Four αI domain-containing integrins (α1β1, α2β1, α10β1, and α11β1) have evolved to recognize various members of the collagen family. A defining structural feature of these receptors is the presence of the αC helix within their αI domains (Glu284-Arg288 in the α2I domain), a structure solely found in collagen-binding integrins, whose functional mechanism has remained unclear. To elucidate the functional role of the αC helix, and to assess the contribution of the mechanistically important Arg288-Glu318 ion pair in α2I domain activation, we created two variants α2IR288A and α2IE318A. Functional solid-phase binding assays and surface plasmon resonance revealed that these variants exhibit strikingly increased avidity for collagens I and IV, whereas affinities for triple-helical GFOGER peptides, representing a single binding motif, were only slightly increased. The variants displayed distinct ligand-binding profiles, including differences in association and dissociation constants. To understand these differences at a structural level, we determined four novel X-ray crystal structures of the variants, including both ligand-free and ligand-bound (succinic acid or malic acid) states. These structures revealed differences in the folding of the αC helix region, suggesting that its conformation regulates α2I domain activation and ligand specificity, consistent with our binding assays. Additionally, the structures captured the movements of the catalytic metal ion in the metal ion dependent adhesion site, providing a detailed view of the sophisticated activation mechanism of the α2I domain.
Hepcidin negatively regulates systemic iron levels. In response to iron overload, the bone morphogenetic protein (BMP) pathway is activated, leading to stimulation of hepcidin transcription in hepatocytes. Here, we evaluated epigenetic regulation of hepcidin expression in low-hepcidin-expressing hepatoma cell lines. The CpGs were hypomethylated at the proximal and distal regions of hepcidin promoters in the liver. In contrast, the methylation of the CpGs in both regions was high in Hepa 1-6 hepatoma cells, and the CpGs in the distal region were hypermethylated in Hep-G2 hepatoma cells. Reporter assays revealed that methylation of CpGs located on the proximal BMP-response element (RE) 1 and the distal BMP-RE2, which are critical elements responsible for BMP-mediated transcription, inhibited not only basal hepcidin transcription but also transactivation of hepcidin by the BMP pathway. DNA demethylation increased hepcidin mRNA levels in a BMP pathway-dependent manner. The heterochromatinization at BMP-RE1 and BMP-RE2 on the hepcidin promoter was higher in Hep-G2 cells than in primary hepatocytes and was negatively associated with hepcidin expression. BMP6 did not modulate abundance of H3K9me2, a repressive histone mark, on the BMP-REs in Hep-G2 cells. The methylation of CpG on the BMP-RE2 was high in some human carcinomas. The present study reveals that DNA methylation and BMP-independent heterochromatinization at the BMP-RE2 are involved in the repression of hepcidin transcription in low-hepcidin-expressing hepatoma cells. In view of the high risk of hepatocarcinoma by iron overload, the recovery of hepcidin expression through epigenetic control may be a target against the onset of hepatocarcinoma.
Protein misfolding of pancreatic secretory enzymes is increasingly recognized as a contributor to chronic pancreatitis. Recently, missense pancreatic lipase (PNLIP) mutations that impair lipase secretion and cause intracellular aggregation and endoplasmic reticulum (ER) stress have been linked to the disease. Among these, A174P, G233E, and C254R are located in the N-terminal domain, whereas V454F is situated in the C-terminal domain of PNLIP. To elucidate how these mutations disrupt PNLIP structure, we performed detailed molecular dynamics simulations and structural modeling of human PNLIP variants. Evolutionary conservation analyses suggested that the mutation sites contribute to PNLIP structural stability. All mutations, regardless of location, altered the residue interaction network within the N-terminal PNLIP domain, consistently affecting Glu270. Detailed structural analysis revealed that the A174P substitution disrupted several helix-stabilizing hydrogen bonds, whereas the G233E mutation introduced multiple local hydrogen bonds and caused structural distortions. The C254R mutation abolished the native Cys254-Cys278 disulfide bond and instead formed an alternative salt bridge destabilizing the lid region. All three mutations altered stabilizing interactions mainly within the N-terminal domain of PNLIP. Interestingly, the V454F substitution exerted long-range allosteric effects, modulating key interactions also within the N-terminal domain. Further structural analysis showed that Phe454 in the variant became solvent-exposed, potentially leading to aggregate formation through the mutation site. These observations provide a molecular basis for PNLIP misfolding and its downstream proteotoxic effects, supporting the development of targeted therapies for chronic pancreatitis.
Homologous recombination (HR) is critical for error-free lesion bypass. This error-free lesion tolerance pathway is initiated by the RAD51 recombinase, which forms nucleoprotein filaments on single-stranded DNA (ssDNA) to facilitate template-directed repair using homologous sister chromatids. RAD51 filaments are tightly regulated by RAD51 mediator proteins. As such mediators, Shu complexes facilitate HR-directed DNA damage tolerance and are evolutionarily conserved from yeast to humans. The Caenorhabditis elegans (C. elegans) Shu complex is a heterotrimer consisting of three protein subunits: RFS-1, RIP-1, and SWS-1. However, the biochemical properties of this trimeric complex remain unclear. Here, we report the biochemical characterization of the C. elegans Shu complex and interactions with DNA, ATP, and RAD-51 filaments. We first revealed that the Shu trimer preferentially binds DNA with an exposed 5' end, particularly favoring a fork-shaped double-stranded DNA (dsDNA). Then, we found that the RFS-1/RIP-1/SWS-1 trimer binds to ATP and exhibits DNA-dependent ATPase activity. Through site-specific mutagenesis, we identified the catalytic residues in the RFS-1 domain and validated the ATPase activity. Using fluorescence-based assays, we further demonstrated that the Shu trimer remodels RAD-51 filaments in an ATP-hydrolysis-dependent manner and stabilizes the filaments in an ATP-binding-dependent manner. Using CRISPR-Cas9-induced disruption of the Walker motif in rfs-1, we demonstrate that the ATPase activity of the C. elegans Shu complex is critical for organismal survival under camptothecin-induced genotoxic stress. These findings provide key mechanistic insights into how the C. elegans Shu complex regulates RAD-51 filament dynamics and primes the filaments for downstream HR-mediated DNA repair processes.
IRE1α (hereafter referred to as IRE1) is one of the sensors implicated in the unfolded protein response that controls the ER protein homeostasis (also known as proteostasis). Alteration of proteostasis is observed in many diseases, making IRE1 a central element of cell adaptability upon disease onset and progression. Upon ER stress, IRE1 initially promotes cell adaptation. Conversely, when proteostasis cannot be restored, IRE1 activation can lead to cell death. IRE1 activity mainly regulates two pathways: the formation of the transcription factor XBP1s; and the regulated IRE1-dependent decay (RIDD) of RNA, which can contribute to both cell adaptation and death. Hence, on one hand, IRE1 favors gene expression, while on the other hand it induces transcript degradation. We have recently identified two genes, CD95 and UBE2D3, which are targeted by both signaling branches downstream of IRE1 RNase's activity, resulting in a dual and opposing regulation of their expression. We propose naming these targets 'DIT' for Dual IRE1 Targets. Interestingly, other IRE1 targets, such as BiP and DGAT2, have previously been reported to be regulated by XBP1s and RIDD in separate studies. We hypothesize that regulation of DIT could be crucial to tilt the balance between the pro-adaptative and pro-death outcomes of IRE1, especially in pathological contexts. Therefore, understanding this regulation could be key to unraveling the IRE1/XBP1s/RIDD signaling network. Here, we explore these hypotheses by highlighting various aspects of the regulation of IRE1 branches, and reviewing the DIT identified in the literature so far.
The free-living ciliate Tetrahymena thermophila avidly acquires exogenous sterols through phagocytosis and pinocytosis. We have previously shown that the aminosteroid U18666A, known to bind the mammalian Niemann-Pick C1 protein (NPC1), completely inhibited both uptake processes, resulting in cholesterol accumulation in phagosome-like vesicles and severe inhibition of sterol esterification. This suggests a blockage of sterol transfer between phagolysosomes and the endoplasmic reticulum (ER). The ciliate possesses a 50% similar ortholog of NPC1 (NPC, TTHERM_00672270), previously localized to the phagosomes. Since U18666A binds to other cholesterol-related proteins, an off-target effect in T. thermophila could not be ruled out. Therefore, we analyzed the role of Tt-NPC in cholesterol transfer using reverse genetics. Tt-NPC lacks the equivalent N-terminal domain of NPC1 (NTD). Using NTD as a query, a protein with 38% similarity (TTHERM_00336030) was retrieved and subsequently named NTD. Knockdown of either the Tt-NPC or Tt-NTD genes produced similar phenotypes. The cell growth was arrested after cholesterol supplementation, with cholesterol accumulating in phagosome-like vesicles. Additionally, the ER processes, sterol esterification and bioconversion, were severely impaired. Transcriptional regulation of two well-characterized sterol-responsive genes was lost in Tt-NTD and significantly delayed in Tt-NPC. This evidence suggests the presence of a functional ortholog of NPC1 in T. thermophila, composed of two peptides and a partial conservation of sterol trafficking across eukaryotic lineages.
The Neurospora crassa RVB-1 and RVB-2 proteins belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are organized in a hetero-hexameric complex essential for multiple cellular processes. Although they have been described as associated with cancer, new molecular functions have recently been described for them. Here, we investigate their participation in the fungus stress response, and our results support that they are components of the regulatory mechanism involved in the cellular stress response. Results of gene and protein expression, as well as cellular localization, revealed that their expression and location are modulated under stressing conditions, such as temperature, salt, and light. Furthermore, they are components of cytoplasmic granules induced by stress. Through immunoprecipitation assays followed by mass spectrometry, we identified many RVB-1 interactors, including components of chromatin remodeling complexes, some of which showed to be involved in stress response. New RVB-1 partners were identified, such as proteins participating in carbohydrate and cell wall metabolism, cell signaling, among other processes. One such new partner is shugoshin (SGO-1), a pericentromeric protein described as a regulator of chromosomal segregation during meiosis. Using genetic and biochemical approaches, we demonstrate the RVB-1 and SGO-1 interaction, and the structural aspects were analyzed by AlphaFold3. This interaction reveals a new RVB-1 function as a protein likely important for the maintenance of the centromeric region. Our findings confirm the relevant function of RVBs as stress sensors in N. crassa and reveal new interactors, highlighting their importance in the context of cellular metabolism.
Xyloglucan (an α-1,6-xylosyl-substituted β-1,4-glucan) is a major hemicellulose of the primary cell wall of many plants and an important growth substrate for biomass-degrading bacteria in diverse ecological niches, including the gut microbiome and hot springs. In Gram-positive bacteria, xyloglucan is deconstructed into soluble oligosaccharides in the extracytoplasmic space before import by ATP-Binding Cassette (ABC) transporters, but the structural basis for this process remains poorly understood. Here, we identified an ABC transporter for xyloglucan uptake (Athe_2052-2054) in the Gram-positive, plant biomass-degrading thermophile Anaerocellum bescii, which is conserved across the Anaerocellum genus. We solved the apo crystal structure of its extracellular substrate-binding protein (SBP), Athe_2052, revealing a unique tertiary fold found only in a small subset of SBPs that bind complex oligosaccharides. To our knowledge, Athe_2052 is the first structurally characterized ABC SBP known to recognize xyloglucan oligosaccharides. Biophysical analysis showed that while Athe_2052 binds unsubstituted β-glucan chains, recognition of xyloglucan side chains in the binding pocket markedly increases affinity (Kd = 14 nm) for xyloglucan heptasaccharide (XXXG), the principal oligosaccharide released during xyloglucan deconstruction. Molecular modeling revealed that xyloglucan heptasaccharide, owing to its branched substitutions, is bound in a distinct conformation compared to unsubstituted β-glucans. This represents a unique mode of xyloglucan recognition driven by α-linked side chain interactions rather than β-glucan backbone recognition alone. Together, these findings provide the first structural basis for xyloglucan oligosaccharide recognition by an ABC transporter in Gram-positive bacteria.
Tropomyosin (Tpm) is an actin-binding protein that, together with troponin (Tn), mediates Ca2+-regulation of cardiac muscle contraction. Tpm coiled-coil dimers bind each other through overlap junctions between their N- and C-termini, forming a continuous strand along the actin filament. Among the many TPM1 mutations identified in cardiac Tpm (Tpm1.1), few substitute canonical residues with proline, the amino acid most disruptive to coiled-coil structure. We examined properties of recombinant cardiac Tpm with L43P or L57P substitutions in both chains and compared them with wild-type (WT) Tpm using differential scanning calorimetry, viscometry, molecular dynamics (MD) simulations, and an in vitro motility assay. Both mutations markedly destabilized the N-terminal part of the Tpm molecule. In MD simulations, Pro43 and Pro57 disrupted nearby backbone hydrogen bonds, and Pro43 promoted N-terminal unfolding, demonstrating a long-range effect within supercoiled Tpm molecules. Both substitutions strongly reduce Tpm's affinity for F-actin in the absence of Tn. In addition, viscometry showed that the L43P Tpm variant polymerizes less efficiently than WT and L57P Tpm, resulting in a reduced ability to assemble into a continuous strand along an actin filament. The L57P substitution increases maximum sliding velocity of thin filaments in in vitro motility assay and enhances Ca2+ sensitivity of actin-myosin interaction, a feature commonly associated with hypertrophic cardiomyopathy. In contrast, the L43P substitution hinders the formation of fully regulatory-competent thin filaments and severely impairs the Ca2+-regulatory function of reconstructed thin filaments in vitro. Our study reveals distinct mechanisms of pathogenic effects for these two largely similar amino acid substitutions.
Proteostasis, the cellular network that governs protein synthesis, folding, trafficking, and degradation, is essential for maintaining cellular and organismal homeostasis. This review series highlights the breadth and impact of European research in the field of proteostasis, spanning fundamental mechanisms, organelle-specific quality control pathways, and emerging therapeutic opportunities. Contributions from leading laboratories across Europe examine key components of the proteostasis network, including translational regulation, molecular chaperones, ubiquitin-dependent protein degradation, organelle communication, and adaptive stress responses. Particular emphasis is placed on proteostasis mechanisms operating within the endoplasmic reticulum and mitochondria as well as on their roles in aging, inflammation, neurodegeneration, and other human diseases. The series also showcases the collaborative efforts that have strengthened the European proteostasis community through major networking initiatives and training programs. Together, these articles provide a comprehensive overview of current advances in proteostasis research and underscore its growing importance as a framework for understanding cellular adaptation and developing innovative therapeutic strategies.
17β-oestradiol (E2) is essential for ovarian development. In teleosts, the current understanding of oestrogen synthesis primarily focuses on Cyp19a, which catalyses the synthesis of E2 from testosterone (T). In contrast, the conversion of oestrone (E1)-to-E2, mediated by Hsd17b12, and the role of this process in ovarian development remain understudied. This study investigated two Hsd17b12 isoforms in the commercially cultured fish, olive flounder (Paralichthys olivaceus). In vitro ovarian assays revealed isoform-specific functions. Hsd17b12a preferentially mediated E2 biosynthesis, whereas Hsd17b12b regulated T metabolism. Subsequent detection in HEK293T cells indicated that Hsd17b12a catalyses E1-to-E2 conversion, whereas Hsd17b12b mediates T-to-androstenedione (A) conversion. Site-directed mutagenesis targeting the conserved YxxxK catalytic motif showed that an alanine-to-serine substitution in Hsd17b12a and a serine-to-threonine substitution in Hsd17b12b significantly reduced enzymatic activity. In vivo overexpression of Hsd17b12a and -12b in the flounder ovaries revealed distinct phenotypes. Hsd17b12a overexpression elevated A and E1 levels without inducing histological changes. In contrast, Hsd17b12b overexpression induced proliferation of oogonium-like cells, significantly upregulated the expression of cyp26b1 and vasa, and increased A and E2 levels. Co-immunoprecipitation assays showed an interaction between Cyp19a and Hsd17b12a, and in vitro experiments showed co-expression of Hsd17b12b and cyp19a. These findings clarify the roles of the flounder Hsd17b12a and -12b in steroidogenesis, and, for the first time, their interaction with Cyp19a in fish.
Entamoeba histolytica, a unicellular protozoan parasite, relies on phagocytosis as a key mechanism of pathogenesis. During this process, actin cytoskeleton remodeling occurs, often mediated by multiple signaling pathways, including the phosphoinositide-regulated Rho signaling cascade. EhFP10, a member of the Dbl homology GEF family, has been previously implicated in phagocytosis and pinocytosis through its interaction with EhMyosin-IB and its role in actin-myosin reorganization. In this study, we report the structural elucidation of the FP10_DH domain at 2.4 Å resolution. Despite low sequence similarity with known DH domains, FP10_DH shares significant structural similarity. Functional assays confirmed that the GDP-GTP exchange activity of FP10, mediated via Rho2, involves the DH (Dbl Homology) domain. Protein-protein interaction studies revealed that the FP10_DH domain directly interacts with Rho2. In contrast, the C-terminal region of FP10, previously known to bind the MyIB_SH3 domain, contributes to signal transmission. Furthermore, we demonstrate that Rho2 itself interacts with the MyIB_SH3 domain, forming a cross-connection. In silico studies of a dimer and tricomplex consisting of Rho2, FP10_DH, and MyIB_SH3-FP10_Cter peptide, suggesting a dynamic but functionally stable signaling module. This indicates two pathways by which FP10 might interact with MyIB: a direct mechanism via FP10_C-ter binding to the MyIB_SH3 domain, and an indirect pathway in which FP10 activates Rho2, which in turn binds to MyIB. Interactions between Rho and PAK4 suggest additional signal transduction, and PAK4 is hypothesized to phosphorylate Myosin-IB during actin--myosin reorganization. These findings propose a novel signaling pathway involving FP10 and Rho2 that facilitates myosin-mediated remodeling of the actin cytoskeleton.
Glucose catabolism in trypanosomatids differs significantly from that in most other eukaryotes. Here, the first few enzymes of the pathway are all located inside glycosomes, that is, the peroxisome-like microbodies uniquely present in the Kinetoplastida and Diplonemida of the Euglenozoa. Glycosomal AMP/ADP/ATP and NAD(H)+ pools cannot freely equilibrate with their corresponding cytosolic pools, and any ATP and NAD+ consumed within the organelles have to be regenerated inside to maintain the redox balance and glycolytic flux. Analyses of the reported end-products of both aerobic and anaerobic glucose catabolism experiments in various trypanosomatids have revealed that the ability to maintain the intra-glycosomal ATP/ADP energy balance and NAD+/NADH redox balance is essentially limited to three pathways. 1) Under aerobic conditions, glycosomal NADH is preferably reoxidized indirectly by molecular oxygen via a dihydroxyacetone phosphate/glycerol-3-phosphate shuttle which links to the mitochondrial respiratory chain, where either an alternative oxidase or a cytochrome oxidase functions as terminal oxidases. 2) Under oxygen starvation or limited activity of enzymes of the DHAP/G3P shuttle, glycosomal NADH is reoxidized by a redirection of part of the cytosolic PEP towards the glycosome, where it is reduced to succinate. 3) A succinate/fumarate/malate cycle permits an exchange of succinate and malate between the glycosome/cytosol and the mitochondrion via a mitochondrial dicarboxylate carrier, whereby succinate is oxidized to fumarate by the mitochondrial succinate dehydrogenase (complex II). In parallel, trypanosomatids have evolved various pathways to form sufficient ATP to satisfy their energy requirements. Some trypanosomatids survive true anaerobiosis through pyruvate dismutation or fermentation of propionic acid.
Despite its clinical importance, the metabolic landscape of MYCN nonamplified neuroblastoma (MYCN-NA NB) remains poorly defined. In this study, we performed integrated metabolic characterization of 22 NB samples by combining single-cell RNA sequencing (scRNA-seq) with proteomics and metabolomics. This was followed by multi-omics integration, two-way orthogonal partial least squares analysis, and biomarker selection using a random forest classifier. The scRNA-seq analysis of NB tumors revealed that, compared to the MYCN-amplified (MYCN-A) group, the MYCN-NA group exhibited higher proportions of T cells, monocytes, and neurons, while lipid metabolism pathways were specifically enriched. Proteomics identified 821 differentially expressed proteins [27 upregulated and 794 downregulated in the high-risk (HR) NB group], and functional analysis indicated that these proteins were primarily involved in biological processes such as the tricarboxylic acid cycle (TCA) and lipid metabolism. Metabolomics further detected 15 significantly altered metabolites associated with pathways such as linoleic acid metabolism. Integrated multiomics analysis identified key molecules, including RPL9 and POLR2G, with potentially critical roles in HR NB. Multi-omics correlation and network analyses revealed significant interactions between key proteins (RPL9 and POLR2G) and metabolites (D-ribose and 9-oxoODE). The biomarker combination determined by random forest and bidirectional orthogonal partial least squares modeling underscored the central role of oxidative lipid metabolism remodeling in high-risk disease progression. This study systematically elucidated the crucial role of lipid metabolic reprogramming in the pathogenesis of HR MYCN-NA NB. These findings provide critical insight for uncovering the mechanisms underlying NB progression and for identifying potential therapeutic targets.
The endoplasmic reticulum (ER) is a cellular organelle frequently subjected to stress under both physiological and pathological circumstances, associated with the accumulation of mis/unfolded proteins in its lumen. To cope with this stress, cells have evolved an adaptive program called the unfolded protein response (UPR), whose primary function is to restore ER proteostasis. When the stress is prolonged, the UPR can also trigger cell death. The UPR controls multiple machineries involved in pre-emptive quality control (QC) of proteins prior to ER entry, ribosome-associated QC, protein folding within the ER, protein degradation through various processes, and export from the ER for secretion. Because the UPR and the machineries it controls play fundamental roles in determining cell fate, they are finely regulated, including through post-translational modifications (PTMs). In this review, we focus on the role of the ubiquitin and ubiquitin-like PTMs in the regulation and mediation of ER proteostasis. We specifically focus on three core processes: the UPR, ER-associated ribosome QC and ER-associated degradation. Lastly, we briefly discuss how Ub and Ubl also control the integrated stress response and the formation of inter-organelle membrane contact sites and thus act as general regulators of responses to cellular stresses beyond ER proteotoxicity.
Mitochondrial proteotoxic stress activates the mammalian UPRmt through a multilayered mechanistic architecture rather than a linear pathway. At its core lies an import-gated sensing logic: reduced preprotein import and mito-nuclear stoichiometric imbalance activates the integrated stress response (ISR) toward the translation of ATF4, CHOP, and the mitochondria-targeted transcription factor ATF5. These factors cooperatively reprogram transcription to expand the chaperone-protease capacity while transiently reducing the nuclear-encoded OXPHOS load. Parallel translational mechanisms that include eIF2α-dependent repression, stress-granule triage, and miRNA-driven selective silencing reduce the mitochondrial precursor import and maintain proteostatic symmetry between the cytosol and mitochondria. Within the organelle, LONP1- and CLPP-dependent proteolysis, mitoribosome pausing, and tRNA-processing checkpoints further dampen nascent chain pressure. Epigenetic licensing by demethylases and acetyltransferases links metabolic and bioenergetic status to promoter accessibility at UPRmt loci. Together, these import-gated, translational, and epigenetic control layers form a coherent mechanistic circuit ensuring that mitochondrial recovery is matched to folding, assembly, and metabolic capacity. We propose a unified framework explaining how these layers cooperate to determine adaptive versus maladaptive outcomes.
Proteostasis, the maintenance of a healthy proteome, is a fundamental pillar of cellular and organismal health that declines with age. While the intracellular proteostasis network (PN) is well-characterised, proteostasis mechanisms acting in the extracellular space remain understudied. Yet, these mechanisms face unique challenges and are critical for ensuring functional systemic signalling, immune surveillance and structural integrity. In contrast to the cytosol, extracellular environments lack ATP-activated chaperones and a comprehensive ubiquitin-proteasome system and instead rely on specialised secreted chaperones, extracellular proteases and receptor-mediated clearance mechanisms. This review examines the emerging landscape of the extracellular proteostasis network (exPN) and its challenges with age. We discuss how age-related remodelling of the extracellular proteome, shifts in extracellular physicochemical properties and disrupted fluid dynamics collectively create a permissive environment for protein misfolding and aggregation. We evaluate current experimental models of extracellular protein damage and examine how exPN factors target specific stages of the aggregation process to cooperatively safeguard extracellular proteome integrity. Analysis of recent human proteomic data spanning the life course uncovers an unexpected upregulation of exPN components with age. We further explore the role of extracellular proteostasis in inflammageing, a defining hallmark of ageing. Finally, we highlight strategies that bolster extracellular proteostasis as a promising frontier for extending healthspan, limiting age-associated protein aggregation and restoring extracellular matrix homeostasis. By adopting an ageing-centred perspective, we move beyond the disease context to present a holistic overview of extracellular proteostasis in organismal health, thereby positioning the exPN as a critical yet under-exploited target for biomedical intervention.
Tumour progression is increasingly recognised as a dynamic, complex ecosystem-driven metabolic plasticity and resource utilisation within the tumour microenvironment. Among metabolic regulators, CD36, an important mediator of lipid uptake, has an emerging role in the lipid-driven metabolic adaptation of cancer. CD36 facilitates the acquisition and use of exogenous fatty acids, enabling tumour cells to maintain bioenergetic homeostasis and growth. This metabolic rewiring supports tumour progression by establishing a complex metabolic symbiosis among heterogeneous cellular populations. CD36 has a significant role in determining the fate of various cancer-associated cells including fibroblasts, adipocytes and immune cells. These tissue-driven, cell type- and environment-dependent lipid metabolic shifts allow the developing tumour to bypass metabolic stress and nutrient deprivation. While CD36-mediated alterations act as a crucial survival promoter for the 'winners' (tumour cells and immunosuppressive myeloid-derived suppressor cells or regulatory T cells), they simultaneously trigger exhaustion, lipid peroxidation and ferroptosis in the 'losers' (cytotoxic T cells and natural killer cells). Consequently, CD36 functions as an essential metabolic 'emergency exit', allowing the tumour to escape starvation and survive despite recent therapies. This review synthesises current knowledge on CD36 as a metabolic bridge linking lipid uptake, metabolic symbiosis and tumour immune ecosystem remodelling. We discuss how CD36-driven lipid metabolism integrates cellular crosstalk, nutrient competition to sustain progression in different cancers. Understanding the role of CD36 in tumour and immunometabolism in the tissue microenvironment highlights the importance of CD36 as a potential target through metabolic adaptations in shaping tumour evolution and clinical outcomes.
Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3 -) and plays an essential role in carbon fixation in marine diatoms. Here we report the structural and functional characterization of a novel CA, θ-CA3, from the diatom Phaeodactylum tricornutum, elucidating its physiological role and catalytic mechanism. AlphaFold prediction, sequence alignment, and metal analysis showed that θ-CA3 is a dimeric enzyme, with each monomer composed of two zinc-binding catalytic domains. High-resolution X-ray crystallographic structures of domain 2 of θ-CA3 in the CO2-bound form revealed the detailed substrate binding pattern in the active site. Site-directed mutagenesis showed that Asp49 and Arg117 in the active site are essential for catalysis. Notably, introducing a negative charge near the active-site entrance resulted in a mutant enzyme with markedly increased activity under acidic pH, suggesting that electrostatic modulation of the active-site environment regulates proton transfer and catalysis. Furthermore, we identified an HCO3 - ion at the dimer interface that contributes to enzyme activation. Collectively, our findings provide fundamental structural insight into how the active-site electrostatic charges and metal environment govern the catalytic efficiency of θ-CA3, offering a new perspective on the molecular basis of carbon fixation in diatoms.
Bacterial biofilms-structured communities of bacteria encased in self-produced polymeric matrices-present formidable challenges in clinical medicine. The resistance of biofilms to conventional antibiotics stems from multiple factors. These include limited drug penetration and the presence of metabolically dormant bacteria that survive treatments, despite their retaining sensitivity under standard laboratory conditions. Bacteriophages (phages), the viruses that infect and kill bacteria, have emerged as promising alternatives or adjuncts to antibiotic therapy, including against bacterial biofilms. Phages, nonetheless, likely evolved to optimize especially their dissemination between spatially separated bacteria, including spatially separated biofilms, rather than to become specialists at eradicating all targeted bacteria from biofilms. By contrast, complete bacterial elimination from the body is the standard goal of antibacterial therapies. Considered here is how an understanding of these competing goals-virion dissemination vs. complete bacterial eradication-can inform our development of phage-based anti-biofilm therapies.