The FEBS journal最新文献

N-terminal nonsynonymous single-nucleotide polymorphisms (SNPs) of G protein-coupled receptors (GPCRs) are common and often affect receptor post-translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human β₁-adrenergic receptor (β₁AR) is O-glycosylated in the N-terminal extracellular domain by polypeptide GalNAc transferase-2 that co-regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O-glycosylation assays, analysis of native receptor N-terminal O-glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O-glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O-glycosites that enhanced or inhibited cleavage at the adjacent sites (P⁵²↓L⁵³ and R³¹↓L³²), respectively, or abolished the major site at R³¹↓L³² (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full-length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol-mediated signaling in an enhanced bystander bioluminescence energy transfer β-arrestin2 recruitment assay in a coordinated manner with the common C-terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a β₁AR gain-of-function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.

Amino acids are important for cellular metabolism. Their uptake across the plasma membrane is mediated by transport proteins. Despite the fact that the organic anion transporting polypeptide 4C1 (OATP4C1, Uniprot: Q6ZQN7) mediates transport of l-arginine and l-arginine derivatives, other members of the OATP family have not been characterized as amino acid transporters. The OATP family member OATP3A1 (gene symbol SLCO3A1, Uniprot: Q9UIG8) is ubiquitously expressed in human cells and highly expressed in many cancer tissues and cell lines. However, only a few substrates are known for OATP3A1. Accordingly, knowledge about its biological relevance is restricted. Our aim was to identify new substrates of OATP3A1 to gain insights into its (patho-)physiological function. In an LC-MS-based untargeted metabolomics assay using untreated OATP3A1-overexpressing HEK293 cells and control cells, we identified several amino acids as potential substrates of OATP3A1. Subsequent uptake experiments using exogenously added substrates revealed OATP3A1-mediated transport of l-tryptophan, l-tyrosine, and l-phenylalanine with 194.8 ± 28.7% (P < 0.05), 226.2 ± 18.7% (P < 0.001), and 235.2 ± 13.5% (P < 0.001), respectively, in OATP3A1-overexpressing cells compared to control cells. Furthermore, kinetic transport parameters (K_m values) were determined (Trp = 61.5 ± 14.2 μm, Tyr = 220.8 ± 54.5 μm, Phe = 234.7 ± 20.6 μm). In summary, we identified the amino acids l-tryptophan, l-tyrosine, and l-phenylalanine as new substrates of OATP3A1. These findings could be used for a better understanding of (patho-)physiological processes involving increased demand of amino acids, where OATP3A1 should be considered as an important uptake transporter of l-tryptophan, l-tyrosine, and l-phenylalanine.

Anti-immunocomplex (Anti-IC) antibodies have been used in developing noncompetitive immunoassays for detecting small molecule analytics (haptens). These antibodies bind specifically to the primary antibody in complex with hapten. Although several anti-IC antibody-based immunoassays have been developed, structural studies of these systems are very limited. In this study, we determined the crystal structures of anti-testosterone Fab220 in complex with testosterone and the corresponding anti-IC antibody FabB12. The structure of the ternary complex of testosterone, Fab220, and FabB12 was predicted using LightDock and AlphaFold. The ternary complex has a large (~ 1100 Ų) interface between antibodies. The A-ring of the testosterone bound by Fab220 also participates in the binding of the anti-IC antibody. The structural analysis was complemented by native mass spectrometry. The affinities for testosterone (TES) and three cross-reactive steroids [dihydrotestosterone (DHT), androstenedione (A4), and dehydroepiandrosterone sulfate (DHEA-S)] were measured, and ternary complex formation was studied. The results clearly show the ternary complex formation in the solution. Although DHT showed significant cross-reactivity, A4 and DHEA-S exhibited minor cross-reactivity.

Because of the association with other complex polysaccharides, extracting and utilizing cellulose from lignocellulosic materials requires the combined action of a broad range of carbohydrate-active enzymes, including multiple glycoside hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs). The interplay between these enzymes and the way in which Nature orchestrates their co-existence and combined action are topics of great scientific and industrial interest. To gain more insight into these issues, we have studied the lignocellulose-degrading abilities of an enzyme from Caldibacillus cellulovorans (CcLPMO10-Man5), comprising an LPMO domain, a GH5 mannanase domain and two family 3 carbohydrate-binding modules (CBM3). Using a natural softwood substrate, we show that this enzyme promotes cellulase activity, i.e., saccharification of cellulose, both by removing mannan covering the cellulose and by oxidatively breaking up the cellulose structure. Synergy with CcLPMO10-Man5 was most pronounced for two tested cellobiohydrolases, whereas effects were smaller for a tested endoglucanase, which is in line with the notion that cellobiohydrolases and LPMOs attack the same crystalline regions of the cellulose, whereas endoglucanases attack semi-crystalline and amorphous regions. Importantly, the LPMO domain of CcLPMO10-Man5 is incapable of accessing the softwood cellulose in absence of the mannanase domain. Considering that LPMOs not bound to a substrate are sensitive to autocatalytic inactivation, this intramolecular synergy provides a perfect rationale for the evolution of modular enzymes such as CcLPMO10-Man5. The intramolecular coupling of the LPMO with a mannanase and two CBMs ensures that the LPMO is directed to areas where mannans are removed and cellulose thus becomes available.

Protein phosphatase 2A (PP2A), one of the most abundant protein phosphatases, has divergent functions in multiple types of cells. Its inactivation has been closely associated with leukemia diseases. However, the physiological function of PP2A for hematopoiesis has been poorly understood in organisms. Drosophila hematopoiesis parallels the vertebrate counterpart in developmental and functional features but involves a much simpler hematopoietic system. Here, utilizing the Drosophila major larval hematopoietic organ lymph gland, we studied the function of PP2A for hematopoiesis in vivo. By knocking down the expression of Pp2A-29B that encodes the scaffold subunit of the PP2A holoenzyme complex, we found that PP2A silencing in the differentiating hemocytes resulted in their excessive proliferation. Furthermore, this PP2A inhibition downregulated the expression of Smoothened (Smo), a crucial component in the Hedgehog pathway, and smo overexpression was able to rescue the phenotypes of PP2A depletion, indicating that Smo functions as a downstream effector of PP2A to restrict the hemocyte proliferation. PDGF/VEGF-receptor (Pvr) overexpression also restored the Smo expression and lymph gland morphology of PP2A silencing, suggesting a PP2A-Pvr-Smo axis to regulate lymph gland growth and hemocyte proliferation. Moreover, inhibiting PP2A activity in the blood progenitor cells promoted their differentiation, but which was independent with Smo. Together, our data suggested that PP2A plays a dual role in the Drosophila lymph gland by preserving the progenitor population and restraining the hemocyte proliferation, to properly regulate the hematopoietic process.

Cellular senescence is described as an irreversible cell cycle arrest for proliferating cells and is associated with the secretion of senescence associated secretory phenotype factors. It has been known to accumulate with age and is regarded as a key driver of aging-associated skin pathologies. However, the lack of markers of skin senescence and partially understood skin cellular senescence mechanisms has limited the exploration of skin aging and anti-skin aging strategies. Recently, intracellular calcium signaling has emerged as an important regulator of cellular senescence and aging. However, little is known about the modulation of skin cellular senescence by calcium-associated factors. Here, we found that the expression of calcium channel transient receptor potential melastatin 7 (TRPM7) is elevated during skin keratinocyte senescence and aging. Importantly, TRPM7 promotes skin keratinocyte senescence by triggering intracellular calcium transfer from the endoplasmic reticulum to the mitochondria; accumulation of mitochondrial calcium then induces a drop in mitochondrial membrane potential and reactive oxygen species production, leading to subsequent nuclear enlargement and DNA damage. Altogether, these findings indicate that TRPM7 controls skin keratinocyte senescence through regulating intracellular calcium signaling, and thus, shed light on novel strategies for anti-skin aging therapy.

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.

The structure of fibrinogen and resulting fibrin formed during the coagulation process have important biological functions in human physiology and pathology. Fibrinogen post-translational modifications (PTMs) increase the complexity of the protein structure and many studies have emphasized the potential associations of post-translationally altered fibrinogen with the formation of a fibrin clot with a prothrombotic phenotype. However, the mechanisms by which PTMs exert their action on fibrinogen, and their causal association with disease pathogenesis are relatively unexplored. Moreover, the significance of fibrinogen PTMs in health has yet to be appreciated. In this review, the impact of fibrinogen PTMs on fibrinogen functionality is discussed from a biochemical perspective, emphasizing the potential mechanisms by which PTMs mediate the acquisition of altered fibrinogen properties. A brief discussion on dysfibrinogenemias of genetic origin, attributed to single point variations of the fibrinogen molecule is also provided, highlighting the influence that amino acid properties have on fibrinogen structure, properties, and molecular interactions that arise during thrombus formation.

The Nipah and Hendra viruses are severe human pathogens. In addition to the P protein, their P gene also encodes the V and W proteins that share with P their N-terminal intrinsically disordered domain (NTD) and possess distinct C-terminal domains (CTDs). The W protein is a key player in the evasion of the host innate immune response. We previously showed that the W proteins are intrinsically disordered and can form amyloid-like fibrils. However, structural information on W CTD (CTD_W) and its potential contribution to the fibrillation process is lacking. In this study, we demonstrate that CTD_WS are disordered and able to form dimers mediated by disulfide bridges. We also show that the NTD and the CTD_W interact with each other and that this interaction triggers both a gain of secondary structure and a chain compaction within the NTD. Finally, despite the lack of intrinsic fibrillogenic properties, we show that the CTD_W favors the formation of fibrils by the NTD both in cis and in trans. Altogether, the results herein presented shed light on the molecular mechanisms underlying Henipavirus pathogenesis and may thus contribute to the development of targeted therapies.

Obesity is a major health challenge due to its high prevalence and associated comorbidities. The excessive intake of a diet rich in fat and sugars leads to a persistent imbalance between energy intake and energy expenditure, which increases adiposity. Here, we provide an update on relevant diet-microbe-host interactions contributing to or protecting from obesity. In particular, we focus on how unhealthy diets shape the gut microbiota and thus impact crucial intestinal neuroendocrine and immune system functions. We describe how these interactions promote dysfunction in gut-to-brain neuroendocrine pathways involved in food intake control and postprandial metabolism and elevate the intestinal proinflammatory tone, promoting obesity and metabolic complications. In addition, we provide examples of how this knowledge may inspire microbiome-based interventions, such as fecal microbiota transplants, probiotics, and biotherapeutics, to effectively combat obesity-related disorders. We also discuss the current limitations and gaps in knowledge of gut microbiota research in obesity.