scholarly journals Biochemical and structural studies of target lectin SapL1 from the emerging opportunistic microfungus Scedosporium apiospermum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dania Martínez-Alarcón ◽  
Viviane Balloy ◽  
Jean-Philippe Bouchara ◽  
Roland J. Pieters ◽  
Annabelle Varrot
Keyword(s):  
Structural Basis ◽  
Soluble Form ◽  
Titration Calorimetry ◽  
Scedosporium Apiospermum ◽  
Dependent Manner ◽  
Bronchial Epithelial ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Conidial Surface

AbstractScedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in humans. Host–pathogen interactions often implicate lectins that have become therapeutic targets for the development of carbohydrate mimics for antiadhesive therapy. Here, we present the first report on the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 was found using bioinformatics as a homolog to the conidial surface lectin FleA from Aspergillus fumigatus known to play a role in the adhesion to host glycoconjugates present in human lung epithelium. In our strategy to obtain recombinant SapL1, we discovered the importance of osmolytes to achieve its expression in soluble form in bacteria. Analysis of glycan arrays indicates specificity for fucosylated oligosaccharides as expected. Submicromolar affinity was measured for fucose using isothermal titration calorimetry. We solved SapL1 crystal structure in complex with α-methyl-L-fucoside and analyzed its structural basis for fucose binding. We finally demonstrated that SapL1 binds to bronchial epithelial cells in a fucose-dependent manner. The information gathered here will contribute to the design and development of glycodrugs targeting SapL1.

scholarly journals SapL1: A New Target Lectin for the Development of Antiadhesive Therapy Against Scedosporium apiospermum

2020 ◽  
Author(s):  
Dania Martínez-Alarcón ◽  
Jean-Philippe Bouchara ◽  
Roland J. Pieters ◽  
Annabelle Varrot
Keyword(s):  
Biochemical Characterization ◽  
Present Report ◽  
Binding Mode ◽  
Host Cells ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Scedosporium Apiospermum ◽  
X Ray Crystallography ◽  
Opportunistic Fungal Pathogen

AbstractScedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised patients. This fungus exhibits limited susceptibility to all current antifungals and, due its emerging character, its pathogeny and virulence factors remain largely unknown. Carbohydrate binding proteins such as lectins are involved in host-pathogen interactions and may constitute valuable therapeutic targets to inhibit microbial adhesion to the host cells by using carbohydrate mimics. However, such lectins are still unidentified in S. apiospermum. Here, we present the first report of the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 is homologous to the conidial surface lectin FleA from Aspergillus fumigatus known to be involved in the adhesion to host glycoconjugates present in human lung epithelium. The present report includes a detailed strategy to achieve SapL1 soluble expression in bacteria, its biochemical characterization, an analysis of its specificity and affinity by glycan arrays and isothermal titration calorimetry (ITC), as well as the structural characterization of its binding mode by X–ray crystallography. The information gathered here contribute to the understanding of glycosylated surface recognition by Scedosporium species and is essential for the design and development of antiadhesive glycodrugs targeting SapL1.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals Thermodynamic Characterization of the Ca2+-Dependent Interaction Between SOUL and ALG-2

2018 ◽  
Vol 19 (12) ◽  
pp. 3802 ◽  
Author(s):  
Taisuke Mikasa ◽  
Masami Kugo ◽  
Seigo Nishimura ◽  
Sigeru Taketani ◽  
Sumio Ishijima ◽  
...  
Keyword(s):  
Exothermic Reaction ◽  
Titration Calorimetry ◽  
Heat Capacity Change ◽  
Dependent Manner ◽  
Recognition Mechanism ◽  
Linked Gene ◽  
Pulldown Assay ◽  
Capacity Change ◽  
Heme Binding Protein

SOUL, a heme-binding protein-2 (HEBP-2), interacts with apoptosis-linked gene 2 protein (ALG-2) in a Ca2+-dependent manner. To investigate the properties of the interaction of SOUL with ALG-2, we generated several mutants of SOUL and ALG-2 and analyzed the recombinant proteins using pulldown assay and isothermal titration calorimetry. The interaction between SOUL and ALG-2 (delta3-23ALG-2) was an exothermic reaction, with 1:1 stoichiometry and high affinity (Kd = 32.4 nM) in the presence of Ca2+. The heat capacity change (ΔCp) of the reaction showed a large negative value (−390 cal/K·mol), which suggested the burial of a significant nonpolar surface area or disruption of a hydrogen bond network that was induced by the interaction (or both). One-point mutation of SOUL Phe100 or ALG-2 Trp57 resulted in complete loss of heat change, supporting the essential roles of these residues for the interaction. Nevertheless, a truncated mutant of SOUL1-143 that deleted the domain required for the interaction with ALG-2 Trp57 still showed 1:1 binding to ALG-2 with an endothermic reaction. These results provide a better understanding of the target recognition mechanism and conformational change of SOUL in the interaction with ALG-2.

scholarly journals Thermodynamic insights into the structural basis governing the donor substrate recognition by human β1,4-galactosyltransferase 7

2009 ◽  
Vol 418 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Franck Daligault ◽  
Sophie Rahuel-Clermont ◽  
Sandrine Gulberti ◽  
Manh-Thong Cung ◽  
Guy Branlant ◽  
...  
Keyword(s):  
Large Scale ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Scale Production ◽  
Binding Studies ◽  
Donor Substrate ◽  
Large Scale Production ◽  
Rate Limiting

Human β1,4-GalT (galactosyltransferase)7 is involved in the biosynthesis of the tetrasaccharide linker protein region (GlcAβ1→3Galβ1→3Galβ1→4Xylβ1) (where GlcA is glucuronic acid and Xyl is xylose) of proteoglycans, by catalysing the transfer of Gal (galactose) from the uridine 5′-diphosphogalactose to a Xyl residue. This reaction is rate-limiting in glycosaminoglycan biosynthesis. In the present study, we established a large-scale production system of β1,4-GalT7 fused with the maltose-binding protein to study substrate recognition. Calorimetric binding studies showed that the binding of the donor substrate UDP-Gal largely promoted binding of the acceptor substrate. To identify the structural basis governing substrate recognition, we used a fragment-based approach involving the artificial breakdown of the donor substrate into smaller fragments and characterization of their respective binding to the enzyme by isothermal titration calorimetry. The β-phosphate, and to a lesser extent the α-phosphate, largely contributed to the binding energy. However, the uridine moiety was found to be essential for the optimal positioning of the donor substrate within the binding site. Unexpectedly, the contribution of the Gal moiety in substrate recognition was found to be negligible. Indeed, UDP-Gal, but also various UDP-sugars, could bind to β1,4-GalT7. Surprisingly, in contrast with other GalTs, soluble β1,4-GalT7 was able to transfer Glc (glucose), Xyl and, to a lesser extent GlcA and GlcNAc (N-acetyl glucosamine), to acceptor sugars, whereas UDP-Man (mannose) and UDP-GalNAc (N-acetyl galactosamine) were not substrates.

Molecular basis for the interaction between stress-inducible phosphoprotein 1 (STIP1) and S100A1

2017 ◽  
Vol 474 (11) ◽  
pp. 1853-1866 ◽  
Author(s):  
Andrzej Maciejewski ◽  
Vania F. Prado ◽  
Marco A.M. Prado ◽  
Wing-Yiu Choy
Keyword(s):  
Calcium Binding ◽  
Molecular Basis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
S100 Family ◽  
Calcium Dependent ◽  
Binding Interface ◽  
Large Conformational Change ◽  
Α Helix

Stress-inducible phosphoprotein 1 (STIP1) is a cellular co-chaperone, which regulates heat-shock protein 70 (Hsp70) and Hsp90 activity during client protein folding. Members of the S100 family of dimeric calcium-binding proteins have been found to inhibit Hsp association with STIP1 through binding of STIP1 tetratricopeptide repeat (TPR) domains, possibly regulating the chaperone cycle. Here, we investigated the molecular basis of S100A1 binding to STIP1. We show that three S100A1 dimers associate with one molecule of STIP1 in a calcium-dependent manner. Isothermal titration calorimetry revealed that individual STIP1 TPR domains, TPR1, TPR2A and TPR2B, bind a single S100A1 dimer with significantly different affinities and that the TPR2B domain possesses the highest affinity for S100A1. S100A1 bound each TPR domain through a common binding interface composed of α-helices III and IV of each S100A1 subunit, which is only accessible following a large conformational change in S100A1 upon calcium binding. The TPR2B-binding site for S100A1 was predominately mapped to the C-terminal α-helix of TPR2B, where it is inserted into the hydrophobic cleft of an S100A1 dimer, suggesting a novel binding mechanism. Our data present the structural basis behind STIP1 and S100A1 complex formation, and provide novel insights into TPR module-containing proteins and S100 family member complexes.

scholarly journals Structural basis of human PDZD8–Rab7 interaction for the ER-late endosome tethering

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Haider Khan ◽  
Lin Chen ◽  
Lingchen Tan ◽  
Young Jun Im
Keyword(s):  
Structural Model ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
Structural Determinants ◽  
Membrane Contact Sites ◽  
Late Endosomes

AbstractThe membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation.

Physical - Chemical Issues of Cephalosporin Intercalated Nanoparticles for Life - Threatening Infections Treatment

2001 ◽  
Vol 71 (3) ◽  
pp. 342-349
Author(s):  
Lucian Eva ◽  
Letitia Doina Duceac ◽  
Liviu Stafie ◽  
Constantin Marcu ◽  
Geta Mitrea ◽  
...  
Keyword(s):  
Chemical Characterization ◽  
Advanced Characterization ◽  
Generation Cephalosporin ◽  
Fourth Generation ◽  
Exchange Method ◽  
Physico Chemical ◽  
Life Threatening ◽  
Double Hydroxide ◽  
Double Hydroxides

The fourth generation cephalosporin antibacterial agent, cefepime, was loaded into layered double hydroxides for enhancing antibiotic efficiency, reducing side effects, as well as achieving the sustained release property. The intercalation of antibiotic into the inter-gallery of ZnAl-layered double hydroxide (LDH) was carried out using ion exchange method, by this constituting a nano-sized organic-inorganic hybrid material for a controlled release novel formulation. Although cefepime is a broad spectrum antibiotic, it has various adverse effects and a significant degradation rate. Thus, the preparation and physico-chemical characterization of nanomaterials able to intercalate this drug is an important study for medical and pharmaceutical field. The antibiotic inclusion into LDHs nanostructure was confirmed by advanced characterization techniques and the release profile of cefepime was analysed with the respect to pH of the simulated media.

scholarly journals A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

2021 ◽  
Vol 22 (14) ◽  
pp. 7390
Author(s):  
Nicole Wesch ◽  
Frank Löhr ◽  
Natalia Rogova ◽  
Volker Dötsch ◽  
Vladimir V. Rogov
Keyword(s):  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Biochemical Characterization ◽  
Effective Interaction ◽  
Regulatory Region ◽  
Titration Calorimetry ◽  
Enzymatic Reactions ◽  
Cellular Processes ◽  
Mechanism Of Interaction

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

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