Hundreds of thousands die annually from malaria caused by Plasmodium falciparum (Pf), with the emergence of drug-resistant parasites hindering eradication efforts. Antimicrobial peptides (AMPs) are known for their ability to disrupt pathogen membranes without targeting specific receptors, thereby reducing the chance of drug resistance. However, their effectiveness and the biophysical mechanisms by which they target the intracellular parasite remain unexplored. Here, by using native and synthetic AMPs, we discovered a selective mechanism that underlies the anti-malaria activity. Remarkably, the AMPs exclusively interact with Pf-infected Red Blood Cells (Pf-iRBCs), disrupting the cytoskeletal network and reaching the enclosed parasites with correlation to their activity. Moreover, we showed that the unique feature of reduced cholesterol content in the membrane of the infected host makes Pf-iRBCs susceptible to AMPs. Overall, this work highlights the Achilles' heel of malaria parasite and demonstrates the power of AMPs as potential antimalarial drugs with reduced risk of resistance.
- Publikační typ
- časopisecké články MeSH
Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Förster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.
Apolipoprotein E (APOE) is distributed across various human tissues and plays a crucial role in lipid metabolism. Recent investigations have uncovered an additional facet of APOE's functionality, revealing its role in host defense against bacterial infections. To assess the antibacterial attributes of APOE3 and APOE4, we conducted antibacterial assays using Pseudomonas aeruginosa and Escherichia coli. Exploring the interaction between APOE isoforms and lipopolysaccharides (LPSs) from E. coli, we conducted several experiments, including gel shift assays, CD, and fluorescence spectroscopy. Furthermore, the interaction between APOE isoforms and LPS was further substantiated through atomic resolution molecular dynamics simulations. The presence of LPS induced the aggregation of APOE isoforms, a phenomenon confirmed through specific amyloid staining, as well as fluorescence and electron microscopy. The scavenging effects of APOE3/4 isoforms were studied through both in vitro and in vivo experiments. In summary, our study established that APOE isoforms exhibit binding to LPS, with a more pronounced affinity and complex formation observed for APOE4 compared with APOE3. Furthermore, our data suggest that APOE isoforms neutralize LPS through aggregation, leading to a reduction of local inflammation in experimental animal models. In addition, both isoforms demonstrated inhibitory effects on the growth of P. aeruginosa and E. coli. These findings provide new insights into the multifunctionality of APOE in the human body, particularly its role in innate immunity during bacterial infections.
- Klíčová slova
- antimicrobial peptides, apolipoprotein E isoforms, endotoxin, host defense, protein aggregation,
- Publikační typ
- časopisecké články MeSH
Accumulation of environmental chitin in the lungs can lead to pulmonary fibrosis, characterized by inflammatory infiltration and fibrosis in acidic chitinase (Chia)-deficient mice. Transgenic expression of Chia in these mice ameliorated the symptoms, indicating the potential of enzyme supplementation as a promising therapeutic strategy for related lung diseases. This study focuses on utilizing hyperactivated human Chia, which exhibits low activity. We achieved significant activation of human Chia by incorporating nine amino acids derived from the crab-eating monkey (Macaca fascicularis) Chia, known for its robust chitin-degrading activity. The modified human Chia retained high activity across a broad pH spectrum and exhibited enhanced thermal stability. The amino acid substitutions associated with hyperactivation of human Chia activity occurred species specifically in monkey Chia. This discovery highlights the potential of hyperactivated Chia in treating pulmonary diseases resulting from chitin accumulation in human lungs.
- Klíčová slova
- acidic chitinase (Chia), amino acid substitutions, chitin, enzyme engineering, evolution, exon swapping, hyperactivation, primate lineage, treating pulmonary diseases,
- MeSH
- aktivace enzymů účinky léků MeSH
- chitin metabolismus chemie MeSH
- chitinasy * metabolismus genetika chemie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- Macaca fascicularis MeSH
- myši MeSH
- plíce metabolismus patologie enzymologie MeSH
- stabilita enzymů MeSH
- substituce aminokyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- CHIA protein, human MeSH Prohlížeč
- chitin MeSH
- chitinasy * MeSH
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.
- Klíčová slova
- Förster resonance energy transfer (FRET), SARS-CoV-2 main protease, activation, autoprocessing, cell-based assay, fluorescence cross-correlation spectroscopy (FCCS), fluorescence life-time imaging, inhibitor, maturation, nsp5, precursor, protease, virus,
- MeSH
- antivirové látky * farmakologie chemie MeSH
- farmakoterapie COVID-19 MeSH
- HEK293 buňky MeSH
- inhibitory proteas farmakologie chemie MeSH
- koronavirové proteasy 3C * metabolismus antagonisté a inhibitory chemie genetika MeSH
- lidé MeSH
- mutace MeSH
- objevování léků * metody MeSH
- proteolýza MeSH
- SARS-CoV-2 * enzymologie účinky léků metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 3C-like proteinase, SARS-CoV-2 MeSH Prohlížeč
- antivirové látky * MeSH
- inhibitory proteas MeSH
- koronavirové proteasy 3C * MeSH
The endoplasmic reticulum is organized into ordered regions enriched in cholesterol and sphingomyelin, and disordered microdomains characterized by more fluidity. Rabbit CYP1A1 and CYP1A2 localize into disordered and ordered microdomains, respectively. Previously, a CYP1A2 chimera containing the first 109 amino acids of CYP1A1 showed altered microdomain localization. The goal of this study was to identify specific residues responsible for CYP1A microdomain localization. Thus, CYP1A2 chimeras containing substitutions from homologous regions of CYP1A1 were expressed in HEK 293T/17 cells, and the localization was examined after solubilization with Brij 98. A CYP1A2 mutant with the three amino acids from CYP1A1 (VAG) at positions 27 to 29 of CYP1A2 was generated that showed a distribution pattern similar to those of CYP1A1/1A2 chimeras containing both the first 109 amino acids and the first 31 amino acids of CYP1A1 followed by remaining amino acids of CYP1A2. Similarly, the reciprocal substitution of three amino acids from CYP1A2 (AVR) into CYP1A1 resulted in a partial redistribution of the chimera into ordered microdomains. Molecular dynamic simulations indicate that the positive charges of the CYP1A1 and CYP1A2 linker regions between the N termini and catalytic domains resulted in different depths of immersion of the N termini in the membrane. The overlap of the distribution of positively charged residues in CYP1A2 (AVR) and negatively charged phospholipids was higher in the ordered than in the disordered microdomain. These findings identify three residues in the CYP1AN terminus as a novel microdomain-targeting motif of the P450s and provide a mechanistic explanation for the differential microdomain localization of CYP1A.
- Klíčová slova
- CYP1A1, CYP1A2, cytochrome P450, membrane charge depth, membrane protein, microdomain localization, microdomain-targeting motif, protein chimera, protein-lipid interaction, structure-function,
- MeSH
- cytochrom P-450 CYP1A1 * genetika metabolismus chemie MeSH
- cytochrom P-450 CYP1A2 * metabolismus genetika chemie MeSH
- endoplazmatické retikulum metabolismus MeSH
- HEK293 buňky MeSH
- králíci MeSH
- lidé MeSH
- membránové mikrodomény metabolismus genetika MeSH
- proteinové domény MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- substituce aminokyselin MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cytochrom P-450 CYP1A1 * MeSH
- cytochrom P-450 CYP1A2 * MeSH
Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography coupled online to sector-field ICP-MS. Our data identified known TM-binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10 to 15 kD containing Cu, Fe, S, and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu, and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.
- Klíčová slova
- glutathione, iron, metallothioneins, phloem sap, size exclusion chromatography, zinc,
- MeSH
- Brassica napus * metabolismus genetika MeSH
- floém * metabolismus MeSH
- metalothionein metabolismus genetika MeSH
- přechodné kovy metabolismus MeSH
- rostlinné proteiny * metabolismus genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- metalothionein MeSH
- přechodné kovy MeSH
- rostlinné proteiny * MeSH
Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.
- Klíčová slova
- A-kinase anchoring protein (AKAP), growth factor receptor-bound protein 2 (GRB2), microtubule associated protein (MAP) 2, nuclear magnetic resonance (NMR), protein kinase A (PKA),
- MeSH
- adaptorový protein Grb2 * metabolismus chemie MeSH
- lidé MeSH
- proteinové domény MeSH
- proteiny asociované s mikrotubuly * metabolismus chemie genetika MeSH
- protoonkogenní proteiny c-fyn metabolismus chemie genetika MeSH
- signální transdukce MeSH
- src homologní domény MeSH
- vazba proteinů * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adaptorový protein Grb2 * MeSH
- GRB2 protein, human MeSH Prohlížeč
- proteiny asociované s mikrotubuly * MeSH
- protoonkogenní proteiny c-fyn MeSH
Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.
- Klíčová slova
- Axin, NanoBRET, Wnt pathway, alternative splicing, casein kinase 1 alpha (CK1α), casein kinase 1 alpha-like (CK1α-like), gene knockout, inhibitor, phosphorylation, β-catenin,
- MeSH
- alternativní sestřih MeSH
- beta-katenin * metabolismus genetika MeSH
- fosforylace MeSH
- GSK3B metabolismus genetika MeSH
- HEK293 buňky MeSH
- kasein kinasa Ialfa * metabolismus genetika MeSH
- kinasa 3 glykogensynthasy metabolismus genetika MeSH
- lidé MeSH
- protein Wnt3A metabolismus genetika MeSH
- signální dráha Wnt * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- beta-katenin * MeSH
- GSK3B MeSH
- kasein kinasa Ialfa * MeSH
- kinasa 3 glykogensynthasy MeSH
- protein Wnt3A MeSH
- WNT3A protein, human MeSH Prohlížeč
Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.
- Klíčová slova
- HPMA copolymer, PD-1, PD-L1, T-cell, antibody mimetic, immune checkpoint, immunosuppression, immunotherapy, inhibitor, tumor immunology,
- MeSH
- antigeny CD274 * antagonisté a inhibitory imunologie metabolismus MeSH
- inhibitory kontrolních bodů * farmakologie chemie MeSH
- lidé MeSH
- monoklonální protilátky chemie farmakologie MeSH
- nádorové buněčné linie MeSH
- polymery chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny CD274 * MeSH
- CD274 protein, human MeSH Prohlížeč
- inhibitory kontrolních bodů * MeSH
- monoklonální protilátky MeSH
- polymery MeSH
