 250 to 260 of vIRF3 were sufficient for PKM2 binding, the researchers designed a short vIRF3-derived peptide, termed VDP, fused to the HIV TAT transduction domain for intracellular delivery. VDP competed with vIRF3 for PKM2 binding, blocked vIRF3-induced PKM2 tetramerization and enzymatic activity, and lowered acetyl-CoA and ATP levels, while a binding-defective mutant peptide, mVDP, did none of these things. Structural modeling with AlphaFold-Multimer and molecular dynamics simulations showed VDP occupying the intersubunit interface of PKM2, and biolayer interferometry measured a dissociation constant of 1.28 micromolar, roughly twenty-nine-fold tighter than the mutant peptide. In chick embryo chorioallantoic membrane assays, TAT-VDP suppressed vIRF3-driven invasion into the endoderm layer, and in xenograft mice the peptide markedly inhibited tumor formation and regressed established tumors without substantial toxicity.

The authors caution that the vIRF3–PKM2–acetyl-CoA–SMAD5 axis still requires validation in patient-derived Kaposi’s sarcoma tissues, which have been difficult to obtain in adequately preserved form, and that their xenograft model does not distinguish true metastatic dissemination from other routes of systemic distribution. Even so, the work provides the most complete mechanistic account to date of how an oncogenic herpesvirus converts a host metabolic enzyme into an engine of tumor progression, linking acetyl-CoA flux through the TCA cycle to epigenetic modification of a developmental transcription factor. By demonstrating that a rationally designed viral peptide can reverse this process in living animals, the study positions PKM2 not merely as a metabolic marker but as an actionable target for treating disseminated Kaposi’s sarcoma.

Subject of Research: KSHV vIRF3–PKM2 interaction, acetyl-CoA metabolic reprogramming and SMAD5 acetylation in Kaposi's sarcoma progression

Article Title: KSHV vIRF3–PKM2 interaction induces SMAD5 acetylation, promoting Kaposi’s sarcoma progression

Article References: Kim, Y. J., Lim, J., Lee, J. J., Kumar, A., Kannappan, S., Zhang, J., Kim, S., Kim, H.-J., Kim, K. K., Toth, Z., Eoh, H., Choi, K.-C., & Lee, H.-R. (2026). KSHV vIRF3–PKM2 interaction induces SMAD5 acetylation, promoting Kaposi’s sarcoma progression. Experimental & Molecular Medicine. https://doi.org/10.1038/s12276-026-01849-2

Image Credits: AI Generated

DOI: 10.1038/s12276-026-01849-2

Keywords: KSHV, Kaposi's sarcoma, vIRF3, PKM2, acetyl-CoA, SMAD5 acetylation, lymphatic endothelial cells, endothelial-to-mesenchymal transition, metabolic reprogramming, p300, TCA cycle, therapeutic peptide

The research, led by investigators at Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, centres on kinesin family member C1, or KIFC1, a motor protein best known for hauling cargo along microtubules and for its role in centrosome clustering during cell division. By mining six publicly available transcriptomic datasets of cervical cancer, the team found that KIFC1 was among the most significantly dysregulated kinesin genes in tumour tissue compared with healthy cervical tissue. Pan-cancer analysis across more than twenty tumour types confirmed widespread overexpression, and examination of clinical databases linked elevated KIFC1 to lymph node metastasis. Crucially, the findings were not confined to computational correlations: immunohistochemical staining of a tissue microarray containing 115 cervical carcinoma specimens and 39 adjacent non-tumour tissues showed markedly higher KIFC1 protein levels in tumours, and patients whose tumours expressed high levels of the protein experienced significantly poorer disease-free and overall survival.

To probe what KIFC1 actually does inside cervical cancer cells, the researchers engineered HeLa and SiHa cell lines with stable KIFC1 knockdown or overexpression using lentiviral vectors. The functional consequences were striking. Depleting KIFC1 curtailed cell proliferation in cell counting and colony formation assays and impaired migration in wound healing and Transwell experiments, while forcing KIFC1 expression produced the opposite effects, enhancing growth and motility. But the most revealing clue came from RNA sequencing of KIFC1-silenced cells, which identified 2,714 differentially expressed genes. Pathway enrichment analysis pointed strongly toward metabolic reprogramming, with glycolysis and gluconeogenesis among the most significantly affected processes. Direct biochemical measurements confirmed the shift: glucose consumption, lactate production and intracellular ATP levels all fell when KIFC1 was silenced and rose when it was overexpressed, hallmarks of the Warburg effect that tumour cells exploit to fuel rapid proliferation.

The mechanistic thread connecting KIFC1 to metabolism runs through pyruvate kinase M, or PKM, the enzyme that catalyses the final, rate-limiting step of glycolysis. The PKM gene undergoes alternative splicing to yield two isoforms with opposing metabolic roles: PKM1 supports oxidative metabolism, whereas PKM2 favours aerobic glycolysis and is the dominant form in most tumours. When the researchers examined isoform expression after KIFC1 knockdown, they found PKM2 levels dropped while PKM1 rose, indicating that KIFC1 helps maintain the pro-glycolytic PKM2 balance that cervical cancer cells depend upon. The question was how a microtubule motor protein could influence a splicing decision buried in the cell’s gene-expression machinery.

The answer emerged from an unbiased proteomic approach. Using co-immunoprecipitation coupled with mass spectrometry, the team catalogued 1,067 proteins that physically associate with KIFC1, and among the enriched functional categories were spliceosome components and mRNA splicing processes. One candidate stood out: SRSF3, a serine/arginine-rich splicing factor previously shown to promote inclusion of PKM exon 10 and thereby tilt splicing toward PKM2. Co-immunoprecipitation experiments confirmed that KIFC1 and SRSF3 interact in cervical cancer cells, and database analysis revealed a robust correlation between the two genes in patient tumours. Intriguingly, KIFC1 knockdown did not change total SRSF3 abundance. Instead, nuclear and cytoplasmic fractionation combined with confocal immunofluorescence microscopy showed that depleting KIFC1 reduced the amount of SRSF3 in the nucleus and increased its cytoplasmic accumulation, suggesting that the motor protein acts as a shuttle that delivers the splicing factor to its site of action rather than as a regulator of its production.

Follow-up experiments cemented SRSF3 as the functional intermediary. Silencing SRSF3 in HeLa and SiHa cells suppressed proliferation, migration and all three glycolytic readouts, mirroring the effects of KIFC1 depletion. RNA immunoprecipitation demonstrated that SRSF3 binds directly to PKM messenger RNA, and its loss reproduced the isoform shift, raising PKM1 and lowering PKM2. Most persuasively, rescue experiments showed that restoring SRSF3 in KIFC1-depleted cells partially reversed the suppression of proliferation, migration, glucose consumption, lactate production and ATP generation, and re-established the PKM2-dominant profile. Taken together, these results delineate a coherent KIFC1–SRSF3–PKM axis in which a transport motor governs the subcellular localisation of a splicing factor, which in turn dictates a metabolic enzyme isoform switch that sustains the glycolytic appetite of the tumour.

What elevates this finding from an interesting piece of cell biology to a potentially transformative therapeutic insight is its connection to radiotherapy. Aerobic glycolysis is increasingly recognised as a driver of radioresistance because it supplies the energy and biosynthetic intermediates needed for DNA repair and helps tumour cells buffer the oxidative stress inflicted by radiation. When the researchers exposed KIFC1-silenced cells to graded doses of irradiation, clonogenic survival dropped significantly, particularly at 4 Gy and above, and radiation-induced apoptosis increased. Pharmacological activation of PKM2 with the selective activator TEPP-46 partially restored proliferative capacity and blunted the apoptotic response, demonstrating that the radiosensitising effect of KIFC1 loss depends on PKM2-driven glycolysis. Restoring SRSF3 produced a similar partial rescue, confirming the pathway’s role in the radiation response.

DNA damage assays provided a mechanistic window into why glycolytic disruption sensitises cells to radiation. After 4 Gy irradiation, KIFC1-depleted cells displayed elevated γ-H2AX fluorescence, a marker of DNA double-strand breaks, along with increased 53BP1 recruitment and reduced RAD51 foci, indicating impaired homologous recombination repair. Both SRSF3 restoration and TEPP-46 treatment partially reversed these changes, reinforcing the idea that a glycolysis-competent state is required for tumour cells to mend radiation-induced damage efficiently. The team then translated these findings into vivo using subcutaneous xenografts in nude mice. KIFC1 knockdown alone suppressed tumour growth, and combining it with a 6 Gy focal dose of irradiation produced significantly slower tumour growth than either intervention alone, accompanied by reduced Ki-67 staining and the same PKM isoform shift observed in culture. The authors candidly note that the in vivo effect was one of tumour growth delay rather than complete regression, a discrepancy they attribute to the complexity of the tumour microenvironment, including hypoxia, stromal interactions and the metabolic plasticity that allows tumours to compensate by activating alternative energy pathways.

The study does carry limitations that the authors themselves acknowledge. Validation in larger clinical cohorts will be needed to firmly establish KIFC1 as a prognostic and predictive biomarker, the precise splicing sites mediating SRSF3’s control of PKM in cervical cancer were not directly mapped, and the molecular determinants of the KIFC1–SRSF3 interaction, including possible adaptor proteins, remain to be identified. Nevertheless, the work expands the functional repertoire of KIFC1 well beyond mitosis, revealing a post-transcriptional mechanism by which intracellular transport machinery shapes metabolic adaptation and therapeutic resistance. If pharmacological inhibitors of this axis can be developed or repurposed, clinicians may one day combine them with radiotherapy to strip cervical tumours of the metabolic armour that currently lets them survive treatment, offering new hope for patients whose disease has stopped responding to conventional care.

Subject of Research: The role of the KIFC1–SRSF3–PKM splicing–metabolic axis in cervical cancer glycolysis and radiosensitivity

Article Title: Targeting the KIFC1‐SRSF3‐PKM Axis Suppresses Cervical Cancer Glycolysis and Enhances Radiosensitivity

Article References: Liu, J., Li, J., Zhou, H., Ha, W., Wu, X., Liu, X., Jiang, Y., Gong, C., Cheng, Y., Liu, Q., Chao, T., & Xiong, H. (2026). Targeting the KIFC 1‐ SRSF 3‐ PKM Axis Suppresses Cervical Cancer Glycolysis and Enhances Radiosensitivity. Journal of Cellular and Molecular Medicine, 30(18), Article e71373. https://doi.org/10.1111/jcmm.71373

Image Credits: AI Generated

DOI: 10.1111/jcmm.71373

Keywords: cervical cancer, KIFC1, SRSF3, PKM2, glycolysis, radioresistance, alternative splicing, Warburg effect, DNA damage repair, kinesin motor protein, radiosensitivity, tumour metabolism

Vascular calcification, the pathological deposition of calcium phosphate minerals within the walls of blood vessels, is one of the most consequential yet least tractable complications of aging, chronic kidney disease, diabetes, and atherosclerosis. Once calcium crystals begin to accumulate in the arterial media or intima, vessels stiffen, lose their elastic recoil, and become far more likely to trigger heart attacks, strokes, and sudden cardiac death. Clinicians can detect calcification readily with imaging, but they have few tools to reverse it. The new work suggests that the body may already possess a natural braking system against this process, one encoded in a stretch of DNA that no one thought produced a protein at all.

The central character in the story is pyruvate kinase M2, or PKM2, an enzyme that sits at a critical junction of glucose metabolism, catalyzing the final step of glycolysis. PKM2 has long been known to moonlight beyond its enzymatic role, participating in gene regulation and cell proliferation, and it has been repeatedly implicated in vascular disease. When vascular smooth muscle cells, the contractile workhorses of the artery wall, are stressed by high phosphate, inflammatory signals, or uremic toxins, they undergo a striking identity change: they downregulate their muscle-like program and begin acting like bone-forming osteoblasts, laying down mineral instead of maintaining vessel elasticity. PKM2 has emerged as a facilitator of this osteogenic switch, making it an attractive target for intervention.

The researchers behind the new study identified PKM2 AP as a micropeptide, a translation product of a short open reading frame that yields a peptide far too small to fold into a conventional enzyme or receptor. For decades, such sequences were considered too short to matter, and they were routinely excluded from genome annotations. That assumption has been steadily dismantled as ribosome profiling and mass spectrometry have revealed hundreds of small peptides actively produced in human cells, many of which carry out discrete regulatory tasks. PKM2 AP now joins this class with a particularly elegant mechanism of action.

According to the study, PKM2 AP functions as what structural and chemical biologists call a molecular glue. Molecular glues are small molecules or peptide-like factors that bind simultaneously to a target protein and to a component of the ubiquitin ligase system, inducing an otherwise nonexistent or fortuitous interaction. The result is that the target protein, in this case PKM2, becomes flagged with ubiquitin chains, the universal degradation tags of the cell, and is delivered to the proteasome for destruction. Rather than blocking PKM2’s activity with an inhibitor, the micropeptide simply causes the enzyme to disappear, an approach that mirrors the strategy behind a new generation of targeted protein degraders now transforming drug development.

The consequences of this degradation cascade through the calcification program. With PKM2 levels reduced, the metabolic and transcriptional signals that push vascular smooth muscle cells toward an osteoblast-like fate lose their momentum. In experimental models, the presence of PKM2 AP correlated with diminished osteogenic marker expression, reduced mineralization of the extracellular matrix, and preserved contractile phenotype of the vessel wall cells. The authors present the micropeptide as a previously unrecognized endogenous inhibitor of vascular calcification, one that operates not at the level of mineral chemistry but at the level of cell fate determination.

What makes the mechanism especially notable is its selectivity. Broadly suppressing PKM2 throughout the body would be undesirable, given the enzyme’s central role in glycolysis in virtually every tissue. A molecular glue strategy that depends on a specific ternary complex, the micropeptide bridging PKM2 and the ubiquitination machinery, offers a degree of precision that small-molecule inhibitors often lack. The interaction surfaces involved are typically more extensive than those of a conventional drug, which can translate into tighter discrimination between the intended target and lookalike proteins. This selectivity principle is precisely what has made molecular glues, from the thalidomide derivatives used in multiple myeloma to engineered degraders in the laboratory, one of the most actively pursued frontiers in pharmacology.

The study also carries implications for how scientists think about the noncoding genome. PKM2 AP arises from a locus that would appear in standard annotations as a noncoding RNA or an untranslated region. Its discovery underscores that the boundary between coding and noncoding is blurrier than textbooks suggest, and that some of the most medically relevant gene products may be hiding in regions cataloged as silent. Similar stories have unfolded in recent years with micropeptides implicated in muscle regeneration, cardiac stress responses, and cancer metabolism, and each new example strengthens the case for systematically annotating small open reading frames across the human genome.

From a translational standpoint, the path forward is demanding but clear. The researchers will need to demonstrate that PKM2 AP, or a stabilized mimic of it, can slow or reverse calcification in animal models of chronic kidney disease and atherosclerosis, and ultimately in patients whose arteries are already stiffening. Micropeptides face familiar delivery challenges: they are small enough to be degraded quickly and may not cross cell membranes without assistance. Yet the molecular glue concept offers a workaround, since a conventional small molecule that reproduces the same PKM2-ligase bridging interaction could, in principle, be developed through screening and structure-based design. If such a compound emerged, it would represent a fundamentally new class of anti-calcification therapy, one that reprograms vascular cell identity rather than chelating calcium after the fact.

For now, the discovery stands as a vivid illustration of biology’s economy: a peptide of a few dozen amino acids, encoded by a genomic footnote, holds the power to decide whether an artery stays supple or turns to stone. As the population ages and calcific vascular disease continues to drive cardiovascular mortality worldwide, the humble micropeptide may prove to be one of the more consequential molecules to emerge from the dark matter of the genome.

Subject of Research: A micropeptide that inhibits vascular calcification through molecular glue-mediated ubiquitination of PKM2

Article Title: PKM2 AP: a micropeptide inhibits vascular calcification via molecular glue-mediated ubiquitination of PKM2

Article References: Wang, M., Cheng, J., Ma, L., Yang, J., Gao, C., Fang, X., Ma, L., Fu, X., Lin, A., Ying, S., Lin, W., & Yang, Y. (2026). PKM2 AP: a micropeptide inhibits vascular calcification via molecular glue-mediated ubiquitination of PKM2. Nature Communications. https://doi.org/10.1038/s41467-026-77693-9

Image Credits: AI Generated

DOI: 10.1038/s41467-026-77693-9

Keywords: vascular calcification, micropeptide, PKM2, molecular glue, ubiquitination, protein degradation, vascular smooth muscle cells, glycolysis, noncoding genome, cardiovascular disease, proteasome, osteogenic switch