AcrB is a member of the resistance-nodulation-cell division (RND) family of efflux transporters expressed in Escherichia coli, and it plays a crucial role in antimicrobial resistance. AcrB has been structurally characterized in a variety of states through observations using diverse methods, and by applying conditions that have not yet been explored, there is potential to reveal new structural features. In this study, we performed cryo-electron microscopy (cryo-EM) structural analysis of AcrB reconstituted into liposomes in the presence of the substrate doxorubicin (DOX). As a result, both the functional LTO state and the resting LLL state were observed on the same EM grid. In the LTO state, substrate binding was observed in the deep binding pocket of the T protomer, whereas the LLL state could be further classified into multiple subclasses. This study not only provides additional structural insight into AcrB but also highlights the importance of observing membrane proteins under a variety of conditions.
l-lysine 6-dehydrogenase (LysDH; EC 1.4.1.18) oxidatively deaminates the ε-amino group of l-lysine. Due to its high substrate specificity, LysDH serves as a valuable tool for l-lysine quantification. However, the molecular basis of this specificity has remained unclear because of the lack of substrate-bound structures. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of LysDH from the thermophilic bacterium Geobacillus stearothermophilus (GstLysDH) in the apo form at 2.9 Å resolution and in complex with NAD+ and l-lysine at 2.5 Å resolution. GstLysDH assembles as a tetramer, which undergoes a global conformational transition upon NAD+ binding. Structural analysis revealed that the α-carboxyl and α-amino groups of l-lysine were coordinated by oppositely charged residues, thereby orienting the ε-amino group toward the nicotinamide ring of NAD+ and anchoring the substrate in the optimal binding mode. This precise recognition mechanism accounts for the enzyme's strict specificity for the ε-amino group of l-lysine. Furthermore, comparative structural analysis with l-phenylalanine dehydrogenase suggests that the oxidative deamination in GstLysDH proceeds through a conserved hydride transfer mechanism. Together, these insights establish a structural framework for the rational design and industrial application of LysDH and related amino acid dehydrogenases.
Co-translational membrane insertion is essential for the efficient integration of mitochondrially encoded proteins into the inner mitochondrial membrane (IMM) and is critical for respiratory chain biogenesis. Mba1 is a mitochondrial ribosome-associated protein implicated in coupling mitochondrial translation with inner-membrane protein biogenesis, but its structural basis of function remains poorly understood. Here, we determined the solution structure of mature Saccharomyces cerevisiae Mba1 (mMba1) using multidimensional nuclear magnetic resonance (NMR) spectroscopy. The structure reveals a compact α + β fold with a central hydrophobic cavity and distinct charged surface regions. Ribosome titration, paramagnetic relaxation enhancement, and Cox2-derived peptide titration identified several regions of mMba1 that are affected by these different interaction conditions. Mapping these regions onto the structure reveals spatially distinct surfaces that may contribute to ribosome association, membrane proximity, and interactions with hydrophobic peptide segments. These findings provide a structural framework for interpreting previous functional studies of Mba1 and support a working model in which Mba1 may function as a peripheral adaptor at the mitoribosome-inner membrane interface. Further structural and biochemical studies will be required to establish the molecular mechanisms underlying these interactions.
Rio Kinase 1 (RioK1) is an anti-cancer target for colorectal cancer. In pursuit of selective inhibitors of RioK1, small drug-like molecules were identified using computer-aided drug design (CADD). CADD made use of a 3D crystal structure of human RioK1 bound to ADP/Mg2+ and a fragment-based computational method termed Site-Identification by Ligand Competitive Saturation (SILCS). Compounds identified via SILCS were selected based on predicted binding affinities and were experimentally confirmed to bind a RioK1 homolog from Archaeoglobus fulgidus via biophysical methods. One newly designed scaffold molecule, KPSH02, had its X-ray crystal structure determined in complex with afRioK1. The structure confirmed that KPSH02 occupies the adenine binding region seen in the Toyocamycin-afRioK1 structure, while also occupying the divalent metal-ion site observed in the hsRioK1-ADP structure. This structure thereby provides novel insights that may be exploited for the design of selective RioK1 inhibitors that may be useful in the future for targeting RioK1 in cancer.
The complex formed between Wiskott-Aldrich syndrome protein (WASP) and WASP-Interacting Protein (WIP) is a potent regulator of cytoskeletal changes in hematopoietic cells. Mutations in the WASP N-terminal domain cause the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Using NMR we determine the structure of the WASP/WIP complex and provide a first molecular view of this key biochemical junction. The central feature of this complex is the extensive binding interface formed by four WIP epitopes that wrap around the canonical EVH1 domain. Phosphoregulation of the WIP chaperone function occurs on two tyrosine residues, and not a distal serine residue as suggested earlier, and involves selective dissociation of the fourth epitope (epiIV), thereby exposing two established WASP ubiquitylation sites. Single-residue WAS-inducing mutations with mild phenotypes all influence the same WASP-epiIV interface, suggesting this is the molecular mechanism behind WAS. This structural viewpoint of WASP/WIP biology creates a much-needed molecular context for understanding hematopoietic cytoskeletal regulation in homeostasis and in WAS/XLT and is expected to be invaluable in the search for new therapeutic approaches to these rare diseases.
Solute carrier proteins (SLCs) are essential membrane protein transporters of small solutes. Among them, the SLC12 family is known to facilitate transport of ions. Members of the SLC12 family ensure cell homeostasis by co-transporting chloride alongside sodium and/or potassium across the plasma membrane. The majority of SLC12 proteins are well described, and a recent surge in structural studies facilitated by cryo-electron microscopy revealed molecular details of their function. These include multiple conformations of the transporters, covering a range of functional states and providing a window into the ion transport mechanism. Yet, only limited knowledge exists regarding their dimerization or higher order oligomerization and its role in regulation, despite SLC12 proteins consistently operating as dimers. In this review, we highlight the structural knowledgebase established in recent years and summarize the varying dimerization mechanisms. Altogether, it is becoming increasingly clear that large conformational changes in dimeric arrangements deserve attention, alongside other understudied areas like lipid interactions, nucleotide or N-terminal binding to the dimerization domains, and potential roles of the less described member SLC12A9.
DNA methyltransferase DNMT3A is a key enzyme responsible for establishing DNA methylation patterns during mammalian development. T-cell leukemia/lymphoma 1 A (TCL1A) is a proto-oncogene expressed mainly in embryonic and fetal tissues, as well as in specific lymphocyte populations. In this study, we determined the structure of the murine DNMT3A-TCL1A complex using single-particle cryo-electron microscopy. The complex adopts a linear conformation, with two TCL1A dimers bound to the catalytic domain of DNMT3A to form a heterohexamer. TCL1A competitively binds to the same structural interface on DNMT3A as DNMT3L, but produces an inhibitory-rather than an activating-effect on the catalytic activity of DNMT3A. Furthermore, comparative analysis with previously reported assembly modes of murine TCL1A revealed that the TCL1A dimer complex we resolved adopts distinct molecular conformations and interaction mechanisms. Our findings elucidate the allosteric mechanism by which murine TCL1A inhibits DNMT3A activity, providing a structural basis for understanding mammalian epigenetic reprogramming.
AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH predominantly forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AFG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function.
Encapsulins are self-assembling protein nanocompartments found in bacteria and archaea that encapsulate cargo enzymes to protect the cell from their toxic reaction products or intermediates. Developments in cryo-electron microscopy (cryo-EM) data processing strategies have enabled encapsulins and their cargo proteins to be investigated together in greater detail. In this study, we present the single particle cryo-EM structure of the Rhodospirillum rubrum encapsulin in both the presence and absence of its partner encapsulated ferritin (EncFtn). Single particle icosahedral reconstructions of empty and loaded encapsulins revealed a higher degree of conformational flexibility at the five-fold pore in the cargo loaded encapsulin. We applied a new non-point group averaging workflow to analyze the encapsulated ferritins within the encapsulin nanocompartment, to produce the first fully refined in situ atomic model of the EncFtn at 2.8 Å resolution. Masked 2D classification and particle subtraction demonstrate that cargo loading is heterogeneous in this recombinant complex, with the encapsulin able to house up to five of the decameric EncFtn complexes. Our data provides new insights into the dynamics and cargo arrangement in encapsulins and demonstrates an adaptable workflow for high resolution reconstruction of encapsulin cargoes.
Kolaviron, flaunted for its anticancer potential, has driven our exploration of its mechanism of interaction with DNA, and further shedding light on its therapeutic promise. Herein, the binding efficacy and thermodynamics of kolaflavanone, a component of kolaviron, with double-stranded calf thymus DNA (ctDNA), were investigated using a range of spectroscopic techniques and molecular docking simulations. Syllogistically, spectroscopic techniques, viscometric analysis, and thermal denaturation revealed that kolaflavanone binds to the double-stranded ctDNA minor groove via electrostatic interactions, with binding constants of 105 L·mol-1. The interaction was both enthalpically and entropically driven, with a ΔH value of -18.16 kJ/mol and a ΔS value of 38.60 J/mol/K at 25 °C. This resulted in subtle but negligible structural perturbations of the ctDNA native B-conformation. ctDNA has one binding site for the flavonoid. Molecular docking and dynamics simulation analyses confirmed that kolaflavanone binds to the minor groove of DNA with a preference for the A-T region and a binding affinity of -7.4 kcal/mol. The conjugate's stability, conferred by minor groove binding and non-intercalative interactions, suggests a promising strategy for designing DNA-targeted therapeutics with enhanced stability and tumor-specific gene regulation potential.
Electrostatic interactions between arginines and phosphates are central to numerous biological processes. Here, using an integrated approach combining mutagenesis, activity measurements, molecular dynamics (MD) simulations, and NMR, we demonstrate that arginines present in the C-terminal domain of Biotin protein ligase (BPL) are critical for biotinyl-5'-AMP formation. Using NMR-based assays and a group I BPL from Leishmania major (LmBPL), we selectively monitored the first biotinylation step, i.e., formation of biotinyl-5'-AMP from biotin and ATP. The distinct chemical shifts of ATP and AMP enabled us to quantitatively measure the amount of biotinyl-5'-AMP formed by the wild-type enzyme and a C-terminal domain deletion mutant. The mutant displayed remarkably low biotinyl-5'AMP formation compared to the wild-type enzyme. MD simulations of the apo- and ATP-bound forms of LmBPL further identified key interactions between the C-terminal domain arginines (R224, R229) and the γ-phosphate of ATP. The in silico predictions were validated by biochemical studies using R224A, R229A, and R224A/R229A mutants, which displayed remarkably lower biotinyl-5'-AMP formation compared to the wild-type enzyme. Using pyrophosphate as a γ-phosphate mimic, and 31P NMR as a probe, we demonstrate pyrophosphate binding to the wild-type LmBPL but not to the arginine mutants. Consistent with this, biotinyl-5'-AMP formation was completely inhibited by preincubation with pyrophosphate. Taken together, our findings establish a critical role for the C-terminal domain arginines in recognizing ATP phosphates during biotinylation. Extrapolating these findings to other group I and bifunctional group II BPLs, our study reveals a broadly conserved role for the C-terminal domain arginines in regulating biotinylation across the BPL family.
Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN₂ to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN₂ refill system that is compatible with gas-cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling and might be particularly beneficial in BSL-3 or BSL-4 laboratories where minimizing physical presence is essential for biosafety reasons.
Accurate determination of ligand structures in protein-ligand complexes is essential for elucidating molecular recognition mechanisms and advancing structure-based drug discovery. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful technique for determining macromolecular structures; however, reliable identification of small-molecule ligands from cryo-EM maps remains challenging, particularly in the absence of accurate initial ligand models. Here, we present MLAC (MicroED-assisted Ligand structure Analysis in Complexes), an integrative framework that combines microcrystal electron diffraction (MicroED) with cryo-EM single-particle analysis (SPA). In MLAC, high-resolution ligand structures determined by MicroED from submicrometer-sized crystals are used as initial models for fitting into cryo-EM maps of protein-ligand complexes. As a proof of concept, previously reported hERG-ligand complexes were reanalyzed. MicroED structures of representative hERG ligands-astemizole, pimozide, and E-4031-were determined at resolutions of 0.66-0.92 Å and then used for model fitting and refinement. Several quantitative metrics, including Q-score, atom inclusion, model-to-map correlation coefficients, clash analysis, and Mogul analysis, together with visual inspection, indicated that MicroED-derived ligand structures can facilitate ligand modeling for astemizole and, to a lesser extent, pimozide, whereas no clear advantage was observed for E-4031. Notably, MicroED frequently revealed structural polymorphs that provided alternative ligand conformations and helped resolve modeling ambiguities, including the chair-boat conformational variability of the piperidine ring and alternative ligand placements in the hERG-astemizole complex. Collectively, these findings support MLAC as a proof-of-concept framework that provides experimentally determined starting models to complement computational ligand-generation approaches.
Henneguya piaractus is a myxozoan parasite infecting the gills of Piaractus mesopotamicus, yet its cellular biology remains poorly understood. Here, we investigated mitochondrial organization and the occurrence of autophagy-related processes using an integrated approach combining confocal laser microscopy and transmission electron microscopy. SSU rDNA sequencing (1546 bp) confirmed species identity, showing 99.7% similarity to available H. piaractus sequences. Confocal microscopy revealed clear labeling of nuclei, polar capsules, and valves, whereas no signal indicative of mitochondrial activity was detected in mature myxospores. Ultrastructural analysis showed a plasmodium surrounded by a single membrane with numerous pinocytotic channels and mitochondria with well-developed cristae in the ectoplasmic region. Sporogenesis occurred asynchronously at the periphery, where sporoblasts and immature myxospores were observed. This region also exhibited double-membrane vesicles consistent with autophagosome-like structures, as well as phagophore-like membranes associated with damaged mitochondria. In addition, mitochondria-endoplasmic reticulum contact sites (MERCs) were identified. In contrast, centrally located mature myxospores contained mitochondria lacking cristae. Together, these findings indicate stage-dependent mitochondrial remodeling and suggest an autophagy-related process, possibly involving mitochondrial degradation. However, as these observations are based primarily on morphological evidence, the involvement of canonical autophagy pathways requires further molecular confirmation. This study provides novel insights into organelle dynamics in H. piaractus and contributes to a better understanding of cellular adaptations in myxozoan parasites.
Alkaptonuria (AKU) is an ultra-rare inherited metabolic disorder caused by impaired activity of homogentisate 1,2-dioxygenase (HGD), a Fe(II)-dependent enzyme that catalyzes the oxidative cleavage of homogentisic acid in the tyrosine degradation pathway. Although high-resolution structures of human HGD have been solved, a fundamental mechanistic question has remained unresolved: how molecular oxygen reaches the deeply buried catalytic iron required for catalysis. Here, we identify a previously unreported AKU-associated HGD variant, c.925G>A (p.G309R), and use it as a mechanistic perturbation reference to dissect the structural determinants of oxygen access. By integrating replicated classical and steered molecular dynamics simulations with transient pocket detection, tunnel mapping, O2 spatial-occupancy analysis, residue-level tunnel composition, and PCA/tICA-based dynamic validation, we identify a structurally accessible and dynamically supported O2-translocation architecture connecting the central pore of the hexameric enzyme to the non-heme Fe(II) active sites. This pathway is not intrinsic to a single subunit but emerges from a cooperative arrangement of residues contributed by three protomers, generating six symmetry-related O2-access routes per hexamer. The G309R substitution perturbs the architecture and continuity of this tunnel system, providing a mechanistic explanation for enzyme dysfunction without evidence of active-site structural perturbation or global destabilization. Together, our findings support oxygen-tunnel integrity as a previously unrecognized mechanistic requirement for human HGD activity and introduce disruption of oxygen trafficking as an additional pathogenic mode in AKU, with implications for structure-guided variant interpretation and precision-medicine strategies.
At entheses, tendons and bones are bridged by mineralized fibrocartilage, joined to tissues through dedicated interfaces. Tendons and bones are characterized by cells interconnected thanks to their underlying dense networks. Nanotubes connect tenocytes in tendons, allowing cellular crosstalk and providing biomechanical stability. Osteocytes are involved in bone mechanoresponsiveness and mineralization: they are encased into cavities and their cellular processes run through channels, forming the osteocyte lacunocanalicular network. Here, we explore the structural connectivity between fibrocartilage and bone, exploiting rat enthesis as model system and focusing on two specific regions: the Achilles tendon insertion into calcaneus and the periosteal fibrocartilage, facilitating tendon sliding. Those regions are used to characterize the impact of loading environment on tissue connectivity. Central to our approach is rhodamine staining, employed to trace connections between tissues. This information is interpreted using data on tissue microstructure, organization and composition, acquired combining high-resolution imaging methods. At the enthesis, we observe potential connections between trabecular bone marrow and mineralized fibrocartilage through a subchondral channel network perforating the interface. Direct cellular connections between bone and fibrocartilage cells are rare: canaliculi mostly stop or switch direction at the cement line. Yet, we observed a high density of canaliculi around perforating channels, which reach fibrochondrocyte lacunae. Such connections seem practically absent at the periosteal region. Our findings are preliminary but suggest that inter-tissue connectivity is required to support the enthesis load-bearing function. To understand multi-tissue biochemical cellular crosstalk, the physical infrastructure enabling this communication is also a critical feature to investigate.
Bridging scales in bone research is challenging due to bone's heterogeneities and anisotropy at different scales. High-resolution imaging is often limited to volumes too small to reveal its complete hierarchical structure, a limitation that is further compounded by site-specific variability. Although bone multiscale structural organization has been extensively studied in humans, extrapolating these findings across species remains difficult. The study of bone pathologies or dynamic processes still relies on in vivo models, as these experiments cannot be performed on humans. In particular, rabbit bone represents a common model for bone studies but remains underexplored, especially at small scales. In this work, we investigate bone hierarchical structure and porosities in the rabbit across scales by combining 3D volumetric global (voxel size ∼ 15 μm) and local (voxel size ∼ 2 μm) micro-computed X-ray tomography with high-resolution 2D scanning electron microscopy (pixel size < 0.5 μm) and transmission electron microscopy (pixel size < 50 nm). 2D images were integrated into the spatial context of the 3D data through image registration, to highlight site-specific differences between endosteal, mid-cortex and periosteal regions. Osteocyte lacunae 2D morphology varied between primary osteons, secondary osteons and periosteal bone. These findings motivate further three-dimensional characterization of osteocyte lacunar morphology across osteon types and in larger cohorts, to clarify the role of osteocytes in bone remodeling. More broadly, our work highlights the potential of sequential multimodal workflows in giving a broader spatial context to sub-micron and nanoscale structural findings, reducing the "blindness" compromise that comes with high-resolution detail.
Enzymatic degradation of plastics has been extensively investigated, but its applications have remained limited due to the low stability and efficiency of enzymes in diverse environmental conditions. The present study elucidates the structural and functional characteristics of the thermostable EstS1 Esterase from Sulfobacillus acidophilus DSM10332 in the degradation of bis(2-hydroxyethyl) terephthalate (BHET), the primary intermediate of PET degradation. The co-crystal structure of wild-type EstS1 with BHET revealed binding of BHET and its degradation products, mono(2-hydroxyethyl) terephthalate (MHET), and ethylene glycol in the active site tunnel, with MHET interacting with the catalytic triad. The structure of the EstS1 Ser154Ala mutant with bound substrate showed two BHET molecules, of which one interacted with the mutated catalytic triad and the oxyanion hole, and the other was positioned in front of the first towards cavity 2. Further, structural analysis suggested that the hydrophobic nature of cavity 1, formed by the cap domain, plays a critical role in substrate binding, orientation, and catalysis. Kinetic analyses demonstrated that EstS1 degraded 75% of BHET within 1 h, producing MHET and terephthalate as end products. These findings indicate the remarkable ability of EstS1 to consecutively cleave two ester bonds. Molecular dynamics (MD) simulation revealed highly stable interactions between BHET and the active site of EstS1 throughout the 1 μs trajectory. Overall, this study provides structural insights into the EstS1-BHET interaction mechanism and demonstrates the potential of EstS1 esterase to directly convert BHET into terephthalate. These findings establish a strong foundation for future enzyme engineering efforts aimed at developing efficient PET plastic degradation technologies.
The stomatopod eye is a fascinating biological system capable of detecting both colour and polarization of light, making it a highly complex, mixed-tissue sample. In the investigation of complex biological systems, three-dimensional methods spanning multiple length scales with the power to resolve soft tissues are required. In this study, propagation-based phase contrast X-ray computed tomography with stitching at a 4th generation synchrotron was used to image a full stomatopod eye with sub-micron voxel size to illustrate how this method accommodates these demands. The images are based on natural X-ray contrast and without any added labels or staining agents. Key features of the eye were identified and segmented. Utilizing these segmentations, photo filter volumes, chitin porosity volumes, and muscle fiber periodicities were measured, demonstrating the ability to perform quantitative as well as qualitative investigations. Neural compartments and associated cells were discernable, showing the power of 4th generation synchrotron phase contrast for the study of soft tissues. The illustrated properties along with its non-invasive nature proves phase contrast synchrotron X-ray computed tomography with stitching to be a powerful tool for the investigation of biological materials.
Efficient resolution of neuroinflammation and debris clearance are key determinants of successful central nervous system (CNS) regeneration. Regenerative vertebrates such as Danio rerio often show faster immune resolution and debris clearance than mammals, yet the molecular determinants underlying these differences remain incompletely understood. TAM receptor tyrosine kinases (Tyro3, Axl, and Mertk) and their ligands Gas6 and Protein S are central regulators of phagocytosis and immune resolution in the nervous system, but whether intrinsic structural properties of these receptor-ligand complexes may modulate TAM signaling across species with distinct regenerative capacities has not been systematically explored. Here, I perform a comparative in silico analysis of TAM receptors and ligands from zebrafish, human, and mouse, integrating sequence evolution, high-confidence structural modeling, interface characterization, and electrostatic analysis. Despite substantial sequence divergence between mammals and zebrafish, ligand-binding domains retain strong structural conservation, supporting a conserved global mode of TAM-ligand engagement. At the interface level, zebrafish complexes exhibit enhanced electrostatic contributions and increased salt-bridge density, particularly in the Tyro3-Protein S interaction. Residue-resolved electrostatic analysis identifies clustered interface hotspots that are conserved in spatial organization and physicochemical function across species, despite evolutionary rewiring of individual contacts. Together, these findings suggest that TAM receptor-ligand interfaces are evolutionarily tuned through subtle electrostatic and geometric variation rather than large-scale structural changes. This conserved yet adaptable electrostatic framework supports the hypothesis that interface-level features may modulate TAM receptor-ligand engagement across vertebrates. However, these structural and electrostatic properties should be interpreted as potential modulatory contributions to TAM signaling rather than definitive determinants of regenerative capacity.
