Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

• MeSH
• buněčná membrána metabolismus   chemie   MeSH
• buněčné linie MeSH
• lidé MeSH
• maleáty chemie   MeSH
• membránové mikrodomény metabolismus   chemie   MeSH
• membránové proteiny * chemie   metabolismus   MeSH
• peptidy * chemie   MeSH
• polystyreny chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

An advantageous alternative to the use of detergents in biochemical studies on membrane proteins are the recently developed styrene-maleic acid (SMA) amphipathic copolymers. In our recent study [1] we demonstrated that using this approach, most T cell membrane proteins were fully solubilized (presumably in small nanodiscs), while two types of raft proteins, GPI-anchored proteins and Src family kinases, were mostly present in much larger (>250 nm) membrane fragments markedly enriched in typical raft lipids, cholesterol and lipids containing saturated fatty acid residues. In the present study we demonstrate that disintegration of membranes of several other cell types by means of SMA copolymer follows a similar pattern and we provide a detailed proteomic and lipidomic characterization of these SMA-resistant membrane fragments (SRMs).

• MeSH
• buněčná membrána chemie   MeSH
• maleáty analýza   chemie   MeSH
• mastné kyseliny analýza   MeSH
• membránové mikrodomény MeSH
• membránové proteiny chemie   MeSH
• polystyreny * chemie   MeSH
• proteomika * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

• MeSH
• buněčné linie MeSH
• cytopatogenní efekt virový MeSH
• exprese genu MeSH
• genový knockdown MeSH
• interakce hostitele a patogenu * genetika   imunologie   MeSH
• interakční proteinové domény a motivy MeSH
• interferony metabolismus   MeSH
• klíšťová encefalitida genetika   imunologie   metabolismus   virologie   MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• multigenová rodina MeSH
• náchylnost k nemoci MeSH
• odolnost vůči nemocem genetika   imunologie   MeSH
• replikace viru MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• viry klíšťové encefalitidy fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

• MeSH
• Bacillus thuringiensis enzymologie   ultrastruktura   MeSH
• cytokiny chemie   genetika   MeSH
• Escherichia coli enzymologie   ultrastruktura   MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• leukocyty mononukleární chemie   enzymologie   MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   ultrastruktura   MeSH
• nukleotidy biosyntéza   chemie   genetika   MeSH
• přirozená imunita genetika   MeSH
• substrátová specifita MeSH
• Thermotoga maritima enzymologie   ultrastruktura   MeSH
• Vibrio cholerae enzymologie   ultrastruktura   MeSH
• vztahy mezi strukturou a aktivitou * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.

• MeSH
• bodová mutace * MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• molekulární struktura MeSH
• nukleotidy cyklické chemie   metabolismus   MeSH
• proteinové domény MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane, is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and super-resolution imaging and flow cytometry, we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics are further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of nonpalmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• glycin genetika   metabolismus   MeSH
• interferenční mikroskopie MeSH
• Jurkat buňky MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• mutace MeSH
• prolin genetika   metabolismus   MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• T-lymfocyty metabolismus   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• iontový transport MeSH
• konformace proteinů * MeSH
• mastné kyseliny chemie   metabolismus   MeSH
• membránové proteiny chemie   MeSH
• mitochondriální proteiny chemie   metabolismus   MeSH
• myši MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky * MeSH
• stabilita proteinů MeSH
• uncoupling protein 2 chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Transcript levels for selected ATP synthase membrane FO-subunits-including DAPIT-in INS-1E cells were found to be sensitive to lowering glucose down from 11 mM, in which these cells are routinely cultured. Depending on conditions, the diminished mRNA levels recovered when glucose was restored to 11 mM; or were elevated during further 120 min incubations with 20-mM glucose. Asking whether DAPIT expression may be elevated by hyperglycemia in vivo, we studied mice with hyaluronic acid implants delivering glucose for up to 14 days. Such continuous two-week glucose stimulations in mice increased DAPIT mRNA by >5-fold in isolated pancreatic islets (ATP synthase F1α mRNA by 1.5-fold). In INS-1E cells, the glucose-induced ATP increment vanished with DAPIT silencing (6% of ATP rise), likewise a portion of the mtDNA-copy number increment. With 20 and 11-mM glucose the phosphorylating/non-phosphorylating respiration rate ratio diminished to ~70% and 96%, respectively, upon DAPIT silencing, whereas net GSIS rates accounted for 80% and 90% in USMG5/DAPIT-deficient cells. Consequently, the sufficient DAPIT expression and complete ATP synthase assembly is required for maximum ATP synthesis and mitochondrial biogenesis, but not for insulin secretion as such. Elevated DAPIT expression at high glucose further increases the ATP synthesis efficiency.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• beta-buňky cytologie   účinky léků   metabolismus   MeSH
• buněčné kultury MeSH
• buněčné linie MeSH
• glukosa aplikace a dávkování   farmakologie   MeSH
• konformace proteinů MeSH
• krysa rodu rattus MeSH
• kyselina hyaluronová chemie   MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• mitochondriální DNA účinky léků   genetika   MeSH
• mitochondrie účinky léků   genetika   metabolismus   MeSH
• molekulární modely MeSH
• myši MeSH
• upregulace * MeSH
• variabilita počtu kopií segmentů DNA účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fibroblast activation protein (FAP) is a non-classical serine protease expressed predominantly in conditions accompanied by tissue remodeling, particularly cancer. Due to its plasma membrane localization, FAP represents a promising molecular target for tumor imaging and treatment. The unique enzymatic activity of FAP facilitates development of diagnostic and therapeutic tools based on molecular recognition of FAP by substrates and small-molecule inhibitors, in addition to conventional antibody-based strategies. In this review, we provide background on the pathophysiological role of FAP and discuss its potential for diagnostic and therapeutic applications. Furthermore, we present a detailed analysis of the structural patterns crucial for substrate and inhibitor recognition by the FAP active site and determinants of selectivity over the related proteases dipeptidyl peptidase IV and prolyl endopeptidase. We also review published data on targeting of the tumor microenvironment with FAP antibodies, FAP-targeted prodrugs, activity-based probes and small-molecule inhibitors. We describe use of a recently developed, selective FAP inhibitor with low-nanomolar potency in inhibitor-based targeting strategies including synthetic antibody mimetics based on hydrophilic polymers and inhibitor conjugates for PET imaging. In conclusion, recent advances in understanding of the molecular structure and function of FAP have significantly contributed to the development of several tools with potential for translation into clinical practice.

• MeSH
• dipeptidylpeptidasa 4 metabolismus   MeSH
• fibroblasty metabolismus   MeSH
• katalytická doména MeSH
• lidé MeSH
• membránové proteiny chemie   účinky léků   metabolismus   MeSH
• molekulární struktura MeSH
• nádorové mikroprostředí MeSH
• nádory diagnóza   metabolismus   terapie   MeSH
• prekurzory léčiv MeSH
• serinové endopeptidasy chemie   účinky léků   metabolismus   MeSH
• substrátová specifita MeSH
• želatinasy chemie   účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

• MeSH
• Actinobacillus pleuropneumoniae chemie   patogenita   MeSH
• bakteriální proteiny chemie   genetika   MeSH
• bakteriální toxiny chemie   MeSH
• infekce bakteriemi rodu Actinobacillus veterinární   MeSH
• membránové proteiny chemie   MeSH
• Neisseria meningitidis chemie   MeSH
• posttranslační úpravy proteinů * MeSH
• prasata MeSH
• vápník metabolismus   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH