RESUMO
The structural dynamics of the interactions between defensins or lysozymes and various saccharide chains that are covalently linked to lipids or proteins were analyzed in relation to the sub-molecular architecture of the carbohydrate binding sites of lectins. Using tissue materials from rare and endangered domestic animals as well as from dogs it was possible to compare these results with data obtained from a human glioblastoma tissue. The binding mechanisms were analyzed on a cellular and a sub-molecular size level using biophysical techniques (e.g. NMR, AFM, MS) which are supported by molecular modeling tools. This leads to characteristic structural patterns being helpful to understand glyco-biochemical pathways in which galectins, defensins or lysozymes are involved. Carbohydrate chains have a distinct impact on cell differentiation, cell migration and immunological processes. The absence or the presence of sialic acids and the conformational dynamics in glycans are often correlated with zoonoses such as influenza- and coronavirus-infections. Receptor-sensitive glycomimetics could be a solution. The new findings concerning the function of galectin-3 in the nucleus in relation to differentiation processes can be understood when the binding specificity of neuroleptic molecules as well as the interactions between proteins and nucleic acids are describable on a sub-molecular size level.
RESUMO
Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4PHD6) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4PHD6, and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3PHD7). Analysis of genomic localization of endogenous MLL4 and ectopically expressed TET3 in mouse embryonic stem cells reveals a high degree overlap on active enhancers and suggests a potential functional relationship of MLL4 and TET3.
Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Histona-Lisina N-Metiltransferase , Ligação Proteica , Humanos , Dioxigenases/metabolismo , Dioxigenases/química , Dioxigenases/genética , Animais , Camundongos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Sítios de Ligação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genéticaRESUMO
Small heat shock proteins (sHsps) play diverse roles in the stress response and maintenance of cellular functions. The Ustilago maydis genome codes for few sHsps. Among these, Hsp12 has previously been demonstrated to be involved in the pathogenesis of the fungus by our group. In the present study we further investigated the biological function of the protein in the pathogenic development of U. maydis. Analysis of the primary amino acid sequence of Hsp12 in combination with spectroscopic methods to analyse secondary protein structures revealed an intrinsically disordered nature of the protein. We also carried out detailed analysis on the protein aggregation prevention activity associated with Hsp12. Our data suggest Hsp12 has trehalose-dependent protein aggregation prevention activity. Through assaying the interaction of Hsp12 with lipid membranes in vitro we also showed the ability of U. maydis Hsp12 to induce stability in lipid vesicles. U. maydis hsp12 deletion mutants exhibited defects in the endocytosis process and delayed completion of the pathogenic life cycle. Therefore, U. maydis Hsp12 contributes to the pathogenic development of the fungus through its ability to relieve proteotoxic stress during infection as well as its membrane-stabilizing function.
Assuntos
Basidiomycota , Ustilago , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Agregados Proteicos , Basidiomycota/metabolismo , Ustilago/genética , Ustilago/metabolismo , Lipídeos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the α-helical native human insulin conformers, disrupting their aggregation. Furthermore, it also increases the insulin secretion. This multipolar effect coupled with its non-toxic nature could be useful for developing highly bioactive, targeted and biostable therapeutic insulin.
Assuntos
Insulinas , Corantes de Rosanilina , Humanos , Chaperonas MolecularesRESUMO
Antimicrobial peptides (AMPs) with cell membrane lysing capability are considered potential candidates for the development of the next generation of antibiotics. Designing novel AMPs requires an in-depth understanding of the mechanism of action of the peptides. In this work, we used various biophysical techniques including 31P solid-state NMR to examine the interaction of model membranes with amphipathic de novo-designed peptides. Two such peptides, MSI-78 and VG16KRKP, were designed with different hydrophobicity and positive charges. The model lipid membranes were constituted by mixing lipids of varying degrees of 'area per lipid' (APL), which directly affected the packing properties of the membrane. The observed emergence of the isotropic peak in 31P NMR spectra as a function of time is a consequence of the fragmentation of the membrane mediated by the peptide interaction. The factors such as the charges, overall hydrophilicity of the AMPs, as well as lipid membrane packing, contributed to the kinetics of membrane fragmentation. Furthermore, we anticipate the designed AMPs follow the carpet and toroidal pore mechanisms when lysing the cell membrane. This study highlights the significance of the effect of the overall charges and the hydrophobicity of the novel AMPs designed for antimicrobial activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Peptídeos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Lipídeos , Bicamadas Lipídicas/químicaRESUMO
The treatment of invasive drug-resistant and potentially life-threatening fungal infections is limited to few therapeutic options that are usually associated with severe side effects. The development of new effective antimycotics with a more tolerable side effect profile is therefore of utmost clinical importance. Here, we used a combination of complementary in vitro assays and structural analytical methods to analyze the interaction of the de novo antimicrobial peptide VG16KRKP with the sterol moieties of biological cell membranes. We demonstrate that VG16KRKP disturbs the structural integrity of fungal membranes both invitro and in model membrane system containing ergosterol along with phosphatidylethanolamine lipid and exhibits broad-spectrum antifungal activity. As revealed by systematic structure-function analysis of mutated VG16KRKP analogs, a specific pattern of basic and hydrophobic amino acid side chains in the primary peptide sequence determines the selectivity of VG16KRKP for fungal specific membranes.
Assuntos
Antifúngicos , Ergosterol , Antifúngicos/química , Antifúngicos/farmacologia , Membrana Celular/metabolismo , Ergosterol/química , Peptídeos/química , Peptídeos/farmacologia , Esteróis/metabolismoRESUMO
Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.
Assuntos
Anti-Infecciosos , Infecções Oculares Bacterianas , Ceratite , Antibacterianos/farmacologia , Peptídeos Antimicrobianos , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções Oculares Bacterianas/microbiologia , Humanos , Ceratite/tratamento farmacológico , Ceratite/metabolismo , Ceratite/microbiologia , Pseudomonas , Pseudomonas aeruginosaRESUMO
Global rise of infections and deaths caused by drug-resistant bacterial pathogens are among the unmet medical needs. In an age of drying pipeline of novel antibiotics to treat bacterial infections, antimicrobial peptides (AMPs) are proven to be valid therapeutics modalities. Direct in vivo applications of many AMPs could be challenging; however, works are demonstrating encouraging results for some of them. In this review article, we discussed 3-D structures of potent AMPs e.g., polymyxin, thanatin, MSI, protegrin, OMPTA in complex with bacterial targets and their mode of actions. Studies on human peptide LL37 and de novo-designed peptides are also discussed. We have focused on AMPs which are effective against drug-resistant Gram-negative bacteria. Since treatment options for the infections caused by super bugs of Gram-negative bacteria are now extremely limited. We also summarize some of the pertinent challenges in the field of clinical trials of AMPs.
Assuntos
Peptídeos Antimicrobianos , Bactérias Gram-Negativas , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias , HumanosRESUMO
Substrate-based sirtuin inhibitors target bacterial genome and RNA and provide a promising approach to address bacterial resistance issues, if cellular internalisation can be achieved. We designed N-trifluoroacetyl lysine and N-thioacetyl lysine peptides (KP 13, KP 15 and KP 24) as inhibitors of bacterial sirtuins and their cell-penetrating peptide conjugates Tat KP 13, Tat KP 15 and Tat KP 24. The conjugated peptides were successfully internalised and showed signs of bacterial transcription inhibition resulting in enhanced antibacterial potency against model Gram negative and Gram positive pathogens. Synergistic activity in combination with streptomycin and polymyxin B has also been established. These peptides were effective in inhibiting biofilm formation and eradicating preformed biofilms. Morphological analysis using both SEM and TEM showed bacterial membrane disruption. Calcein dye leakage analysis established the selectivity of these peptides to bacterial membranes. This study documents the first report of the application of substrate-based sirtuin inhibitors as antimicrobial therapeutics.
Assuntos
Antibacterianos/síntese química , Peptídeos Penetradores de Células/química , Lisina/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Desenho de Fármacos , Escherichia coli/fisiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Sirtuínas/antagonistas & inibidores , Sirtuínas/metabolismo , Staphylococcus aureus/fisiologia , Lipossomas Unilamelares/metabolismoRESUMO
Alzheimer's disease (AD) is a severe neurodegenerative disorder caused by abnormal accumulation of toxic amyloid plaques of the amyloid-beta (Aß) or the tau proteins in the brain. The plaque deposition leading to the collapse of the cellular integrity is responsible for a myriad of surface phenomena acting at the neuronal lipid interface. Recent years have witnessed dysfunction of the blood-brain barriers (BBB) associated with AD. Several studies support the idea that BBB acts as a platform for the formation of misfolded Aß peptide, promoting oligomerization and fibrillation, compromising the overall integrity of the central nervous system. While the amyloid plaque deposition has been known to be responsible for the collapse of the BBB membrane integrity, the causal effect relationship between BBB and Aß amyloidogenesis remains unclear. In this study, we have used physiologically relevant synthetic model membrane systems to gain atomic insight into the functional aspects of the lipid interface. Here, we have used a minimalist BBB mimic, POPC/POPG/cholesterol/GM1, to compare with the native BBB (total lipid brain extract (TLBE)), to understand the molecular events occurring in the membrane-induced Aß40 amyloid aggregation. Our study showed that the two membrane models accelerated the Aß40 aggregation kinetics with differential secondary structural transitions of the peptide. The observed structural transitions are defined by the lipid compositions, which in turn undermines the differences in lipid surface phenomena, leading to peptide induced cellular toxicity in the neuronal membrane.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Humanos , Placa AmiloideRESUMO
The formation and accumulation of amyloid aggregates are the phenomena that accompany amyloidoses, which are currently untreatable and include Alzheimer's and Parkinson's diseases, diabetes mellitus, non-neuropathic lysozyme systemic amyloidosis, and others. One of the very promising therapeutic approaches seems to be an inhibition of amyloid formation and/or clearance of amyloid aggregates. Small molecules have a great potential to interfere with amyloid fibrillation of peptides and polypeptides, which can be improved by connection of cyclic structures into single multicyclic molecules and their dimerization. In our study, we focused on heterodimers consisting of 7-methoxytacrine (7-MEOTA) and 2-aminobenzothiazole (BTZ) parent molecules connected by an aliphatic linker. Using in vitro and in silico methods, we investigated the ability of studied compounds to inhibit the amyloid aggregation of hen egg white lysozyme. Heterodimerization led to significant improvement of inhibitory activity compared to that of the parent molecules. The efficiency of the heterodimers varied; the most effective inhibitor contained the longest linker, eight carbons long. We suggest that binding of a heterodimer to a lysozyme blocks the interaction between the ß-domain and C-helix region essential for the formation of amyloid cross-ß structure. Elongation of the linker ultimately enhances the compound's ability to prevent this interaction by allowing the BTZ part of the heterodimer to bind more effectively, increasing the compound's binding affinity, and also by greater steric obstruction. This study represents an important contribution to the recent rational design of potential lead small molecules with anti-amyloid properties, and the heterodimers studied are prospective candidates for the treatment of systemic lysozyme amyloidosis and other amyloid-related diseases.
Assuntos
Amiloide , Amiloidose , Proteínas Amiloidogênicas , Humanos , Estudos Prospectivos , Tacrina/análogos & derivadosRESUMO
The emergence of multidrug-resistant pathogens and their rapid adaptation against new antibiotics is a major challenge for scientists and medical professionals. Different approaches have been taken to combat this problem, which includes rationally designed potent antimicrobial peptides (AMPs) and several nanoparticles and quantum dots. AMPs are considered as a new generation of super antibiotics that hold enormous potential to fight against bacterial resistance by the rapidly killing planktonic as well as their biofilm form while keeping low toxicity profile against eukaryotic cells. Various nanoparticles and quantum dots have proved their effectiveness against a vast array of infections and diseases. Conjugation and functionalization of nanoparticles with potentially active antimicrobial peptides have added advantages that widen their applications in the field of drug discovery as well as delivery system including imaging and diagnostics. This article reviews the current progress and implementation of different nanoparticles and quantum dots conjugated antimicrobial peptides in terms of bio-stability, drug delivery, and therapeutic applications.
Assuntos
Nanopartículas/química , Nanotecnologia/métodos , Proteínas Citotóxicas Formadoras de Poros/química , Medicina de Precisão/métodos , Pontos Quânticos/química , Animais , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Bibliometria , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Diagnóstico por Imagem/métodos , Estabilidade de Medicamentos , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , História do Século XX , História do Século XXI , Humanos , Camundongos , Nanopartículas/toxicidade , Nanotecnologia/história , Plâncton/efeitos dos fármacos , Plâncton/crescimento & desenvolvimento , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Medicina de Precisão/história , Pontos Quânticos/toxicidadeRESUMO
Pseudomonas aeruginosa is an opportunistic bacterial pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis. Several clinically isolated P. aeruginosa strains were found to be resistant to three or more antimicrobial classes indicating the importance of identifying new antimicrobials active against this pathogen. Here, we characterized the antimicrobial activity and the action mechanisms against P. aeruginosa of two natural isoforms of the antimicrobial peptide cecropin B, both isolated from the silkworm Bombyx mori. These cecropin B isoforms differ in a single amino acid substitution within the active portion of the peptide, so that the glutamic acid of the E53 CecB variant is replaced by a glutamine in the Q53 CecB isoform. Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa, with Q53 CecB displaying greater activity compared with the E53 CecB isoform. Biophysical analyses, live-cell NMR, and molecular-dynamic-simulation studies indicated that both peptides might act as membrane-interacting elements, which can disrupt outer-membrane organization, facilitating their translocation toward the inner membrane of the bacterial cell. Our data also suggest that the amino acid variation of the Q53 CecB isoform represents a critical factor in stabilizing the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. Its high stability to pH and temperature variations, tolerance to high salt concentrations, and low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa.
Assuntos
Substituição de Aminoácidos , Anti-Infecciosos/farmacologia , Bombyx/metabolismo , Proteínas de Insetos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Membrana Externa Bacteriana/efeitos dos fármacos , Bombyx/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Estabilidade de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Insetos/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Dinâmica Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , TermodinâmicaRESUMO
A synthetic antimicrobial peptide library based on the human autophagy 16 polypeptide has been developed. Designed acetylated peptides bearing lipids of different chain lengths resulted in peptides with enhanced potency compared to the parent Atg16. A 21-residue fragment of Atg16 conjugated to 4-methylhexanoic acid (K30) emerged as the most potent antibacterial, with negligible hemolysis. Several studies, including microscopy, dye leakage, and ITC, were conducted to gain insight into the antibacterial mechanism of action of the peptide. Visual inspection using both SEM and TEM revealed the membranolytic effect of the peptide on bacterial cells. The selectivity of the peptide against bacterial cell membranes was also proven using dye leakage assays. ITC analysis revealed the exothermic nature of the binding interaction of the peptide to D8PG micelles. The three-dimensional solution NMR structure of K30 in complex with dioctanoylphosphatidylglycerol (D8PG) micelles revealed that the peptide adopts a helix-loop-helix structure in the presence of anionic membrane lipids mimicking bacterial membranes. Intermolecular NOEs between the peptide and lipid deciphered the location of the peptide in the bound state, which was subsequently supported by the paramagnetic relaxation enhancement (PRE) NMR experiment. Collectively, these results describe the structure-function relationship of the peptide in the bacterial membrane.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/farmacologia , Acilação , Sequência de Aminoácidos , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacosRESUMO
Two-dimensional (2D) tungsten disulfide (WS2) quantum dots offer numerous promising applications in materials and optoelectronic sciences. Additionally, the catalytic and photoluminescence properties of ultra-small WS2 nanoparticles are of potential interest in biomedical sciences. Addressing the use of WS2 in the context of infection, the present study describes the conjugation of two potent antimicrobial peptides with WS2 quantum dots, as well as the application of the resulting conjugates in antimicrobial therapy and bioimaging. In doing so, we determined the three-dimensional solution structure of the quantum dot-conjugated antimicrobial peptide by a series of high-resolution nuclear magnetic resonance (NMR) techniques, correlating this to the disruption of both model lipid and bacterial membranes, and to several key biological performances, including antimicrobial and anti-biofilm effects, as well as cell toxicity. The results demonstrate that particle conjugation enhances the antimicrobial and anti-biofilm potency of these peptides, effects inferred to be due to multi-dendate interactions for the conjugated peptides. As such, our study provides information on the mode-of-action of such conjugates, laying the foundation for their potential use in treatment and monitoring of infections.
Assuntos
Anti-Infecciosos/farmacologia , Diagnóstico por Imagem , Dissulfetos/química , Peptídeos/química , Pontos Quânticos/química , Tungstênio/química , Sequência de Aminoácidos , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/ultraestruturaRESUMO
Antimicrobial peptides have gained widespread attention as an alternative to the conventional antibiotics for combating microbial infections. Here, we report a detailed structure-function correlation of two nontoxic, nonhemolytic, and salt-tolerant de novo designed seven-residue leucine-lysine-based peptides, NH2LKWLKKLCONH2 (P4) and NH2LRWLRRLCONH2 (P5), with strong antimicrobial and antifungal activity. Biological experiments, low- and high-resolution spectroscopic techniques in conjunction with molecular dynamics simulation studies, could establish the structure-function correlation. The peptides are unstructured both in water and in bacterial membrane mimicking environment, suggesting that the secondary structure does not play a major role in their activity. Our studies could justify the probable membranolytic mode of action for killing the pathogens. Attempts to understand the mode of action of these small AMPs is fundamental in the rational design of more potential therapeutic molecules beyond serendipity in the future.