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1. Click Conjugation of Boron Dipyrromethene (BODIPY) Fluorophores to EGFR-Targeting Linear and Cyclic Peptides

   https://doi.org/10.3390/molecules26030593  

   Williams, Tyrslai M. ; Kaufman, Nichole E. ; Zhou, Zehua ; Singh, Sitanshu S. ; Jois, Seetharama D. ; Vicente, Maria da ( February 2021 , Molecules)

   null (Ed.)

   Through a simple 1,3-cycloaddition reaction, three BODIPY-peptide conjugates that target the extracellular domain of the epidermal growth factor receptor (EGFR) were prepared and their ability for binding to EGFR was investigated. The peptide ligands K(N3)LARLLT and its cyclic analog cyclo(K(N3)larllt, previously shown to have high affinity for binding to the extracellular domain of EGFR, were conjugated to alkynyl-functionalized BODIPY dyes 1 and 2 via a copper-catalyzed click reaction. This reaction produced conjugates 3, 4, and 5 in high yields (70–82%). In vitro studies using human carcinoma HEp2 cells that overexpress EGFR demonstrated high cellular uptake, particularly for the cyclic peptide conjugate 5, and low cytotoxicity in light (~1 J·cm−2) and darkness. Surface plasmon resonance (SPR) results show binding affinity of the three BODIPY-peptide conjugates for EGFR, particularly for 5 bearing the cyclic peptide. Competitive binding studies using three cell lines with different expressions of EGFR show that 5 binds specifically to EGFR-overexpressing colon cancer cells. Among the three conjugates, 5 bearing the cyclic peptide exhibited the highest affinity for binding to the EGFR protein. 

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2. Probing the Interactions of LRP1 Ectodomain-Derived Peptides with Fibrillar Tau Protein and Its Impact on Cellular Internalization

   https://doi.org/10.3390/app13020853  

   Boder, E. Josephine ; Goncalves, Beatriz G. ; Lebedenko, Charlotta G. ; Banerjee, Ipsita A. ( January 2023 , Applied Sciences)

   Cellular internalization and the spreading of misfolded tau have become increasingly important for elucidating the mechanism of Tau pathology involved in Alzheimer’s disease (AD). The low-density lipoprotein-related receptor 1 (LRP1) has been implicated in the internalization of fibrillar tau. In this work, we utilized homology modeling to model the Cluster 2 domain of LRP1 and determined that a 23-amino-acid sequence is involved in binding to paired helical filaments (PHF) of Tau. Fourteen short peptide segments derived from this ectodomain region were then designed and docked with PHF Tau. Molecular dynamics studies of the optimal peptides bound to PHF Tau demonstrated that the peptides formed critical contacts through Lys and Gln residues with Tau. Based on the computational results, flow cytometry, AFM, SPR analysis and CD studies were conducted to examine binding and cellular internalization. The results showed that the peptide sequence TauRP (1–14) (DNSDEENCES) was not only associated with fibrillar Tau but was also able to mitigate its cellular internalization in LRP1-expressed HEK-293 cells. Preliminary docking studies with Aβ (1–42) revealed that the peptides also bound to Aβ (1–42). While this study focused on the CCR2 domain of LRP1 to design peptide sequences to mitigate Tau internalization, the work can be extended to other domains of the LRP1 receptor or other receptors to examine if the cellular internalization of fibrillar Tau can be deterred. These findings show that short peptides derived from the LRP1 receptor can alter the internalization of its ligands. 

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3. “Two‐Point” Self‐Assembly and Photoinduced Electron Transfer in meso ‐Donor‐Carrying Bis(styryl crown ether)‐BODIPY–Bis(alkylammonium)fullerene Donor–Acceptor Conjugates

   https://doi.org/10.1002/asia.201700662  

   Shao, Shuai ; Gobeze, Habtom B. ; Karr, Paul A. ; D'Souza, Francis ( August 2017 , Chemistry – An Asian Journal)

   BF₂‐chelated dipyrromethene, BODIPY, was functionalized to carry two styryl crown ether tails and a secondary electron donor at themesoposition. By using a “two‐point” self‐assembly strategy, a bis‐alkylammonium‐functionalized fullerene (C₆₀) was allowed to self‐assemble the crown ether voids of BODIPY to obtain multimodular donor–acceptor conjugates. As a consequence of the two‐point binding, the 1:1 stoichiometric complexes formed yielded complexes of higher stability in which fluorescence of BODIPY was found to be quenched; this suggested the occurrence of excited‐state processes. The geometry and electronic structure of the self‐assembled complexes were derived from B3LYP/3‐21G(*) methods in which no steric constraints between the entities was observed. An energy‐level diagram was established by using spectral, electrochemical, and computational results to help understand the mechanistic details of excited‐state processes originating from¹bis‐styryl‐BODIPY*. Femtosecond transient absorbance studies were indicative of the formation of an exciplex state prior to the charge‐separation process to yield a bis‐styryl‐BODIPY^.+–C₆₀^.−radical ion pair. The time constants for charge separation were generally lower than charge‐recombination processes. The present studies bring out the importance of multimode binding strategies to obtain stable self‐assembled donor–acceptor conjugates capable of undergoing photoinduced charge separation needed in artificial photosynthetic applications.

    

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4. Production of Class II MHC Proteins in Lentiviral Vector‐Transduced HEK‐293T Cells for Tetramer Staining Reagents

   https://doi.org/10.1002/cpz1.36  

   Willis, Richard A. ; Ramachandiran, Vasanthi ; Shires, John C. ; Bai, Ge ; Jeter, Kelly ; Bell, Donielle L. ; Han, Lixia ; Kazarian, Tamara ; Ugwu, Kyla C. ; Laur, Oskar ; et al ( February 2021 , Current Protocols)

   Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed inE. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.

   This article was corrected on 19 July 2022. See the end of the full text for details.

   Basic Protocol 1: Lentivirus production and expression line creation

   Support Protocol 1: Six‐well assay for estimation of production cell line yield

   Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

   Basic Protocol 2: Cultures for production of Class II MHC proteins

   Basic Protocol 3: Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatography

   Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

   Support Protocol 3: Protein concentration measurements and adjustments

   Support Protocol 4: Polishing purification by anion‐exchange chromatography

   Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

   Basic Protocol 4: Peptide exchange

   Basic Protocol 5: Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry

   Alternate Protocol 2: Native isoelectric focusing to validate MHC‐II peptide loading

   Basic Protocol 6: Multimerization

   Basic Protocol 7: Staining cells with Class II MHC tetramers

    

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5. Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5

   https://doi.org/10.3390/v14122696  

   Shi, Deling ; Bu, Changkai ; He, Peng ; Song, Yuefan ; Dordick, Jonathan S. ; Linhardt, Robert J. ; Chi, Lianli ; Zhang, Fuming ( December 2022 , Viruses)

   The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus–host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein–glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD–heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin. 

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