Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

• MeSH
• DNA vazebné proteiny MeSH
• endopeptidasy metabolismus   chemie   genetika   MeSH
• Escherichia coli metabolismus   genetika   MeSH
• kardiolipiny * metabolismus   chemie   MeSH
• membránové proteiny * metabolismus   chemie   genetika   MeSH
• proteinové inženýrství * MeSH
• proteiny z Escherichia coli * metabolismus   chemie   genetika   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Targeting ubiquitin E3 ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches for identifying inhibitors. In a large, unbiased biochemical screen, we discover inhibitors that bind a cryptic cavity distant from the catalytic cysteine of the homologous to E6-associated protein C terminus domain (HECT) E3 ligase, SMAD ubiquitin regulatory factor 1 (SMURF1). Structural and biochemical analyses and engineered escape mutants revealed that these inhibitors restrict an essential catalytic motion by extending an α helix over a conserved glycine hinge. SMURF1 levels are increased in pulmonary arterial hypertension (PAH), a disease caused by mutation of bone morphogenetic protein receptor-2 (BMPR2). We demonstrated that SMURF1 inhibition prevented BMPR2 ubiquitylation, normalized bone morphogenetic protein (BMP) signaling, restored pulmonary vascular cell homeostasis, and reversed pathology in established experimental PAH. Leveraging this deep mechanistic understanding, we undertook an in silico machine-learning-based screen to identify inhibitors of the prototypic HECT E6AP and confirmed glycine-hinge-dependent allosteric activity in vitro. Inhibiting HECTs and other glycine-hinge proteins opens a new druggable space.

• MeSH
• alosterická regulace účinky léků   MeSH
• lidé MeSH
• myši MeSH
• plicní arteriální hypertenze farmakoterapie   MeSH
• receptory morfogenetických kostních proteinů typu II MeSH
• signální transdukce účinky léků   MeSH
• ubikvitinace účinky léků   MeSH
• ubikvitinligasy * antagonisté a inhibitory   metabolismus   chemie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA * chemie   metabolismus   MeSH
• flavinmononukleotid * chemie   metabolismus   MeSH
• flaviny chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• oxidace-redukce * MeSH
• simulace molekulární dynamiky MeSH
• světlo * MeSH
• Thermosynechococcus metabolismus   MeSH
• transkripční faktory metabolismus   chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Tunnels in enzymes with buried active sites are key structural features allowing the entry of substrates and the release of products, thus contributing to the catalytic efficiency. Targeting the bottlenecks of protein tunnels is also a powerful protein engineering strategy. However, the identification of functional tunnels in multiple protein structures is a non-trivial task that can only be addressed computationally. We present a pipeline integrating automated structural analysis with an in-house machine-learning predictor for the annotation of protein pockets, followed by the calculation of the energetics of ligand transport via biochemically relevant tunnels. A thorough validation using eight distinct molecular systems revealed that CaverDock analysis of ligand un/binding is on par with time-consuming molecular dynamics simulations, but much faster. The optimized and validated pipeline was applied to annotate more than 17,000 cognate enzyme-ligand complexes. Analysis of ligand un/binding energetics indicates that the top priority tunnel has the most favourable energies in 75% of cases. Moreover, energy profiles of cognate ligands revealed that a simple geometry analysis can correctly identify tunnel bottlenecks only in 50% of cases. Our study provides essential information for the interpretation of results from tunnel calculation and energy profiling in mechanistic enzymology and protein engineering. We formulated several simple rules allowing identification of biochemically relevant tunnels based on the binding pockets, tunnel geometry, and ligand transport energy profiles.Scientific contributionsThe pipeline introduced in this work allows for the detailed analysis of a large set of protein-ligand complexes, focusing on transport pathways. We are introducing a novel predictor for determining the relevance of binding pockets for tunnel calculation. For the first time in the field, we present a high-throughput energetic analysis of ligand binding and unbinding, showing that approximate methods for these simulations can identify additional mutagenesis hotspots in enzymes compared to purely geometrical methods. The predictor is included in the supplementary material and can also be accessed at https://github.com/Faranehhad/Large-Scale-Pocket-Tunnel-Annotation.git . The tunnel data calculated in this study has been made publicly available as part of the ChannelsDB 2.0 database, accessible at https://channelsdb2.biodata.ceitec.cz/ .

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.

• MeSH
• antigeny CD279 * metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory plic diagnostické zobrazování   patologie   metabolismus   genetika   MeSH
• nemalobuněčný karcinom plic diagnostické zobrazování   patologie   metabolismus   MeSH
• pozitronová emisní tomografie * metody   MeSH
• proteinové inženýrství * MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• chronická lymfatická leukemie * genetika   patologie   metabolismus   farmakoterapie   MeSH
• lidé MeSH
• myši MeSH
• NF-kappa B * metabolismus   MeSH
• piperidiny farmakologie   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

"Genetics and Genomics in Medicine is a new textbook written for undergraduate and graduate students, as well as medical researchers, which explains the science behind the uses of genetics and genomics in medicine today. It is not just about rare inherited and chromosomal disorders, but how genetics affects the whole spectrum of human health and disease. DNA technologies are explained, with emphasis on the modern techniques that have revolutionized the use of genetic information in medicine and are indicating the role of genetics in common complex diseases. The detailed, integrative coverage of genetic approaches to treatment and prevention includes pharmacogenomics and the prospects for personalized medicine. Cancers are essentially genetic diseases and are given a dedicated chapter that includes new insights from cancer genome sequencing. Clinical disorders are covered throughout and there are extensive end-of-chapter questions and problems"--Provided by publisher.

Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.

• MeSH
• inzulin metabolismus   MeSH
• lidé MeSH
• peptidy * chemie   MeSH
• polyethylenglykoly MeSH
• protein - isoformy MeSH
• receptor inzulinu * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Inebilizumab, a humanized, glycoengineered, IgG1 monoclonal antibody that depletes CD19+ B-cells, is approved to treat aquaporin 4 (AQP4) IgG-seropositive neuromyelitis optica spectrum disorder (NMOSD). Inebilizumab is afucosylated and engineered for enhanced affinity to Fc receptor III-A (FCGR3A) receptors on natural killer cells to maximize antibody-dependent cellular cytotoxicity. Previously, the F allele polymorphism at amino acid 158 of the FCGR3A gene (F158) was shown to decrease IgG-binding affinity and reduce rituximab (anti-CD20) efficacy for NMOSD attack prevention. In contrast, our current findings from inebilizumab-treated NMOSD patients indicate similar clinical outcomes between those with F158 and V158 allele genotypes.

• MeSH
• akvaporin 4 genetika   MeSH
• humanizované monoklonální protilátky terapeutické užití   MeSH
• imunoglobulin G MeSH
• lidé MeSH
• neuromyelitis optica * farmakoterapie   genetika   MeSH
• receptory IgG genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The cytokine IL-23 activates the IL-23 receptor (IL-23R) and stimulates the differentiation of naïve T helper (Th) cells into a Th17 cell population that secretes inflammatory cytokines and chemokines. This IL-23/Th17 proinflammatory axis drives inflammation in Crohn's disease and ulcerative colitis and represents a therapeutic target of monoclonal antibodies. Non-immunoglobulin binding proteins based on the Streptococcus albumin-binding domain (ABD) provide a small protein alternative to monoclonal antibodies. They can be readily expressed in bacteria. Lactococcus lactis is a safe lactic acid bacterium that has previously been engineered as a vector for the delivery of recombinant therapeutic proteins to mucosal surfaces. Here, L. lactis was engineered to display or secrete ABD-variants against the IL-17 receptor (IL-17R). Its expression and functionality were confirmed with flow cytometry using specific antibody and recombinant IL-17R, respectively. In addition, L. lactis were engineered into multifunctional bacteria that simultaneously express two binders from pNBBX plasmid. First, binders of IL-17R were combined with binder of IL-17. Second, binders of IL-23R were combined with binders of IL-23. The dual functionality of the bacteria was confirmed by flow cytometry using corresponding targets, namely the recombinant receptors IL-17R and IL-23R or the p19 subunit of IL-23. Binding of IL-17 was confirmed by ELISA. With the latter, 97% of IL-17 was removed from solution by 2 × 109 recombinant bacteria. Moreover, multifunctional bacteria targeting IL-17/IL-17R prevented IL-17A-mediated activation of downstream signaling pathways in HEK-Blue IL-17 cell model. Thus, we have developed several multifunctional L. lactis capable of targeting multiple factors of the IL-23/Th17 proinflammatory axis. This represents a novel therapeutic strategy with synergistic potential for the treatment of intestinal inflammations.

• MeSH
• albuminy metabolismus   MeSH
• cytokiny * metabolismus   MeSH
• imunologické faktory MeSH
• interleukin-17 metabolismus   MeSH
• interleukin-23 chemie   metabolismus   MeSH
• Lactococcus lactis * genetika   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• rekombinantní proteiny metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH