Biophysical and Biochemical Characterization of the Receptor Binding Domain of SARS-CoV-2 Variants

The Protein Journal - The newly emerging SARS-CoV-2 variants are potential threat and posing new challenges for medical intervention due to high transmissibility and escaping neutralizing antibody...     

Genome Mining in Sorangium cellulosum So ce56: IDENTIFICATION AND CHARACTERIZATION OF THE HOMOLOGOUS ELECTRON TRANSFER PROTEINS OF A MYXOBACTERIAL CYTOCHROME P450*

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.The cytochrome P450 (CYP)² enzymes constitute a superfamily of external monooxygenases. The catalytic versatility of the family members explains their involvement in such diverse biological processes as biosynthesis of steroid hormones, carbon source assimilation, and metabolism of xenobiotics. In addition, cytochrome P450 enzymes have been reported to be involved in the biosynthesis of many pharmaceutically interesting secondary metabolites from a variety of microorganisms (1–4). Cytochromes P450 are usually dependent on an external electron donor. With respect to their electron transport system they can be divided into several classes, with class I (the mitochondrial/bacterial cytochrome P450 systems) being the predominant form in prokaryotes (5). In this system the electrons required for the enzymatic reaction originate from NAD(P)H and are delivered to the cytochrome P450 via a ferredoxin reductase and a ferredoxin. In a number of examples, the heterologous reconstitution of the electron transfer chain has been shown to be ineffective, if possible at all (5). Thus, it is desirable to identify the natural redox partners, especially if genomic sequence information is available. However, even then the identification of the correct interaction partners remains challenging because the encoding genes are frequently located at genomic loci distant to the cytochrome P450 genes (6, 7). Interestingly, members of both the [2Fe-2S] and the non-[2Fe-2S] ferredoxins have been reported to sustain cytochrome P450 catalyzed reactions. The latter group is further subdivided into mono- and dicluster ferredoxins (i.e. the [3Fe-4S] or [4Fe-4S] and the [3Fe-4S] + [4Fe-4S] or [4Fe-4S] + [4Fe-4S] ferredoxins). Remarkably, cytochrome P450 systems depending on non-[2Fe-2S] ferredoxins have been found exclusively in bacteria to date (8, 9).To fulfill the role as electron mediator, the ferredoxin component of any given cytochrome P450 system has to be reduced. This reduction is achieved by a ferredoxin reductase, which in turn takes up electrons from NAD(P)H. The ferredoxin reductase is often the least characterized constituent of the cytochrome P450 system because these flavoproteins may be unstable (i.e. easily lose their cofactor) and usually show a relatively low level of expression (10).Sorangium cellulosum So ce56 is a genome-sequenced myxobacterial model strain. Because of their biotechnological potential as producers of secondary metabolites, the myxobacteria attract attention from both the academic community and the pharmaceutical industry. To date, more than 100 new basic structures and some 500 derivatives have been reported (11), with almost half of the newly discovered natural products being isolated from the genus Sorangium (11, 12). The potent anti-cancer agent epothilone, for example, was discovered from S. cellulosum So ce90 (13, 14). Epothilone is one of so far seven known myxobacterial compounds, the biosynthesis of which involves cytochromes P450 (15). Besides the epothilones, these are the antifungal leupyrrins (16) and the cytotoxic spirangienes (17) (also from S. cellulosum), the antibiotic myxovirescin from Myxococcus (18), the electron transport inhibitor stigmatellin (19) and the antibiotic aurafuron (20) from Stigmatella aurantiaca, and the antifungal ajudazols from Chondromyces crocatus (21).The recently genome-sequenced myxobacterium S. cellulosum So ce56 (12) shows great potential for biotechnological applications, as judged on the basis of its capacity for the production of secondary metabolites. Three biologically active compounds have been described so far, namely the fungicidal chivosazoles, the macrolide antibiotic etnangien, and the iron chelator myxochelin (12). Moreover, the bioinformatic analysis of the So ce56 genome has revealed numerous biosynthetic gene clusters of yet unknown function (11, 12). With a size of more than 13 Mbp, the genome of S. cellulosum So ce56 is to date the largest sequenced prokaryotic genome (12). It has been shown to harbor 21 cytochrome P450 genes. In light of the significance of S. cellulosum as a viable source of bioactive secondary metabolites (14) and the role of cytochromes P450 in the synthesis of natural products (2), it is of great interest to elucidate the function of these enzymes.Therefore, the investigation of the S. cellulosum So ce56 cytochrome P450 systems opens a fascinating field not only with regard to basic research but also to exploit the biotechnological potential of this model strain. To achieve this goal it is important to provide a functional electron transport chain. Thus, the main objective of this work was to identify a myxobacterial ferredoxin/ferredoxin reductase couple able to support reactions catalyzed by S. cellulosum So ce56 cytochromes P450.     

The HIF signaling pathway in osteoblasts directly modulates erythropoiesis through the production of EPO

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.     

AMPA receptor tetramerization is mediated by Q/R editing

AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain.     

Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities

Although there is increasing evidence that virus-specific cytotoxic-T-lymphocyte (CTL) responses play an important role in the control of human immunodeficiency virus (HIV) replication in vivo, only scarce CTL data are available for the ethnic populations currently most affected by the epidemic. In this study, we examined the CD8(+)-T-cell responses in African-American, Caucasian, Hispanic, and Caribbean populations in which clade B virus dominates and analyzed the potential factors influencing immune recognition. Total HIV-specific CD8(+)-T-cell responses were determined by enzyme-linked immunospot assays in 150 HIV-infected individuals by using a clade B consensus sequence peptide set spanning all HIV proteins. A total of 88% of the 410 tested peptides were recognized, and Nef- and Gag-specific responses dominated the total response for each ethnicity in terms of both breadth and magnitude. Three dominantly targeted regions within these proteins that were recognized by >90% of individuals in each ethnicity were identified. Overall, the total breadth and magnitude of CD8(+)-T-cell responses correlated with individuals' CD4 counts but not with viral loads. The frequency of recognition for each peptide was highly correlated with the relative conservation of the peptide sequence, the presence of predicted immunoproteasomal cleavage sites within the C-terminal half of the peptide, and a reduced frequency of amino acids that impair binding of optimal epitopes to the restricting class I molecules. The present study thus identifies factors that contribute to the immunogenicity of these highly targeted and relatively conserved sequences in HIV that may represent promising vaccine candidates for ethnically heterogeneous populations.     

Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes

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