23 research outputs found
The spike-ACE2 interaction underlying SARS-CoV-2 infection and inhibition is enhanced by intermolecular cross linking
A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint
Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 âWYâ conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 ÎŒM binding affinity for CtUGGTGT24 in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 ÎŒM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors
Tumor cell and carcinoma-associated fibroblast interaction regulates matrix metalloproteinases and their inhibitors in oral squamous cell carcinoma
AbstractCo-culture of periodontal ligament (PDL) fibroblasts and SCC-25 oral squamous carcinoma cells (OSCC), results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs). Paracrin circuits between CAFs and OSCC cells were hypothesized to regulate the gene expression of matrix remodeling enzymes in their co-culture, which was performed for 7days, followed by analysis of the mRNA/protein expression and activity of metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and other relevant genes. Interleukin1-ÎČ, transforming growth factor-ÎČ1, fibronectin and αvÎČ6 integrin have shown to be involved in the regulation of the MMP and TIMP gene expression in co-culture of CAFs and tumor cells. In addition, these cells also cooperated in activation of MMP pro-enzymes. It is particularly interesting that the fibroblast-produced inactive MMP-2 has been activated by the tumor-cell-produced membrane-type 1 matrix metalloproteinase (MT1-MMP). The crosstalk between cancer- and the surrounding fibroblast stromal-cells is essential for the fine tuning of cancer cells invasivity
Crystal polymorphism in fragment-based lead discovery of ligands of the catalytic domain of UGGT, the glycoprotein folding quality control checkpoint
None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDPâglucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystalsâwith the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA (crystal polymorph and ligand likelihood-based assignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple/REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement Rfree statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT
SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues
Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to
genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility
and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component.
Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci
(eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene),
including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform
genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer
SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the
diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types
Patterns of distribution and abundance of larval phosichthyidae (actinopterygii, stomiiformes) in southeastern Brazilian waters
Structural and functional studies of the antiviral target proteins HCV p7 and ER alpha-glucosidase II
To accomplish the correct folding of N-glycosylated proteins eukaryotes rely on their endoplasmic reticulum quality control pathway. This pathway which consists of glycosyl- hydrolases and -transferases, lectins and chaperones, assists the folding of immature glycoproteins post their translation into the ER lumen. Glycoproteins are processed based on glycan and protein misfold recognition. While correctly matured glycoproteins proceed through the secretory pathway to their final destination, immature glycoproteins are discarded eventually by the ER associated degradation pathway.
Many viruses hijack the glycoprotein quality control pathway after host cell infection in order to produce their own viral glycoproteins, which makes them vulnerable to the interference with the correct performance of the cycle.
Iminosugars which are glucose mimetics inhibit N-linked glycan processing enzymes such as alpha-glucosidase I and II of the glycoprotein quality control pathway, resulting in decreased virion secretion from infected cells and/or virus infectivity.
Presented in this thesis is the protein X-ray crystallography work carried out on ER alpha- glucosidase II in combination with a new class of N-alkyloxy iminosugars revealing binding motifs of the latter within the active site of the enzyme. Furthermore, the study demonstrates a new approach of potential alpha-glucosidase II inhibition and the development and use of a fluorescence energy resonance transfer-based high-throughput screen for new drug discovery. The findings enable future studies aimed at improving alpha-glucosidase II inhibition both by developing more specific active site inhibitors as well as via a novel strategy that aims to prevent or disrupt GluII heterodimerisation by targeting the enzymeâs -/- subunit interface. Long alkyl chain iminosugars do not only inhibit certain host cell enzymes such as ER alpha- glucosidase II but also the hepatitis C virus p7 ion channel, but it is not yet known how this inhibition is achieved. The analysis of how iminosugars interact with their target protein p7 is hindered by the lack of knowledge of the protein channelâs oligomeric structure and its location in the ER membrane. Work in this thesis uses electron paramagnetic resonance spectroscopy in combination with spin labelled HCV p7 to analyse the oligomerisation behaviour of the p7 protomer in lipid bilayers. The structural information gained provides the basis for future experiments to better understand drug inhibition of the p7 ion channel and contributes to the still very limited information about viral ion channels in lipid environments
RECORD DYNAMICS AND THE OBSERVED TEMPERATURE PLATEAU IN THE MAGNETIC CREEP-RATE OF TYPE-II SUPERCONDUCTORS
Molecular basis of MHC I quality control in the peptide loading complex.
Major histocompatibility complex class I (MHC I) molecules are central to adaptive immunity. Their assembly, epitope selection, and antigen presentation are controlled by the MHC I glycan through a sophisticated network of chaperones and modifying enzymes. However, the mechanistic integration of the corresponding processes remains poorly understood. Here, we determine the multi-chaperone-client interaction network of the peptide loading complex (PLC) and report the PLC editing module structure by cryogenic electron microscopy at 3.7âĂ
resolution. Combined with epitope-proofreading studies of the PLC in near-native lipid environment, these data show that peptide-receptive MHC I molecules are stabilized by multivalent chaperone interactions including the calreticulin-engulfed mono-glucosylated MHC I glycan, which only becomes accessible for processing by α-glucosidase II upon loading of optimal epitopes. Our work reveals allosteric coupling between peptide-MHC I assembly and glycan processing. This inter-process communication defines the onset of an adaptive immune response and provides a prototypical example of the tightly coordinated events in endoplasmic reticulum quality control