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Dysregulation of the CXCL12/CXCR4 axis is implicated in autoimmune, inflammatory, and oncogenic diseases, positioning CXCR4 as a pivotal therapeutic target. We evaluated optimized variants of the specific endogenous CXCR4 antagonist, EPI-X4, addressing existing challenges in stability and potency. Our structure-activity relationship study investigates the conjugation of EPI-X4 derivatives with long-chain fatty acids, enhancing serum albumin interaction and receptor affinity. Molecular dynamic simulations revealed that the lipid moieties stabilize the peptide-receptor interaction through hydrophobic contacts at the receptor's N-terminus, anchoring the lipopeptide within the CXCR4 binding pocket and maintaining essential receptor interactions. Accordingly, lipidation resulted in increased receptor affinities and antagonistic activities. Additionally, by interacting with human serum albumin lipidated EPI-X4 derivatives displayed sustained stability in human plasma and extended circulation times in vivo. Selected candidates showed significant therapeutic potential in human retinoblastoma cells in vitro and in ovo, with our lead derivative exhibiting higher efficacies compared to its non-lipidated counterpart. This study not only elucidates the optimization trajectory for EPI-X4 derivatives but also underscores the intricate interplay between stability and efficacy, crucial for delineating their translational potential in clinical applications.
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Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent invasive infection. Mycobacterium tuberculosis (Mtb) enters its host via the airways. AMPs released into the airways are therefore likely candidates to contribute to the clearance of Mtb immediately after infection. Since lysozyme is detectable in airway secretions, we evaluated its antimicrobial activity against Mtb. We demonstrate that lysozyme inhibits the growth of extracellular Mtb, including isoniazid-resistant strains. Lysozyme also inhibited the growth of non-tuberculous mycobacteria. Even though lysozyme entered Mtb-infected human macrophages and co-localized with the pathogen we did not observe antimicrobial activity. This observation was unlikely related to the large size of lysozyme (14.74 kDa) because a smaller lysozyme-derived peptide also co-localized with Mtb without affecting the viability. To evaluate whether the activity of lysozyme against extracellular Mtb could be relevant in vivo, we incubated Mtb with fractions of human serum and screened for antimicrobial activity. After several rounds of sub-fractionation, we identified a highly active fraction-component as lysozyme by mass spectrometry. In summary, our results identify lysozyme as an antimycobacterial protein that is detectable as an active compound in human serum. Our results demonstrate that the activity of AMPs against extracellular bacilli does not predict efficacy against intracellular pathogens despite co-localization within the macrophage. Ongoing experiments are designed to unravel peptide modifications that occur in the intracellular space and interfere with the deleterious activity of lysozyme in the extracellular environment.
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Macrófagos , Muramidasa , Mycobacterium tuberculosis , Muramidasa/farmacología , Muramidasa/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/metabolismo , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacosRESUMEN
Systemic AL amyloidosis is one of the most frequently diagnosed forms of systemic amyloidosis. It arises from mutational changes in immunoglobulin light chains. To explore whether these mutations may affect the structure of the formed fibrils, we determine and compare the fibril structures from several patients with cardiac AL amyloidosis. All patients are affected by light chains that contain an IGLV3-19 gene segment, and the deposited fibrils differ by the mutations within this common germ line background. Using cryo-electron microscopy, we here find different fibril structures in each patient. These data establish that the mutations of amyloidogenic light chains contribute to defining the fibril architecture and hence the structure of the pathogenic agent.
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Microscopía por Crioelectrón , Cadenas Ligeras de Inmunoglobulina , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Mutación , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/genética , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/patología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/química , Amiloide/metabolismo , Amiloide/genética , Amiloide/ultraestructura , Masculino , Femenino , Persona de Mediana EdadRESUMEN
Introduction: In septic patients the damage of the endothelial barrier is decisive leading to circulatory septic shock with disseminated vascular coagulation, edema and multiorgan failure. Hemadsorption therapy leads to rapid resolution of clinical symptoms. We propose that the isolation of proteins adsorbed to hemadsorption devices contributes to the identification of mediators responsible for endothelial barrier dysfunction. Material and methods: Plasma materials enriched to hemadsorption filters (CytoSorb®) after therapy of patients in septic shock were fractionated and functionally characterized for their effect on cell integrity, viability, proliferation and ROS formation by human endothelial cells. Fractions were further studied for their contents of oxidized nucleic acids as well as peptides and proteins by mass spectrometry. Results: Individual fractions exhibited a strong effect on endothelial cell viability, the endothelial layer morphology, and ROS formation. Fractions with high amounts of DNA and oxidized DNA correlated with ROS formation in the target endothelium. In addition, defined proteins such as defensins (HNP-1), SAA1, CXCL7, and the peptide bikunin were linked to the strongest additive effects in endothelial damage. Conclusion: Our results indicate that hemadsorption is efficient to transiently remove strong endothelial damage mediators from the blood of patients with septic shock, which explains a rapid clinical improvement of inflammation and endothelial function. The current work indicates that a combination of stressors leads to the most detrimental effects. Oxidized ssDNA, likely derived from mitochondria, SAA1, the chemokine CXCL7 and the human neutrophil peptide alpha-defensin 1 (HNP-1) were unique for their significant negative effect on endothelial cell viability. However, the strongest damage effect occurred, when, bikunin - cleaved off from alpha-1-microglobulin was present in high relative amounts (>65%) of protein contents in the most active fraction. Thus, a relevant combination of stressors appears to be removed by hemadsorption therapy which results in fulminant and rapid, though only transient, clinical restitution.
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Estrés del Retículo Endoplásmico , Choque Séptico , Humanos , Choque Séptico/metabolismo , Choque Séptico/terapia , Choque Séptico/sangre , Biomarcadores , alfa-Globulinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Supervivencia Celular , Células Endoteliales/metabolismo , MasculinoRESUMEN
Amyloid resistance is the inability or the reduced susceptibility of an organism to develop amyloidosis. In this study we have analysed the molecular basis of the resistance to systemic AApoAII amyloidosis, which arises from the formation of amyloid fibrils from apolipoprotein A-II (ApoA-II). The disease affects humans and animals, including SAMR1C mice that express the C allele of ApoA-II protein, whereas other mouse strains are resistant to development of amyloidosis due to the expression of other ApoA-II alleles, such as ApoA-IIF. Using cryo-electron microscopy, molecular dynamics simulations and other methods, we have determined the structures of pathogenic AApoAII amyloid fibrils from SAMR1C mice and analysed the structural effects of ApoA-IIF-specific mutational changes. Our data show that these changes render ApoA-IIF incompatible with the specific fibril morphologies, with which ApoA-II protein can become pathogenic in vivo.
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Amiloide , Amiloidosis , Apolipoproteína A-II , Animales , Ratones , Amiloide/química , Amiloide/genética , Amiloidosis/genética , Amiloidosis/metabolismo , Apolipoproteína A-II/química , Apolipoproteína A-II/genética , Microscopía por Crioelectrón , Alelos , Simulación de Dinámica Molecular , Mutación , Ratones MutantesRESUMEN
Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.
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Interferón Tipo I , Humanos , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transducción de SeñalRESUMEN
Macroautophagy/autophagy is a tightly regulated cellular process integral to homeostasis and innate immunity. As such, dysregulation of autophagy is associated with cancer, neurodegenerative disorders, and infectious diseases. While numerous factors that promote autophagy have been characterized, the key mechanisms that prevent excessive autophagy are less well understood. Here, we identify CSNK2/CK2 (casein kinase 2) as a negative regulator of autophagy. Pharmacological inhibition of CSNK2 activity or siRNA-mediated depletion of CSNK2 increased basal autophagic flux in cell lines and primary human lung cells. Vice versa, ectopic expression of CSNK2 reduced autophagic flux. Mechanistically, CSNK2 interacted with the FLN (filamin)-NHL domain-containing tripartite motif (TRIM) family members TRIM2, TRIM3 and TRIM71. Our data show that recruitment of CSNK2 to the C-terminal NHL domain of TRIM3 lead to its robust phosphorylation at serine 661 by CSNK2. A phosphorylation-defective mutant of TRIM3 was unable to reduce autophagosome numbers indicating that phosphorylation by CSNK2 is required for TRIM-mediated autophagy inhibition. All three TRIMs facilitated inactivation of the ULK1-BECN1 autophagy initiation complex by facilitating ULK1 serine 757 phosphorylation. Inhibition of CSNK2 promoted autophagy upon influenza A virus (IAV) and measles virus (MeV) infection. In line with this, targeting of CSNK2 or depletion of TRIM2, TRIM3 or TRIM71 enhanced autophagy-dependent restriction of IAV, MeV and human immunodeficiency virus 1 (HIV-1). Thus, our results identify the CSNK2-TRIM2, -TRIM3, -TRIM71 axis as a key regulatory pathway that limits autophagy. Targeting this axis may allow for therapeutic induction of autophagy against viral infections and in diseases associated with dysregulated autophagy.
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EPI-X4, a natural peptide CXCR4 antagonist, shows potential for treating inflammation and cancer, but its short plasma stability limits its clinical application. We aimed to improve the plasma stability of EPI-X4 analogues without compromising CXCR4 antagonism. Our findings revealed that only the peptide N-terminus is prone to degradation. Consequently, incorporating d-amino acids or acetyl groups in this region enhanced peptide stability in plasma. Notably, EPI-X4 leads 5, 27, and 28 not only retained their CXCR4 binding and antagonism but also remained stable in plasma for over 8 h. Molecular dynamic simulations showed that these modified analogues bind similarly to CXCR4 as the original peptide. To further increase their systemic half-lives, we conjugated these stabilized analogues with large polymers and albumin binders. These advances highlight the potential of the optimized EPI-X4 analogues as promising CXCR4-targeted therapeutics and set the stage for more detailed preclinical assessments.
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Infecciones por VIH , VIH-1 , Humanos , VIH-1/metabolismo , Péptidos/química , Receptores CXCR4/metabolismo , Albúminas/metabolismo , Transducción de Señal , Aminas/metabolismoRESUMEN
Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.
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Células Asesinas Naturales , Linfocitos T , Transducción Genética , Péptidos , Inmunoterapia Adoptiva/métodosRESUMEN
Systemic ATTR amyloidosis is an increasingly important protein misfolding disease that is provoked by the formation of amyloid fibrils from transthyretin protein. The pathological and clinical disease manifestations and the number of pathogenic mutational changes in transthyretin are highly diverse, raising the question whether the different mutations may lead to different fibril morphologies. Using cryo-electron microscopy, however, we show here that the fibril structure is remarkably similar in patients that are affected by different mutations. Our data suggest that the circumstances under which these fibrils are formed and deposited inside the body - and not only the fibril morphology - are crucial for defining the phenotypic variability in many patients.
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Neuropatías Amiloides Familiares , Deficiencias en la Proteostasis , Humanos , Amiloide/metabolismo , Neuropatías Amiloides Familiares/metabolismo , Microscopía por Crioelectrón , Prealbúmina/metabolismoRESUMEN
The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.
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Médula Ósea , Células Madre Mesenquimatosas , Humanos , Medios de Cultivo/farmacología , Suplementos Dietéticos , Ácido LácticoRESUMEN
Whooping cough is a severe childhood disease, caused by the bacterium Bordetella pertussis, which releases pertussis toxin (PT) as a major virulence factor. Previously, we identified the human antimicrobial peptides α-defensin-1 and -5 as inhibitors of PT and demonstrated their capacity to inhibit the activity of the PT enzyme subunit PTS1. Here, the underlying mechanism of toxin inhibition was investigated in more detail, which is essential for developing the therapeutic potential of these peptides. Flow cytometry and immunocytochemistry revealed that α-defensin-5 strongly reduced PT binding to, and uptake into cells, whereas α-defensin-1 caused only a mild reduction. Conversely, α-defensin-1, but not α-defensin-5 was taken up into different cell lines and interacted with PTS1 inside cells, based on proximity ligation assay. In-silico modeling revealed specific interaction interfaces for α-defensin-1 with PTS1 and vice versa, unlike α-defensin-5. Dot blot experiments showed that α-defensin-1 binds to PTS1 and even stronger to its substrate protein Gαi in vitro. NADase activity of PTS1 in vitro was not inhibited by α-defensin-1 in the absence of Gαi. Taken together, these results suggest that α-defensin-1 inhibits PT mainly by inhibiting enzyme activity of PTS1, whereas α-defensin-5 mainly inhibits cellular uptake of PT. These findings will pave the way for optimization of α-defensins as novel therapeutics against whooping cough.
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Tos Ferina , Humanos , Niño , Toxina del Pertussis/farmacología , Tos Ferina/microbiología , Bordetella pertussis , Proteínas , Línea CelularRESUMEN
Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-21 screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.
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Amiloide , Ribonucleoproteína Nuclear Heterogénea A1 , Amiloide/química , Microscopía por Crioelectrón/métodos , Ribonucleoproteína Nuclear Heterogénea A1/química , Mutación , Priones/química , Dominios ProteicosRESUMEN
BACKGROUND: Systemic AA amyloidosis is a world-wide occurring protein misfolding disease in humans and animals that arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein and their deposition in multiple organs. OBJECTIVE: To identify new agents that prevent fibril formation from SAA protein and to determine their mode of action. MATERIALS AND METHODS: We used a cell model for the formation of amyloid deposits from SAA protein to screen a library of peptides and small proteins, which were purified from human hemofiltrate. To clarify the inhibitory mechanism the obtained inhibitors were characterised in cell-free fibril formation assays and other biochemical methods. RESULTS: We identified lysozyme as an inhibitor of SAA fibril formation. Lysozyme antagonised fibril formation both in the cell model as well as in cell-free fibril formation assays. The protein binds SAA with a dissociation constant of 16.5 ± 0.6 µM, while the binding site on SAA is formed by segments of positively charged amino acids. CONCLUSION: Our data imply that lysozyme acts in a chaperone-like fashion and prevents the aggregation of SAA protein through direct, physical interactions.
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Amiloidosis , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Animales , Humanos , Proteína Amiloide A Sérica/metabolismo , Muramidasa , Amiloidosis/metabolismo , Amiloide/metabolismoRESUMEN
Semen is an important vector for sexual HIV-1 transmission. Although CXCR4-tropic (X4) HIV-1 may be present in semen, almost exclusively CCR5-tropic (R5) HIV-1 causes systemic infection after sexual intercourse. To identify factors that may limit sexual X4-HIV-1 transmission, we generated a seminal fluid-derived compound library and screened it for antiviral agents. We identified four adjacent fractions that blocked X4-HIV-1 but not R5-HIV-1 and found that they all contained spermine and spermidine, abundant polyamines in semen. We showed that spermine, which is present in semen at concentrations up to 14 mM, binds CXCR4 and selectively inhibits cell-free and cell-associated X4-HIV-1 infection of cell lines and primary target cells at micromolar concentrations. Our findings suggest that seminal spermine restricts sexual X4-HIV-1 transmission.
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Infecciones por VIH , VIH-1 , Humanos , Espermidina/farmacología , Espermina/farmacología , Infecciones por VIH/tratamiento farmacológico , Línea Celular , Receptores CXCR4RESUMEN
Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.
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COVID-19 , Infección por el Virus Zika , Virus Zika , Humanos , Péptidos , Amiloide/química , Antibacterianos/farmacología , HemoglobinasRESUMEN
RNA-binding proteins (RBPs) form a large and diverse class of factors, many members of which are overexpressed in hematologic malignancies. RBPs participate in various processes of messenger RNA (mRNA) metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here, we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in patients with acute myeloid leukemia (AML) and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. Instead, it largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates the human myeloid progenitor signature and leukemia stem cell (LSC)-associated genes and upregulates DNA damage signaling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells.
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Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/patología , Células Madre Hematopoyéticas/metabolismo , Proliferación Celular , Genómica , ARN Mensajero/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
Mollusks have been widely investigated for antimicrobial peptides because their humoral defense against pathogens is mainly based on these small biomolecules. In this report, we describe the identification of three novel antimicrobial peptides from the marine mollusk Nerita versicolor. A pool of N. versicolor peptides was analyzed with nanoLC-ESI-MS-MS technology, and three potential antimicrobial peptides (Nv-p1, Nv-p2 and Nv-p3) were identified with bioinformatical predictions and selected for chemical synthesis and evaluation of their biological activity. Database searches showed that two of them show partial identity to histone H4 peptide fragments from other invertebrate species. Structural predictions revealed that they all adopt a random coil structure even when placed near a lipid bilayer patch. Nv-p1, Nv-p2 and Nv-p3 exhibited activity against Pseudomonas aeruginosa. The most active peptide was Nv-p3 with an inhibitory activity starting at 1.5 µg/mL in the radial diffusion assays. The peptides were ineffective against Klebsiella pneumoniae, Listeria monocytogenes and Mycobacterium tuberculosis. On the other hand, these peptides demonstrated effective antibiofilm action against Candida albicans, Candida parapsilosis and Candida auris but not against the planktonic cells. None of the peptides had significant toxicity on primary human macrophages and fetal lung fibroblasts at effective antimicrobial concentrations. Our results indicate that N. versicolor-derived peptides represent new AMP sequences and have the potential to be optimized and developed into antibiotic alternatives against bacterial and fungal infections.
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Antiinfecciosos , Gastrópodos , Animales , Humanos , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Antimicrobianos , Antiinfecciosos/farmacología , Antibacterianos/farmacología , Moluscos , Pruebas de Sensibilidad MicrobianaRESUMEN
BACKGROUND: Systemic AL amyloidosis arises from the misfolding of patient-specific immunoglobulin light chains (LCs). Potential drivers of LC amyloid formation are mutational changes and post-translational modifications (PTMs). However, little information is available on the exact primary structure of the AL proteins and their precursor LCs. OBJECTIVE: We analyse the exact primary structure of AL proteins extracted from 10 λ AL amyloidosis patients and their corresponding precursor LCs. MATERIALS AND METHODS: By cDNA sequencing of the precursor LC genes in combination with mass spectrometry of the AL proteins, the exact primary structure and PTMs were determined. This information was used to analyse their biochemical properties. RESULTS: All AL proteins comprise the VL and a small part of the CL with a common C-terminal truncation region. While all AL proteins retain the conserved native disulphide bond of the VL, we found no evidence for presence of other common PTMs. The analysis of the biochemical properties revealed that the isoelectric point of the VL is significantly increased due to introduced mutations. CONCLUSION: Our data imply that mutational changes influence the surface charge properties of the VL and that common proteolytic processes are involved in the generation of the cleavage sites of AL proteins.
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Amiloidosis , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/genética , Amiloidosis/genética , Amiloidosis/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Amiloide/genética , Amiloide/metabolismo , Espectrometría de Masas , Grasa Abdominal/metabolismoRESUMEN
TFIIH is a complex essential for transcription of protein-coding genes by RNA polymerase II, DNA repair of UV-lesions and transcription of rRNA by RNA polymerase I. Mutations in TFIIH cause the cancer prone DNA-repair disorder xeroderma pigmentosum (XP) and the developmental and premature aging disorders trichothiodystrophy (TTD) and Cockayne syndrome. A total of 50% of the TTD cases are caused by TFIIH mutations. Using TFIIH mutant patient cells from TTD and XP subjects we can show that the stress-sensitivity of the proteome is reduced in TTD, but not in XP. Using three different methods to investigate the accuracy of protein synthesis by the ribosome, we demonstrate that translational fidelity of the ribosomes of TTD, but not XP cells, is decreased. The process of ribosomal synthesis and maturation is affected in TTD cells and can lead to instable ribosomes. Isolated ribosomes from TTD patients show an elevated error rate when challenged with oxidized mRNA, explaining the oxidative hypersensitivity of TTD cells. Treatment of TTD cells with N-acetyl cysteine normalized the increased translational error-rate and restored translational fidelity. Here we describe a pathomechanism that might be relevant for our understanding of impaired development and aging-associated neurodegeneration.