Antimikrobiális peptidek szerkezetének és hatásmechanizmusának vizsgálata szilárd fázisú mágneses magrezonanciával = Investigation of the Structure and Mode of Action of Antimicrobial Peptides by Solid-State Nuclear Magnetic Resonance

Az antimikrobiális peptidek (AMP) rövid, 15-40 aminosavból álló peptidláncok, amelyek a kórokozók sejtmembránjában ioncsatornákat, pórusokat képezve fejtik ki baktériumölő hatásukat. Szilárdfázisú NMR spektroszkópiai vizsgálatok segítségével megállapítottuk, hogy a maximin 4 nevű 27 aminosavból álló (GIGGVLLSAGKAALKGLAKVLAEKYAN-NH2), baktériumsejtek iránt nagyfokú szelektivitást mutató peptid lipid kettősrétegekben alfa-hélix szerkezetet ölt. A peptid és a lipid fejcsoportok közötti távolságmeghatározásra irányuló, valamint a peptid lipid kettősrétegben való orientációjának meghatározását célzó vizsgálatok a molekula mély penetrációját jelzik a lipid alkil láncok közé mind negatív töltésű, mind zwitterionos membránban. A szilárd fázisú NMR mérésekkel párhuzamosan negatív töltésű SDS micellákban és metanolos közegben végzett oldatfázisú NMR mérések alapján a hélix az aminosavszekvencia közepén megtörik, és V alakra emlékeztető konformációt vesz fel. Bár az így előálló hélix-kanyar-hélix motívum a kétféle környezetben hasonló, markáns eltérések tapasztalhatók a kanyart stabilizáló kölcsönhatásokban, valamint a molekulafelszín töltéseloszlásában. Feltételezhetően ez utóbbi tényező az, ami meghatározó módon befolyásolja a peptidnek a negatív töltésű (prokarióta-típusú) illetve elektromosan semleges (eukarióta-típusú) membránokkal való kölcsönhatását, és ezáltal a peptid bakteriális szelektivitását. | Antimicrobial peptides (AMP) are short, 15-40-residue peptide chains, whose mode of action is thought to be ion channel or pore formation in the bacterial cytoplasmic membrane. Using solid-state NMR methodologies, we have established that maximin 4, a 27-residue AMP (GIGGVLLSAGKAALKGLAKVLAEKYAN-NH2) of high bacterial selectivity, in lipid bilayers adopts an alpha-helical conformation. Peptide-lipid distance measurements and the determination of peptide orientation in lipid bilayers show a deep penetration of maximin 4 into both zwitterionic and negatively charged membranes. In combination with the solid-state NMR measurements, solution NMR study of maximin 4 in negatively charged SDS micelles and in the presence of methanol show a helix-break-helix conformation and a V-shaped structure. Even though the overall topology of the peptide is similar, the atomic details of the structure in SDS and in methanol are markedly different. Major differences are observed in terms of the forces stabilizing the kink region as well as the charge distribution of the structures. The latter should have a profound effect on the interaction of maximin 4 with negatively charged (prokaryotic) vs. electronically neutral (eukaryotic) membranes and likely have implications for the bacterial selectivity of the peptide

A mechanistic view of lipid membrane disrupting effect of PAMAM dendrimers

The effect of 5th generation polyamidoamine (PAMAM G5) dendrimers on multilamellar dipalmitoylphosphocholine (DPPC) vesicles was investigated. PAMAM was added in two different concentration to the lipids (10-3 and 10-2 dendrimer/lipid molar ratios). The thermal behavior of the evolved systems was characterized by DSC; while the structure and the morphology were investigated with small- and wide-angel X-ray scattering (SWAXS), freeze-fracture electron microscopy (FFTEM) and phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy, respectively. IR spectroscopy was used to study the molecular interactions between PAMAM and DPPC. The obtained results show that the dendrimers added in 10-3 molar ratio to the lipids generate minor perturbations in the multilamellar structure and thermal character of liposomes, while added in 10-2 molar ratio dendrimers cause major disturbance in the vesicular system. The terminal amino groups of the dendrimers are in strong interaction with the phosphate headgroups and through this binding dendrimers disrupt the regular multilamellar structure of DPPC. Besides highly swollen, fragmented bilayers, small vesicles are formed

Change of the kinetics of inclusion in cucurbit[7]uril upon hydrogenation and methylation of palmatine

The inclusion of protonated (-)-tetrahydropalmatine (THP+) and dehydrocorydaline (DHC+), natural alkaloids, in the cavity of cucurbit[7]uril was monitored in real time by a spectrofluorimetric method in water at various temperatures. Both guests produced 1:1 complexes in enthalpy controlled processes without any detectable intermediates. The tight entrance of CB7 imposed substantial steric hindrance for encapsulation making the entry into the host several orders of magnitude slower than diffusion. Despite the approximate to 6 kJ mol(-1) lower activation enthalpy, the rate constant of THP+ ingression into CB7 was about 44-fold smaller at 298 K than that of DHC+ as a consequence of the considerably negative activation entropy of the former binding. The egression rates of the two studied alkaloids differed to a much lesser extent because the lower energy barrier of THP+ release was almost compensated by the unfavourable activation entropy. In comparison with the kinetics of the reversible confinement of the palmatine parent compound, the presence of the methyl substituent on the aromatic heterocyclic ring in DHC+ barely modified the rate constant of entry into CB7 but caused about 10-fold increase in the dissociation rate at 298 K

Different modes of barrel opening suggest a complex pathway of ligand binding in human gastrotropin

Gastrotropin, the intracellular carrier of bile salts in the small intestine, binds two ligand molecules simultaneously in its internal cavity. The molecular rearrangements required for ligand entry are not yet fully clear. To improve our understanding of the binding process we combined molecular dynamics simulations with previously published structural and dynamic NMR parameters. The resulting ensembles reveal two distinct modes of barrel opening with one corresponding to the transition between the apo and holo states, whereas the other affecting different protein regions in both ligation states. Comparison of the calculated structures with NMR-derived parameters reporting on slow conformational exchange processes suggests that the protein undergoes partial unfolding along a path related to the second mode of the identified barrel opening motion. Introductio

Structural plasticity of the Salmonella FliS flagellar export chaperone

The Salmonella FliS flagellar export chaperone is a highly α-helical protein. Proteolytic experiments suggest that FliS has a compact core. However, the calorimetric melting profile of FliS does not show any melting transition in the 25-110 °C temperature range. CD measurements reveal that FliS is losing its helical structure over a broad temperature range upon heating. These observations indicate that FliS unfolds in a non-cooperative way and its native state shows features reminiscent of the molten globule state of proteins possessing substantial structural plasticity. As FliS has several binding partners within the cell, conformational adaptability seems to be an essential requirement to fulfill its multiple roles. This article is protected by copyright. All rights reserved

In Silico Model Estimates the Clinical Trial Outcome of Cancer Vaccines

Over 30 years after the first cancer vaccine clinical trial (CT), scientists still search the missing link between immunogenicity and clinical responses. A predictor able to estimate the outcome of cancer vaccine CTs would greatly benefit vaccine development. Published results of 94 CTs with 64 therapeutic vaccines were collected. We found that preselection of CT subjects based on a single matching HLA allele does not increase immune response rates (IRR) compared with non-preselected CTs (median 60% vs. 57%, p = 0.4490). A representative in silico model population (MP) comprising HLA-genotyped subjects was used to retrospectively calculate in silico IRRs of CTs based on the percentage of MP-subjects having epitope(s) predicted to bind ≥ 1–4 autologous HLA allele(s). We found that in vitro measured IRRs correlated with the frequency of predicted multiple autologous allele-binding epitopes (AUC 0.63–0.79). Subgroup analysis of multi-antigen targeting vaccine CTs revealed correlation between clinical response rates (CRRs) and predicted multi-epitope IRRs when HLA threshold was ≥ 3 (r = 0.7463, p = 0.0004) but not for single HLA allele-binding epitopes (r = 0.2865, p = 0.2491). Our results suggest that CRR depends on the induction of broad T-cell responses and both IRR and CRR can be predicted when epitopes binding to multiple autologous HLAs are considered

A Peptide Vaccine Candidate Tailored to Individuals' Genetics Mimics the Multi-Targeted T Cell Immunity of COVID-19 Convalescent Subjects

Long-term immunity to coronaviruses likely stems from T cell activity. We present here a novel approach for the selection of immunoprevalent SARS-CoV-2-derived T cell epitopes using an in silico cohort of HLA-genotyped individuals with different ethnicities. Nine 30-mer peptides derived from the four major structural proteins of SARS-CoV-2 were selected and included in a peptide vaccine candidate to recapitulate the broad virus-specific T cell responses observed in natural infection. PolyPEPI-SCoV-2-specific, polyfunctional CD8+ and CD4+ T cells were detected in each of the 17 asymptomatic/mild COVID-19 convalescents' blood against on average seven different vaccine peptides. Furthermore, convalescents' complete HLA-genotype predicted their T cell responses to SARS-CoV-2-derived peptides with 84% accuracy. Computational extrapolation of this relationship to a cohort of 16,000 HLA-genotyped individuals with 16 different ethnicities suggest that PolyPEPI-SCoV-2 vaccination will likely elicit multi-antigenic T cell responses in 98% of individuals, independent of ethnicity. PolyPEPI-SCoV-2 administered with Montanide ISA 51 VG generated robust, Th1-biased CD8+, and CD4+ T cell responses against all represented proteins, as well as binding antibodies upon subcutaneous injection into BALB/c and hCD34+ transgenic mice modeling human immune system. These results have implications for the development of global, highly immunogenic, T cell-focused vaccines against various pathogens and diseases

Structural insights into the tyrosine phosphorylation-mediated inhibition of SH3 domain-ligand interactions.

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains have been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr-89 and Tyr-134 in ABL1, or the homologous residues Tyr-116 and Tyr-161 in ABL2 induce only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine-phosphorylation induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed that the residues phosphorylated in our model systems are not only well conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome

Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

Background: Glutamate (Glu) and c-aminobutyric acid (GABA) transporters play important roles in regulating neuronal activity. Glu is removed from the extracellular space dominantly by glial transporters. In contrast, GABA is mainly taken up by neurons. However, the glial GABA transporter subtypes share their localization with the Glu transporters and their expression is confined to the same subpopulation of astrocytes, raising the possibility of cooperation between Glu and GABA transport processes. Methodology/Principal Findings: Here we used diverse biological models both in vitro and in vivo to explore the interplay between these processes. We found that removal of Glu by astrocytic transporters triggers an elevation in the extracellular level of GABA. This coupling between excitatory and inhibitory signaling was found to be independent of Glu receptor-mediated depolarization, external presence of Ca2+ and glutamate decarboxylase activity. It was abolished in the presence of non-transportable blockers of glial Glu or GABA transporters, suggesting that the concerted action of these transporters underlies the process. Conclusions/Significance: Our results suggest that activation of Glu transporters results in GABA release through reversal of glial GABA transporters. This transporter-mediated interplay represents a direct link between inhibitory and excitatory neurotransmission and may function as a negative feedback combating intense excitation in pathological conditions such as epilepsy or ischemia