The FEBS journal最新文献

Mucus in the colon is crucial for intestinal homeostasis by forming a barrier that separates microbes from the epithelium. This is achieved by the structural arrangement of the major mucus proteins, such as MUC2 and FCGBP, both of which are comprised of several von Willebrand D domains (vWD) and assemblies. Numerous disulfide bonds stabilise these domains, and intermolecular bonds generate multimers of MUC2. The oligomeric nature of FCGBP is not known. Human hFCGBP contains 13 vWD domains whereas mouse mFCGBP consists of only 7. We found unpaired cysteines in the vWD1 (human and mouse) and vWD5 (mouse)/vWD11 (human) assemblies which were not involved in disulfide bonds. However, the most C-terminal vWD domains, vWD7 (mouse)/vWD13 (human), formed disulfide-linked dimers. The intermolecular bond between C₅₂₈₄ and C₅₄₀₃ of human hFCGBP was observed by using mass spectrometry to generate the dimer. Cryo-EM structure analysis of recombinant mouse mFCGBP revealed a compact dimer with two symmetric intermolecular disulfide bonds between C₂₄₆₂ and C₂₅₈₁, corresponding to the dimerising cysteines in the human hFCGBP. This compact conformation involves interactions between the vWD assemblies, but although the domains involved at the interface are the same, the nature of the interactions differ. Mouse mFCGBP was also found to exist in a semi-extended conformation. These different interactions offer insights into the dynamic nature of the FCGBP homodimer.

The emergence of new coronavirus variants and concerns about vaccine effectiveness against these novel variants emphasize the need for broad-spectrum therapeutics targeting conserved coronaviral non-structural proteins. Accordingly, a virtual library of 178 putative inhibitors targeting SARS-CoV-2 Papain-like protease (PL^pro) was compiled through a systematic review of published literature and subsequently screened using molecular docking. Selected hits were analyzed for protease inhibitory activities, binding strength, and antiviral activities against HCoV229E-based surrogate system and subsequently against SARS-CoV-2 for validation. Differences in potential modes of action were investigated using an HCoV229E-based system, combined with in silico and biophysical methods against SARS-CoV-2 system. Of the 178 hits, 13 molecules showed superior docking scores against PL^pro and met the inclusion criteria for further investigations. Of these, seven showed notable inhibitory activities against PL^pro. Particularly, both Psoralidin and Corylifol-A exhibited superior and, importantly, dual activities against SARS-CoV-2 M^pro. Both molecules were found to be biologically active against HCoV229E and SARS-CoV-2; however, Psoralidin exhibited more consistent effects and was relatively well-tolerated. Detailed in silico analyses of their interactions with the two proteases identified differences in their modes of action, primarily due to differences in their binding of PL^pro. Based on these findings, we propose Psoralidin as a potential candidate for further development as a broad-spectrum antiviral and Corylifol-A as an ideal candidate for lead optimization.

Over 400 different types of post-translational modifications (PTMs) have been reported and over 200 various types of PTMs have been discovered using mass spectrometry (MS)-based proteomics. MS-based proteomics has proven to be a powerful method capable of global PTM mapping with the identification of modified proteins/peptides, the localization of PTM sites and PTM quantitation. PTMs play regulatory roles in protein functions, activities and interactions in various heart related diseases, such as ischemia/reperfusion injury, cardiomyopathy and heart failure. The recognition of PTMs that are specific to cardiovascular pathology and the clarification of the mechanisms underlying these PTMs at molecular levels are crucial for discovery of novel biomarkers and application in a clinical setting. With sensitive MS instrumentation and novel biostatistical methods for precise processing of the data, low-abundance PTMs can be successfully detected and the beneficial or unfavorable effects of specific PTMs on cardiac function can be determined. Moreover, computational proteomic strategies that can predict PTM sites based on MS data have gained an increasing interest and can contribute to characterization of PTM profiles in cardiovascular disorders. More recently, machine learning- and deep learning-based methods have been employed to predict the locations of PTMs and explore PTM crosstalk. In this review article, the types of PTMs are briefly overviewed, approaches for PTM identification/quantitation in MS-based proteomics are discussed and recently published proteomic studies on PTMs associated with cardiovascular diseases are included.

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.

The carbohydrate sulfotransferase 6 (chst6) gene is linked to macular corneal dystrophy (MCD), a rare disease that leads to bilateral blindness due to the accumulation of opaque aggregates in the corneal stroma. chst6 encodes for a keratan sulfate proteoglycan (KSPG) specific sulfotransferase. MCD patients lose sulfated KSPGs (cKS) in the cornea and the serum. The significance of serum cKS loss has not been understood. Zebrafish cornea structure is similar to that of humans and it contains high levels of sulfated cKS in the stroma. Here, zebrafish chst6 is shown to be expressed in the cornea and head structures of the embryos. An animal model of MCD is developed by generating chst6 mutant animals with CRISPR/Cas9-mediated gene editing. The dramatic decrease in cKS epitopes in the mutants was shown with ELISA and immunofluorescence. Morphological defects or alterations of jaw cartilage were detected in a minor fraction of the mutant larvae. Loss of cKS epitopes and morphological defects was fully rescued with wild-type chst6. Mutant adult zebrafish displayed all clinical manifestations of MCD, while a fraction also displayed jaw and skeleton defects. Opaque accumulations formed in the eye, which were alcian blue positive. Loss of cKS in the corneal stroma and a decrease in corneal thickness were shown. Interestingly, alteration of transforming growth factor beta-induced (BIGH3) expression which was not described in patients was also observed. This is the first report of an MCD model in a genetically tractable organism, providing a preclinical model and insight into the importance of KSPG sulfation for proper skeletal morphogenesis.

The hematopoietic system of Drosophila is a well-established genetic model for studying hematopoiesis mechanisms, which are strictly regulated by multiple signaling pathways. Autophagy-related 2 (Atg2) protein is involved in autophagosome formation through its lipid transfer function; however, other functions in animal development, especially the role of Atg2 in maintaining hematopoietic homeostasis, are unclear. Here, we show that Atg2 knockdown in the cortical zone (CZ) induced the proliferation and differentiation of mature plasmatocytes and disrupted progenitor maintenance in the medullary zone (MZ). We also observed the differentiation of lamellocytes among circulating hemocytes and in the lymph gland, which is rarely observed in healthy larvae. The above results on hematopoiesis disorders are due to Atg2 regulating the Drosophila PDGF/VEGF receptor (PVR) and target of rapamycin (TOR) in the CZ of lymph gland. In conclusion, we identified Atg2 as a previously undescribed regulator of hematopoiesis. Understanding the mechanism of maintenance of hematopoietic homeostasis in Drosophila will help us better evaluate human blood disorder-related diseases.

TRIM2 belongs to the TRIM-NHL class of ubiquitin E3-ligases and inhibits apoptosis by a dual function. Liao et al. reported in the recent issue that under glutamine deprivation, TRIM2 transcription is activated by ATF4 to increase the uptake of long fatty acids into mitochondria. Here, TRIM2 acts as a direct activator of CPT1 independent of its E3 ubiquitin ligase activity and prevents apoptosis otherwise triggered by starvation. On the contrary, TRIM E3-ubiquitin ligase has been described to ubiquitinate and thus target proapoptotic BIM for its degradation in the proteasome. Thus, TRIM2 inhibits apoptosis classically via its ligase activity but also independent of this stimulating energy metabolism.

Trypanosoma cruzi, the causative agent of Chagas disease, depends on acquiring nutrients and cofactors, such as copper (Cu), from different hosts. Cu is essential for aerobic organisms, but it can also be toxic, and so its transport and storage must be regulated. In the present study, we characterized the effects of changes in Cu availability on growth behavior, intracellular ion content and oxygen consumption. Our results show that copper is essential for epimastigote proliferation and for the metacyclogenesis process. On the other hand, intracellular amastigotes suffered copper stress during infection. In addition, we identify gene products potentially involved in copper metabolism. Orthologs of the highly conserved P-type Cu ATPases involved in copper export and loading of secreted enzymes were identified and named T. cruzi Cu P-type ATPase (TcCuATPase). TcCuATPase transcription is upregulated during infective stages and following exposure to copper chelators in the epimastigote stage. Homolog sequences for the high affinity import protein CTR1 were not found. Instead, we propose that the T. cruzi iron transporter (TcIT), a ZIP family transporter, could be involved in copper uptake based on transcriptional response to copper availability. Further canonical copper targets (based on homology to yeast and mammals) such as the T. cruzi ferric reductase (TcFR) and the cupro-oxidase TcFet3 are upregulated during infective stages and under conditions of intracellular copper deficiency. In sum, copper metabolism is essential for the life cycle of T. cruzi. Even though cytosolic copper chaperons were not identified, we propose a previously undescribed model for copper transport and intracellular distribution in T. cruzi, including some conserved factors such as TcCuATPase, as well as others such as TcFR and TcIT, playing novel functions.

White and brown adipocytes are central mediators of lipid metabolism and thermogenesis, respectively. Their function is tightly regulated by all three β-adrenergic receptor (β-AR) subtypes which are coupled to the production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). While known for decades in other cell types, compartmentation of adipocyte β-AR/cAMP signaling by spatial organization of the pathway and by cAMP degrading phosphodiesterases (PDEs) as well as its role in the regulation of lipolysis is only beginning to emerge. Here, we provide a short overview of recent findings which shed light on compartmentalized signaling using live cell imaging of cAMP in adipocytes and discuss possible future directions of research which could open up new avenues for the treatment of metabolic disorders.

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.