Dependence receptors are distinctive type of receptors that induce a cellular state of dependence towards ligand availability. UNC5H receptors are notable examples of these receptors. They promote cell survival upon binding to their ligands, including netrin-1, whereas the absence of the ligand induces apoptosis. Unbound UNC5B promotes apoptosis through death-associated protein kinase (DAPK), while cell survival requires ligand binding. Previous studies have also shown the requirement of protein phosphatase 2A (PP2A) for this UNC5B-mediated apoptosis. Netrin-1 and its receptors are involved in nervous development, angiogenesis, and homeostatic maintenance of intestinal epithelium. UNC5H receptors are often down-regulated in various tumors, whereas several cancers have up-regulated netrin-1 expression, and both these conditions promote tumor cell survival. Interestingly, the interaction between netrin-1 and its receptors has been targeted in cancers to promote cancer cell death, reverse epithelial-to-mesenchymal transition, and inhibit metastasis and anti-cancer drug resistance. Here we discuss the mechanisms of signaling through UNC5H receptors and their role in normal cellular physiology and carcinogenesis. Furthermore, in the present review, we highlight the strategies that are currently being tested to target the netrin-UNC5H pathway for the treatment of cancer.

E2 conjugating enzymes function in the ubiquitin-mediated proteolysis pathway by accepting ubiquitin (Ub) from an E1 enzyme (Uba1), forming an E2~Ub thioester conjugate, and transferring Ub to a substrate or to an E3 ligase enzyme. Fluorescent or affinity tags are often used as sensitive readouts for catalytic variability and specificity of E2 enzymes and their variants. Here we examined how the size or placement of fluorescent tags compromise Ub conjugation for the E2 enzymes UbcH7, UbcH5a and Ubc13. Large green fluorescent protein (GFP) based fluorophores were placed at the N- or C-terminus of the E2 enzyme UbcH7 and the N-terminus of Ub, and compared to Alexa-based dyes, approximately 30 times smaller, at the same positions. While all modified proteins formed E2~Ub conjugates, placing a smaller Alexa dye at the C-terminus of the E2 enzyme improves the initial rate of E2~Ub conjugate formation by up to 10-fold compared to a larger GFP-based tag (YPet) at the N-terminus. In the case of UbcH5a, YPet labelling at its N-terminus significantly slowed formation of an E2~Ub conjugate and Ub unloading to the HECT domain of HUWE1 when compared to UbcH7. Overall, we find that small Alexa dye-based tags, especially when placed at the C-terminus of E2 enzymes, are an optimal choice for monitoring E2 ubiquitination using fluorescence-based experiments.

TYRO3, AXL, and MERTK, the TAM receptor tyrosine kinases (RTKs), play roles in phagocytosis and negative regulation of immune responses, contributing to homeostasis in many adult tissues. Aberrant TAM signaling leads to diseases including autoimmune disorders, viral infection, inflammation and cancer. Development of successful drugs targeting TAM receptors is hindered by incomplete knowledge of the receptor activation mechanism. Receptor dimerization is assumed to play a role, as seen for most RTKs. However, full biological responses require the TAM ligands to engage phosphatidylserine in an opposing cell or virus membrane, suggesting a more complex mechanism, possibly involving higher-order receptor oligomerization or clustering. Here, we present an X ray crystal structure of the ectodomain of MERTK, which adopts a highly curved conformation with key interdomain boundaries stabilized by N-linked glycan moieties. We show that this MERTK extracellular region (ECR) forms a similar conformation in solution using small-angle X ray scattering (SAXS), which also suggests a flexible ECR. We compare these crystallographic and solution data for MERTK with SAXS and AlphaFold structural predictions for the other two TAM receptors. SAXS data confirm that each TAM ECR has a distinct curvature, with TYRO3 the most bent and AXL relatively straight. This leads us to speculate that these different ectodomain conformations guide receptor-specific functions. The MERTK X-ray crystal structure also suggests a head-to-tail intermolecular interaction between the Ig2 domain of one MERTK and the FNIIIb domain of a second that has intriguing parallels with interactions that have been proposed to regulate Eph receptor clustering.

The cysteine-mediated catalytic reactions are essential for redox homeostasis in photosynthetic organisms. In the genome of cyanobacterium Anabaena PCC 7120 (Anabaena), the genes encoding thioredoxin reductase (TR; alr2204) and the atypical thioredoxin (Trx; alr2205) are contiguously located, but whether these proteins function as a redox pair remains unknown. Interestingly, Alr2204 and Alr2205, respectively, show high homology to the TR and Trx domain of the chloroplast NADPH-Thioredoxin Reductase C (NTRC). To delve into its possible role, Alr2204 was overexpressed in Anabaena. The resulting strain displayed elevated TR activity and demonstrated enhanced resistance to oxidative stresses. Biochemical characterization showed Alr2204 to be a FAD-associated, predominantly alpha helical protein with NADPH-dependent TR activity. Notably, Alr2204 supported the function of thioredoxin Alr2205, indicating that Alr2204-Alr2205 formed a cognate redox pair wherein Alr2204 donated electrons to Alr2205. When co-expressed in Anabaena, Alr2204 copurified with Alr2205, and together these proteins enhanced the cellular levels of the reduced form of 2-Cys-Prx, a physiologically vital anti-oxidant protein. Importantly, Alr2204-Alr2205 also sustained peroxidase activity of 2-Cys-Prx. This is the first study to show that Alr2204-Alr2205, though independently synthesized, are functionally analogous to the NTRC protein, and thioredoxin systems can be modulated to enhance stress resistance in organisms.

As one of the most abundant and diverse post-translational modifications in biology, glycosylation has a significant influence on the functions of proteins in health and disease. To untangle the complexity of glycan biosynthetic pathways, the process of studying glycosylation has been expedited by specialised, chemical labelling techniques. In the present review, we explore the development of metabolic glycan labelling from early biochemical methods to current approaches that use interdisciplinary techniques. We highlight work that has informed our understanding of glycan biosynthesis through pioneering chemical biology approaches. Moreover, we provide guidance for experimental design that can be tailored to the specific needs of those investigating glycosylation in the context of their own work. Our 'Design Rules' consolidate the findings of several groups in a simple roadmap to make metabolic glycan labelling as effective as possible.

Linear, nonreducing terminal a-galactopyranosyl (a-Gal) epitopes are displayed on the major surface glycosylphosphatidylinositol (GPI)-anchored glycoconjugates of infective Trypanosoma cruzi trypomastigotes, the agent of Chagas disease (CD). These immunodominant epitopes elicit abundant (3-5% of circulating IgG), trypanolytic CD anti-a-Gal antibodies, yet their role on the parasite surface remains elusive. Here, affinity purification of macrophage extracts on the immobilized Galα1-3Galβ1-4GlcNAcβ (Galili's aGal epitope) identified Galectin-3 (Gal-3) and the annexin A2-S100A10 heterotetramer (A2t) as candidate host-cell receptors, whose specificities were mapped against glycoconjugates and parasite-derived molecules. Gal-3 bound a-Gal-bearing neoglycoproteins (α-Gal-NGPs), the glycosphingolipid (GSL) iGb3 (Galα1-3Galβ1-4Glcβ1-1-Cer), trypomastigote-derived GPI-mucins (tGPI-mucins), and glycoinositolphospholipids (tGIPLs). α-Galactosidase abolished Gal-3 binding to trypomastigotes; epimastigote binding was reduced only by β-galactosidase. Conversely, A2t bound galactosylated GSLs in calcium-dependent manner, most strongly Gb4 (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1-Cer), but not α-Gal-NGPs, and only two of 442 glycans on a printed array gave signals above background, indicating lipid-dependent recognition. A2t recognized the phospholipid-enriched fractions of both developmental stages equally, the tGIPL fraction of trypomastigotes only, and not tGPI-mucins. Antibody blockade of surface Anx2, S100A10, or Gal-3, competition with the soluble proteins, and RNA interference reduced macrophage infection by 29 to 50%, about 53%, and 42 to 59%, respectively. Terminal a-Gal epitopes on major trypomastigote GPI-anchored glycoconjugates therefore contribute to host-cell invasion, chiefly through Gal-3, whereas A2t engages a broader set of lipid-associated ligands; both contribute to a redundant entry repertoire rather than acting as obligate receptors. These interactions may offer targets for vaccine and chemotherapeutic development and serve as diagnostic and prognostic biomarkers in CD.

Hypoxia-inducible factors (HIFs) are key regulators of cellular responses to low oxygen (hypoxia), controlling the expression of genes required for survival and adaptation. KDM2B, a chromatin-modifying enzyme, is a direct target of HIF-1α, but its precise role in regulating HIF and the hypoxia response remains unclear. Here, we investigated the role of KDM2B in the response to hypoxia in a variety of cell lines. Our analysis reveals that KDM2B depletion regulates HIF activity in a cell type-dependent manner, with KDM2B depletion decreasing HIF activity in U2OS and MDA-MB-231 cells and increasing HIF activity in HeLa cells. We show that KDM2B depletion also reduces HIF-1α protein and RNA expression and reduces HIF-1α binding at hypoxia-response elements of its target genes in U2OS and MDA-MB-231 cells. Conversely, overexpression of KDM2B enhances HIF activity and HIF-1α levels in both U2OS and HEK293 cells. Mechanistically, we find that KDM2B requires its jumonji-C demethylase and CxxC DNA-binding domains for HIF regulation. Furthermore, we demonstrate that KDM2B is required for RNA Pol II recruitment to the promoter of HIF1A. At the cellular level, KDM2B supports cell proliferation, with its depletion impairing proliferation and reducing cell numbers under hypoxic conditions. Our work highlights a new function of KDM2B as a key regulator of HIF-1α expression, acting through its demethylase and DNA-binding functions. Our data indicate that KDM2B is essential for cellular adaptation to hypoxia, impacting both HIF-dependent gene expression and cell survival, and has important implications for our understanding of HIF regulation.

For more than 50 years, the linear sequence and the multiple sequence alignment have been the foundational data structures of protein science, and they remain central to homology search, phylogenetic inference, covariance-based contact prediction, and modern protein language models. However, relational and graph-based representations are increasingly being adopted alongside sequence-based methods to capture biological relationships that linear data structures express only implicitly. Proteins fold as three-dimensional residue interaction networks, evolve through high-dimensional genotype networks defined by mutational connectivity, and operate within cellular protein-protein interaction graphs. Here, we review how graph theory is being used to describe and understand these relationships across protein science, with an emphasis on what these methods offer biochemists working on enzyme superfamilies, protein engineering, drug targets, and functional annotation. We trace the development of these ideas from early theoretical topologies, through statistical coupling and the structural network analyses, to the geometric and graph-like representations used in recent machine-learning-driven advances. Throughout, we emphasise that graphs do not replace sequences or MSAs but provide a complementary representation for biochemical relationships that are difficult to express in one dimension.

Biomolecular condensates are membrane-less compartments formed through phase separation that concentrate and organize biomolecules within cells. Far from being static droplets, they are dynamic molecular assemblies whose internal dynamics, structure, mechanics, and composition can evolve over time during cellular aging, disease-associated aggregation, or engineered material formation. These changes are often described using state descriptors such as liquid-like, gel-like, aged, or solid-like, yet similar apparent states can be interpreted differently depending on whether they are assessed in terms of morphology, dynamics, structure, mechanical response, or composition. The present review article summarizes an experimental framework used to probe biomolecular condensates and their transitions across complementary dimensions while minimizing perturbation to the system. A diverse suite of label-free optical, scattering, spectroscopic, mechanical, and microfluidic approaches is discussed, highlighting how integrated strategies can connect these readouts to enable more rigorous interpretations of condensate behavior. This framework is intended to guide researchers to move beyond broad material descriptors toward understanding the mechanisms underlying condensate evolution, the resolution of phase transitions, and their implications for biological function, pathology, and biomaterial design.

N 6-Methyladenosine (m6A) is a prevalent RNA modification that regulates multiple aspects of RNA metabolism, including RNA localization, stability, decay, and translation. m6A deposition is catalyzed by distinct methyltransferase complexes, including the METTL3/14 complex and METTL16, which recognize different RNA sequence motifs. The biological effects of m6A are mediated by effector proteins that selectively recognize m6A-modified RNA. To identify previously uncharacterized m6A-binding proteins, we developed an RNA-binding protein domain array to systematically screen for candidate m6A effectors. Using this approach, we identified the spliceosomal protein SF3B4 as a potential m6A reader. RNA pulldown assays using m6A-modified RNA probes demonstrated selective enrichment of endogenous SF3B4, supporting its ability to recognize m6A-modified RNA. To define the RNA targets of SF3B4, we performed SF3B4 RIP-seq alongside m6A RIP-seq, followed by RIP-qPCR validation of overlapping targets. Motif analysis revealed that SF3B4 preferentially associates with the conserved GRAGRA (R = A/G) motif, consistent with the RNA sequence recognized by the METTL16 methyltransferase. Notably, transcripts of the BCR and MET oncogenes were identified as shared targets of SF3B4 and METTL16. Together, these findings identify SF3B4 as a previously unrecognized m6A effector and suggest that it participates in RNA metabolic processes downstream of METTL16-mediated m6A modification.

The cyclin-dependent kinases (CDKs) regulate cell cycle progression subject to strict control by activating cyclin subunits and by site-specific phosphorylation on the activation loop. These mechanisms are highly conserved across eukaryotic organisms. The non-canonical CDK activator Speedy A (SPY1) is unrelated in sequence to the cyclins and activates CDK2 in G1/S phase in somatic cells and promotes meiosis in germ cells without requiring activation-loop phosphorylation of CDK2 on T160. This has been attributed to a single aspartic acid residue of SPY1 (D136) that mimics T160 phosphorylation by forming similar salt-bridging interactions that stabilize the active conformation of the SPY1-CDK2 complex. Here, we show that SPY1 activates both phosphorylated and unphosphorylated CDK2 with similar efficacy to its canonical activator CyclinA. FRET experiments show that SPY1 binding causes a similar magnitude of CDK2 conformational shift to that triggered by CyclinA binding to phosphorylated CDK2, but regardless of the kinase phosphorylation state. SPY1 binding allosterically enhances binding of ATP-competitive CDK2 inhibitors to a similar degree as CyclinA, resulting in similarly high-affinity inhibitor binding and inhibition. Remarkably, we found that the D136A mutant of SPY1, while inactive towards unphosphorylated CDK2, can still activate phosphorylated CDK2 as effectively as WT SPY1, demonstrating functional non-equivalence of its two activation mechanisms.

Bisecting GlcNAc, a central branch in N-glycans synthesized by the N-acetylglucosaminyltransferase III (MGAT3; also known as GnT-III), is highly expressed in neurons. Although pathological roles of MGAT3 and bisecting GlcNAc have been demonstrated in Alzheimer's disease, their physiological functions in neurons remain unclear. To identify their physiological functions, here we examine the morphology of neurons in knockout (Mgat3-/-) mouse brains and investigate the expression and localization of bisecting GlcNAc-bearing glycoproteins involved in neuronal activity and morphogenesis. We found impaired neurite extension and spine maturation in cultured cortical neurons of Mgat3-/- mice, along with reduced stimulus-induced in vivo neuronal activity. Moreover, key glycoproteins for these processes (specifically α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid [AMPA]-type glutamate receptors) showed reduced accumulation in postsynaptic density membrane fractions and a weakened interaction with transmembrane AMPA receptor regulatory protein 8 in Mgat3-/- mouse brain. These results suggest that bisecting GlcNAc plays important roles in synaptic maturation, highlighting a new mechanism for neuronal activity regulated by specific N-glycans.

Stress granules are transient biomolecular condensates of proteins and RNA formed during stress. Stress granules support cell survival by sequestering components of the translational machinery, suppressing pro-apoptotic signaling, and conserving cellular resources. O-GlcNAcylation, a dynamic modification of cytoplasmic proteins, is an important regulator of stress responses, including stress-granule assembly. O-GlcNAcylation is protective in acute myocardial stress; however, whether O-GlcNAcylation regulates stress granules to protect cardiomyocytes remains unclear. Here, we investigated whether O-GlcNAcylation regulates stress granule formation in cardiomyocytes exposed to sodium arsenite stress or ischemia/reperfusion (I/R) injury. G3BP1 is an RNA-binding protein that is essential in stress granule formation. Using an EGFP-G3BP1 reporter, we found that OGT, the enzyme responsible for installing O-GlcNAc on target proteins, is necessary for efficient stress-granule formation in cardiomyocytes. Importantly, we identified T268 of G3BP1 as an O-GlcNAcylation site in these cells and demonstrated by mutagenesis its functional importance for stress granule assembly. In primary cardiomyocytes subjected to simulated I/R, stress granules formed transiently during ischemia, and this response was decreased by OGT-knockdown. Similarly, stress granules were transiently detected at the ischemic phase of I/R injury, and their numbers were reduced in OGT-deficient hearts. Furthermore, pretreatment with sodium arsenite before I/R was cardioprotective. Likewise, pretreatment with sodium arsenite protected cells from staurosporine-induced death, and this effect was decreased by OGT knockdown. Together, these findings identify OGT and O-GlcNAcylation as important regulators of stress-granule assembly in cardiomyocytes, define G3BP1 as an O-GlcNAc-modified effector, and demonstrate that stress granules can act as cytoprotective mediators during acute cardiac injury.

Flavonoids are plant-derived natural compounds belonging to the large group of polyphenols. These molecules have a wide range of applications in the pharmaceutical, nutraceutical, and cosmetic industries due to their well-documented antioxidant, anti-inflammatory, and antimicrobial properties. However, their extraction from plant sources is limited by low yields, seasonal variability, and complex purification processes, making large-scale production challenging. Chemical synthesis is often not a viable alternative because of high production costs and the environmental impact associated with the use of toxic reagents and solvents. Consequently, microbial bioproduction through metabolic engineering has emerged as a sustainable and efficient alternative. In this context, Escherichia coli has become a leading microbial chassis for flavonoid production. The present review summarizes the advances in engineering E. coli for the de novo heterologous biosynthesis of flavonoids, including de novo production of flavanones, flavones, flavonols, prenylflavonoids, glycosylated flavonoids, and methylated flavonoids. Furthermore, the present review discusses the major metabolic and enzymatic bottlenecks that currently limit industrial-scale de novo flavonoid production. These challenges include insufficient precursor supply, suboptimal performance of plant-derived enzymes, cofactor imbalance, accumulation of toxic intermediates, and product toxicity. Strategies to overcome these limitations and enhance flavonoid titers, yields, and structural diversity are also highlighted. By integrating these approaches, E. coli emerges as a robust, sustainable, and economically competitive platform for large-scale flavonoid production.