RESUMEN
Alzheimer's disease (AD) is a progressive neurological disorder featuring abnormal protein aggregation in the brain, including the pathological hallmarks of amyloid plaques and hyperphosphorylated tau. Despite extensive research efforts, understanding the molecular intricacies driving AD development remains a formidable challenge. This study focuses on identifying key protein conformational changes associated with the progression of AD. To achieve this, we employed quantitative cross-linking mass spectrometry (XL-MS) to elucidate conformational changes in the protein networks in cerebrospinal fluid (CSF). By using isotopically labeled cross-linkers BS3d0 and BS3d4, we reveal a dynamic shift in protein interaction networks during AD progression. Our comprehensive analysis highlights distinct alterations in protein-protein interactions within mild cognitive impairment (MCI) states. This study accentuates the potential of cross-linked peptides as indicators of AD-related conformational changes, including previously unreported site-specific binding between α-1-antitrypsin (A1AT) and complement component 3 (CO3). Furthermore, this work enables detailed structural characterization of apolipoprotein E (ApoE) and reveals modifications within its helical domains, suggesting their involvement in MCI pathogenesis. The quantitative approach provides insights into site-specific interactions and changes in the abundance of cross-linked peptides, offering an improved understanding of the intricate protein-protein interactions underlying AD progression. These findings lay a foundation for the development of potential diagnostic or therapeutic strategies aimed at mitigating the negative impact of AD.
Asunto(s)
Enfermedad de Alzheimer , Apolipoproteínas E , Espectrometría de Masas , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/diagnóstico , Humanos , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Reactivos de Enlaces Cruzados/química , Conformación Proteica , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo , Disfunción Cognitiva/metabolismoRESUMEN
Mutants of alpha-1-antitrypsin cause the protein to self-associate and form ordered aggregates ('polymers') that are retained within hepatocytes, resulting in a predisposition to the development of liver disease. The associated reduction in secretion, and for some mutants, impairment of function, leads to a failure to protect lung tissue against proteases released during the inflammatory response and an increased risk of emphysema. We report here a novel deficiency mutation (Gly192Cys), that we name the Sydney variant, identified in a patient in heterozygosity with the Z allele (Glu342Lys). Cellular analysis revealed that the novel variant was mostly retained as insoluble polymers within the endoplasmic reticulum. The basis for this behaviour was investigated using biophysical and structural techniques. The variant showed a 40% reduction in inhibitory activity and a reduced stability as assessed by thermal unfolding experiments. Polymerisation involves adoption of an aggregation-prone intermediate and paradoxically the energy barrier for transition to this state was increased by 16% for the Gly192Cys variant with respect to the wild-type protein. However, with activation to the intermediate state, polymerisation occurred at a 3.8-fold faster rate overall. X-ray crystallography provided two crystal structures of the Gly192Cys variant, revealing perturbation within the 'breach' region with Cys192 in two different orientations: in one structure it faces towards the hydrophobic core while in the second it is solvent-exposed. This orientational heterogeneity was confirmed by PEGylation. These data show the critical role of the torsional freedom imparted by Gly192 in inhibitory activity and stability against polymerisation.
Asunto(s)
alfa 1-Antitripsina , Humanos , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo , Cristalografía por Rayos X , Mutación , Modelos Moleculares , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Conformación Proteica , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/genéticaRESUMEN
The rising demand for novel cosmeceutical ingredients has highlighted peptides as a significant category. Based on the collagen turnover modulation properties of SA1-III, a decapeptide derived from a serine protease inhibitor (serpin A1), this study focused on designing shorter, second-generation peptides endowed with improved properties. A tetrapeptide candidate was further modified employing the retro-inverso approach that uses d-amino acids aiming to enhance peptide stability against dermal enzymes. Surprisingly, the modified peptide AAT11RI displayed notably high activity in vitro, as compared to its precursors, and suggested a mode of action based on the inhibition of collagen degradation. It is worth noting that AAT11RI showcases stability against dermal enzymes contained in human skin homogenates due to its rationally designed structure that hampers recognition by most proteases. The rational approach we embraced in this study underscored the added value of substantiated claims in the design of new cosmeceutical ingredients, representing a rarity in the field.
Asunto(s)
Cosmecéuticos , alfa 1-Antitripsina , Humanos , alfa 1-Antitripsina/química , alfa 1-Antitripsina/farmacología , Péptidos/farmacología , Péptidos/química , Colágeno , Adyuvantes InmunológicosRESUMEN
Conjugation to polyethylene glycol (PEG) is a widely used approach to improve the therapeutic value of proteins essentially by prolonging their body residence time. PEGylation may however induce changes in the structure and/or the stability of proteins and thus on their function(s). The effects of PEGylation on the thermodynamic stability can either be positive (stabilization), negative (destabilization), or neutral (no effect). Moreover, various factors such as the PEG length and PEGylation site can influence the consequences of PEGylation on the structure and stability of proteins. In this study, the effects of PEGylation on the structure, stability, and polymerization of alpha1-antitrypsin (AAT) were investigated, using PEGs with different lengths, different structures (linear or 2-armed) and different linking chemistries (via amine or thiol) at two distinct positions of the sequence. The results show that whatever the size, position, and structure of PEG chains, PEGylation (a) does not induce significant changes in AAT structure (either at the secondary or tertiary level); (b) does not alter the stability of the native protein upon both chemical- and heat-induced denaturation; and (c) does not prevent AAT to fully refold and recover its activity following chemical denaturation. However, the propensity of AAT to aggregate upon heat treatment was significantly decreased by PEGylation, although PEGylation did not prevent the irreversible inactivation of the enzyme. Moreover, conjugation to PEG, especially 2-armed 40 kDa PEG, greatly improved the proteolytic resistance of AAT. PEGylation of AAT could be a promising strategy to prolong its half-life after infusion in AAT-deficient patients and thereby decrease the frequency of infusions.
Asunto(s)
Polietilenglicoles , alfa 1-Antitripsina/química , Humanos , Polietilenglicoles/química , Proteolisis , TermodinámicaRESUMEN
Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked.
Asunto(s)
Apolipoproteínas/metabolismo , Caspasas/metabolismo , Proteínas del Sistema Complemento/metabolismo , Citocinas/metabolismo , alfa 1-Antitripsina/metabolismo , Sitios de Unión/genética , COVID-19/metabolismo , COVID-19/virología , Glicosilación , Humanos , Mutación , Unión Proteica , Dominios Proteicos , SARS-CoV-2/fisiología , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/metabolismoRESUMEN
C-terminal peptides (CAAPs) of the highly abundant serine protease alpha-1-antitrypsin (A1AT) have been identified at various lengths in several human materials and have been proposed to serve as putative biomarkers for a variety of diseases. CAAPs are enzymatically formed and these enzymatic activities are often associated with excessive immune responses (e.g. sepsis, allergies). However, most of those CAAPs have been either detected using in vitro incubation experiments or in human materials which are not easily accessible. To gain a comprehensive understanding about the occurrence and function of CAAPs in health and disease, a LC-MS/MS method for the simultaneous detection of nine CAAPs was developed and validated for human plasma (EDTA and lithium-heparin) and serum. Using this newly developed method, we were able to detect and quantify five CAAPs in healthy individuals thereby providing an initial proof for the presence of C36, C37, C40 and C44 in human blood. Concentrations of four CAAPs in a clinical test cohort of patients suffering from sepsis were significantly higher compared to healthy controls. These results reveal that in addition to C42 other fragments of A1AT seem to play a crucial role during systemic infections. The proposed workflow is simple, rapid and robust; thus this method could be used as diagnostic tool in routine clinical chemistry as well as for research applications for elucidating the diagnostic potential of CAAPs in numerous diseases. To this end, we also provide an overview about the current state of knowledge for CAAPs identified in vitro and in vivo.
Asunto(s)
Inflamación , alfa 1-Antitripsina , Cromatografía Liquida , Humanos , Péptidos/química , Espectrometría de Masas en Tándem , alfa 1-Antitripsina/químicaRESUMEN
SP16 is an innovative peptide derived from the carboxyl-terminus of α1-Antitrypsin (AAT), corresponding to residues 364-380, and contains recognition sequences for the low-density lipoprotein receptor-related protein-1 (LRP1). LRP1 is an endocytic and cell-signaling receptor that regulates inflammation. Deletion of Lrp1 in Schwann cells increases neuropathic pain; however, the role of LRP1 activation in nociceptive and neuropathic pain regulation remains unknown. Herein, we show that SP16 is bioactive in sensory neurons in vitro. Neurite length and regenerative gene expression were increased by SP16. In PC12 cells, SP16 activated Akt and ERK1/2 cell-signaling in an LRP1-dependent manner. When formalin was injected into mouse hind paws, to model inflammatory pain, SP16 dose-dependently attenuated nociceptive pain behaviors in the early and late phases. In a second model of acute pain using capsaicin, SP16 significantly reduced paw licking in both male and female mice (p < .01) similarly to enzymatically inactive tissue plasminogen activator, a known LRP1 interactor. SP16 also prevented development of tactile allodynia after partial nerve ligation and this response was sustained for nine days (p < .01). Immunoblot analysis of the injured nerve revealed decreased CD11b (p < .01) and Toll-like receptor-4 (p < .005). In injured dorsal root ganglia SP16 reduced CD11b+ cells (p < .05) and GFAP (p < .005), indicating that inflammatory cell recruitment and satellite cell activation were inhibited. In conclusion, administration of SP16 blocked pain-related responses in three distinct pain models, suggesting efficacy against acute nociceptive, inflammatory, and neuropathic pain. SP16 also attenuated innate immunity in the PNS. These studies identify SP16 as a potentially effective treatment for pain.
Asunto(s)
Dolor Agudo/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas , Neuralgia/tratamiento farmacológico , Péptidos/farmacología , alfa 1-Antitripsina/química , Dolor Agudo/inducido químicamente , Dolor Agudo/genética , Dolor Agudo/metabolismo , Animales , Modelos Animales de Enfermedad , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Neuralgia/inducido químicamente , Neuralgia/genética , Neuralgia/metabolismo , Neuritas/metabolismo , Células PC12 , Péptidos/química , Ratas , Ratas Sprague-Dawley , Células de Schwann/metabolismo , Células Receptoras Sensoriales/metabolismo , alfa 1-Antitripsina/genéticaRESUMEN
Human alpha-1-antitrypsin (A1AT), a native serine-protease inhibitor that protects tissue damage from excessive protease activities, is used as an augmentation therapy to treat A1AT-deficienct patients. However, A1AT is sensitive to oxidation-mediated deactivation and has a short circulating half-life. Currently, there is no method that can effectively protect therapeutic proteins from oxidative damage in vivo. Here we developed a novel biocompatible selenopolypeptide and site-specifically conjugated it with A1AT. The conjugated A1AT fully retained its inhibitory activity on neutrophil elastase, enhanced oxidation resistance, extended the serum half-life, and afforded long-lasting protective efficacy in a mouse model of acute lung injury. These results demonstrated that conjugating A1AT with the designed selenopolymer is a viable strategy to improve its pharmacological properties, which could potentially further be applied to a variety of oxidation sensitive biotherapeutics.
Asunto(s)
Materiales Biocompatibles/farmacología , Elastasa de Leucocito/antagonistas & inhibidores , Péptidos/farmacología , Selenio/farmacología , Inhibidores de Serina Proteinasa/farmacología , alfa 1-Antitripsina/farmacología , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Humanos , Elastasa de Leucocito/metabolismo , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Péptidos/química , Selenio/química , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , alfa 1-Antitripsina/químicaRESUMEN
Human α1-antitrypsin (hAAT) has two distinguishing functions: anti-protease activity and regulation of the immune system. In the present study we hypothesized that those two protein functions are mediated by different structural domains on the hAAT surface. Indeed, such biologically active immunoregulatory sites (not associated with canonical anti-protease activity) on the surface of hAAT were identified by in silico methods. Several peptides were derived from those immunoregulatory sites. Four peptides exhibited impressive biological effects in pharmacological concentration ranges. Peptidomimetic (14) was developed, based on the structure of the most druggable and active peptide. The compound exhibited a potent anti-inflammatory activity in vitro and in vivo. Such a compound could be used as a basis for developing novel anti-inflammatory drug candidates and as a research tool for better understanding hAAT functions.
Asunto(s)
Antiinflamatorios/farmacología , Desarrollo de Medicamentos , Peptidomiméticos/farmacología , alfa 1-Antitripsina/metabolismo , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Relación Estructura-Actividad , alfa 1-Antitripsina/químicaRESUMEN
Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.
Asunto(s)
Hepatocitos/citología , Deficiencia de alfa 1-Antitripsina/patología , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico , Mutación con Ganancia de Función , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Transgénicos , Transporte de Proteínas , Proteolisis , Análisis de Secuencia de ARN , alfa 1-Antitripsina/química , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/metabolismoRESUMEN
Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson's disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.
Asunto(s)
Afibrinogenemia/genética , Fibrinógeno/química , Enfermedad de Parkinson/genética , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/química , alfa-Sinucleína/química , Afibrinogenemia/tratamiento farmacológico , Afibrinogenemia/metabolismo , Afibrinogenemia/patología , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Coagulantes/uso terapéutico , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Fibrinógeno/genética , Fibrinógeno/metabolismo , Regulación de la Expresión Génica , Humanos , Hígado/metabolismo , Hígado/patología , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Inhibidores de Proteasas/uso terapéutico , Agregado de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacos , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , Deficiencia de alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/patología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
During biosynthesis, proteins can begin folding co-translationally to acquire their biologically-active structures. Folding, however, is an imperfect process and in many cases misfolding results in disease. Less is understood of how misfolding begins during biosynthesis. The human protein, alpha-1-antitrypsin (AAT) folds under kinetic control via a folding intermediate; its pathological variants readily form self-associated polymers at the site of synthesis, leading to alpha-1-antitrypsin deficiency. We observe that AAT nascent polypeptides stall during their biosynthesis, resulting in full-length nascent chains that remain bound to ribosome, forming a persistent ribosome-nascent chain complex (RNC) prior to release. We analyse the structure of these RNCs, which reveals compacted, partially-folded co-translational folding intermediates possessing molten-globule characteristics. We find that the highly-polymerogenic mutant, Z AAT, forms a distinct co-translational folding intermediate relative to wild-type. Its very modest structural differences suggests that the ribosome uniquely tempers the impact of deleterious mutations during nascent chain emergence. Following nascent chain release however, these co-translational folding intermediates guide post-translational folding outcomes thus suggesting that Z's misfolding is initiated from co-translational structure. Our findings demonstrate that co-translational folding intermediates drive how some proteins fold under kinetic control, and may thus also serve as tractable therapeutic targets for human disease.
Asunto(s)
Biosíntesis de Proteínas , Pliegue de Proteína , Ribosomas/metabolismo , Deficiencia de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/química , Algoritmos , Secuencia de Aminoácidos , Animales , Western Blotting , Dicroismo Circular , Endopeptidasa K/metabolismo , Humanos , Cinética , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Conejos , Reticulocitos/citología , Reticulocitos/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins ("spike"). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1ß mRNA expression and protein release were significantly inhibited (by about 46-50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.
Asunto(s)
Antiinflamatorios/farmacología , Leucocitos Mononucleares/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , alfa 1-Antitripsina/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/inmunología , Células CHO , COVID-19/terapia , Células Cultivadas , Cricetulus , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , alfa 1-Antitripsina/química , alfa 1-Antitripsina/inmunologíaRESUMEN
Pancreatic cancer (PC) is an aggressive cancer with a high mortality rate, necessitating the development of effective diagnostic, prognostic and predictive biomarkers for disease management. Aberrantly fucosylated proteins in PC are considered a valuable resource of clinically useful biomarkers. The main objective of the present study was to identify novel plasma glycobiomarkers of PC using the iTRAQ quantitative proteomics approach coupled with Aleuria aurantia lectin (AAL)-based glycopeptide enrichment and isotope-coded glycosylation site-specific tagging, with a view to analyzing the glycoproteome profiles of plasma samples from patients with non-metastatic and metastatic PC and gallstones (GS). As a result, 22 glycopeptides with significantly elevated levels in plasma samples of PC were identified. Fucosylated SERPINA1 (fuco-SERPINA1) was selected for further validation in 121 plasma samples (50 GS and 71 PC) using an AAL-based reverse lectin ELISA technique developed in-house. Our analyses revealed significantly higher plasma levels of fuco-SERPINA1 in PC than GS subjects (310.7 ng/mL v.s. 153.6 ng/mL, p = 0.0114). Elevated fuco-SERPINA1 levels were associated with higher TNM stage (p = 0.024) and poorer prognosis for overall survival (log-rank test, p = 0.0083). The increased plasma fuco-SERPINA1 levels support the utility of this protein as a novel prognosticator for PC.
Asunto(s)
Biomarcadores de Tumor/sangre , Fucosa/química , Glicoproteínas/sangre , Lectinas/química , Neoplasias Pancreáticas/diagnóstico , Proteoma/metabolismo , alfa 1-Antitripsina/sangre , Estudios de Casos y Controles , Cromatografía de Afinidad , Femenino , Fucosa/metabolismo , Humanos , Lectinas/metabolismo , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Neoplasias Pancreáticas/sangre , Proteoma/análisis , Tasa de Supervivencia , alfa 1-Antitripsina/químicaRESUMEN
Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells.
Asunto(s)
Adenina , Sistemas CRISPR-Cas , Edición Génica , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Biomarcadores , Diferenciación Celular/genética , Células Cultivadas , Estrés del Retículo Endoplásmico , Expresión Génica , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Mutación , alfa 1-Antitripsina/químicaRESUMEN
Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.
Asunto(s)
Mutación Missense , Deficiencia de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina , Sustitución de Aminoácidos , Células HEK293 , Humanos , Dominios Proteicos , alfa 1-Antitripsina/biosíntesis , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
Expression levels of CX3CR1 (C-X3-C motif chemokine receptor 1) on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host's misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, a novel example demonstrates that polymers of human ZZ alpha-1 antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This parallels with increase of intracellular levels of CX3CR1 and Z-AAT proteins. Presented data indicate the involvement of the CX3CR1 pathway in the Z-AAT-related disorders and further support the role of misfolded proteins in CX3CR1 regulation.
Proteins can lose their structure and form polymers because of mutations or changes in their immediate environment which can lead to cell damage and disease. Interestingly, polymers formed by a variety of proteins can reduce the levels of CX3C chemokine receptor 1 (CX3CR1 for short) that controls the behaviour of immune cells and is implicated in a range of illnesses. Inherited ZZ alpha-1 antitrypsin deficiency is a rare genetic condition that highly increases the risk of liver and lung diseases. This disorder is characterised by mutant alpha-1 antitrypsin proteins (AAT for short) reacting together to form polymers; yet it remains unclear how the polymers affect different cells or organs, and lead to diseases. To investigate this question, Tumpara et al. examined whether polymers of mutant AAT influence the level of the CX3CR1 protein in specific classes of immune cells. Experiments revealed that in people with AAT deficiency, certain blood immune cells express lower levels of CX3CR1. Regardless of age, clinical diagnosis, or treatment regimen, all individuals with ZZ alpha-1 antitrypsin deficiency had AAT polymers circulating in their blood: the higher the levels of polymers measured, the lower the expression of CX3CR1 recorded in the specific immune cells. When Tumpara et al. added polymers of mutant AAT to the immune cells of healthy donors, the expression of CX3CR1 dropped in a manner dependent on the polymer concentration. According to microscopy data, AAT polymers occurred inside cells alongside the CX3CR1 protein, suggesting that the two molecular actors interact. In the future, new drugs that remove these polymers, either from inside cells or as they circulate in the body, could help patients suffering from conditions associated with this abnormal protein aggregation.
Asunto(s)
Receptor 1 de Quimiocinas CX3C/metabolismo , Mutación , Polímeros , alfa 1-Antitripsina/genética , Adulto , Receptor 1 de Quimiocinas CX3C/genética , Quimiocina CX3CL1/metabolismo , Femenino , Humanos , Leucocitos Mononucleares , Masculino , Persona de Mediana Edad , Conformación Proteica , ARN Mensajero/metabolismo , alfa 1-Antitripsina/químicaRESUMEN
Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2'-P3' was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3', PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis.
Asunto(s)
Factor XIa/química , Expresión Génica , Mutagénesis , Biblioteca de Péptidos , alfa 1-Antitripsina/química , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , alfa 1-Antitripsina/biosíntesis , alfa 1-Antitripsina/genéticaRESUMEN
Loss of insulin-secreting pancreatic ß cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains ß cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce ß cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. ß cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by ß cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of ß cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents ß cell death and metabolic abnormalities. These data uncover a pathway for ß cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.
Asunto(s)
Apoptosis , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/patología , Proteínas del Tejido Nervioso/metabolismo , alfa 1-Antitripsina/química , Animales , Proteínas Reguladoras de la Apoptosis/fisiología , Citoplasma/metabolismo , Proteínas del Citoesqueleto/genética , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Musculares/fisiología , Proteínas del Tejido Nervioso/genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
Based on previous results demonstrating that complexes of a mutant α1-antitrypsin with the heat shock proteins (HSP)70 and glucose-regulated protein94 (Grp94) circulate in the blood of patients with type 1 diabetes, we raised the hypothesis that these complexes could represent the primary antigen capable of triggering the autoimmune reactions leading to overt diabetes. As a first approach to this issue, we searched whether A1AT and HSPs had a sequence similarity to major islet antigen proteins so as to identify among the similar sequences those with potential relevance for the pathogenesis of diabetes. A thorough in silico analysis was performed to establish the score of similarity of the human proteins: A1AT, pro-insulin (INS), GAD65, IAPP, IA-2, ICA69, Grp94, HSP70 and HSP60. The sequences of A1AT and HSPs with the highest score of similarity to the islet peptides reported in the literature as the main autoantigens in human diabetes were recorded. At variance with other HSPs, also including HSP90 and Grp78, Grp94 contained the highest number and the longest sequences with structural similarity to A1AT and to well-known immunogenic peptides/epitopes of INS, GAD65, and IA-2. The similarity of A1AT with Grp94 and that of Grp94 with INS also suggested a functional relationship among the proteins. Specific sequences were identified in A1AT, Grp94 and HSP70, with the highest score of cross-similarity to a pattern of eight different islet protein epitopes. The similarity also involved recently discovered autoantigens in type 1 diabetes such as a hybrid peptides of insulin and the defective ribosomal insulin gene product. The significant similarity displayed by specific sequences of Grp94 and A1AT to the islet peptides considered main antigens in human diabetes, is a strong indication for testing these sequences as new peptides of immunogenic relevance in diabetes.