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Mucopolysaccharidosis type I (MPS I) is a rare genetic disorder caused by mutations in the IDUA gene, leading to alpha-L-iduronidase enzyme deficiency and resulting in the accumulation of glycosaminoglycans (GAG; heparan and dermatan sulfate) in lysosomes. The consequent GAG accumulation within cells leads to organ dysfunction and a range of debilitating symptoms. Enzyme replacement therapy (ERT) is the prevailing treatment, but its limitations (including high cost, time requirements, inefficiency in treatment of central nervous system (CNS), and immunogenicity) necessitate exploration of alternative therapeutic strategies. This research propose a novel approach leveraging the synergistic effects of ERT and resveratrol-induced autophagy. Resveratrol, with its immunomodulatory and GAG degradation-stimulating properties, holds a promise in mitigating immune responses triggered by ERT. Moreover, its ability to penetrate the blood-brain barrier presents a potential solution for addressing CNS manifestations. This study employed cells from MPS I patients to investigate the combined effects of resveratrol and the enzyme. Evaluation of the therapeutic impact involved assessing GAG accumulation, enzyme testing, and examining lysosome functionality and the autophagy process through fluorescence microscopy and Western blotting. The combined therapy stimulated the lysosomal mannose-6-phosphate receptor (M6PR) and lysosome biogenesis through the transcription factor EB (TFEB). Additionally, initial block of autophagy in autophagosome formation was relieved after the combined therapy and resveratrol alone. Together with increased enzyme activity through stimulation of the receptor, this synergistic therapy can be considered a new potential treatment for MPS I patients, improving their overall quality of life.
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Mucopolysaccharidoses (MPS) comprise a group of 12 metabolic disorders where defects in specific enzyme activities lead to the accumulation of glycosaminoglycans (GAGs) within lysosomes. This classification expands to 13 when considering MPS IIIE. This type of MPS, associated with pathogenic variants in the ARSG gene, has thus far been described only in the context of animal models. However, pathogenic variants in this gene also occur in humans, but are linked to a different disorder, Usher syndrome (USH) type IV, which is sparking increasing debate. This paper gathers, discusses, and summarizes arguments both for and against classifying dysfunctions of arylsulfatase G (due to pathogenic variants in the ARSG gene) in humans as another subtype of MPS, called MPS IIIE. Specific difficulties in diagnostics and the classification of some inherited metabolic diseases are also highlighted and discussed.
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Over the past decades, there has been a significant increase in the burden of neurological diseases, including neurodegenerative disorders, on a global scale. This is linked to a widespread demographic trend in which developed societies are aging, leading to an increased proportion of elderly individuals and, concurrently, an increase in the number of those afflicted, posing one of the main public health challenges for the coming decades. The complex pathomechanisms of neurodegenerative diseases and resulting varied symptoms, which differ depending on the disease, environment, and lifestyle of the patients, make searching for therapies for this group of disorders a formidable challenge. Currently, most neurodegenerative diseases are considered incurable. An important aspect in the fight against and prevention of neurodegenerative diseases may be broadly understood lifestyle choices, and more specifically, what we will focus on in this review, a diet. One proposal that may help in the fight against the spread of neurodegenerative diseases is a diet rich in flavonoids. Flavonoids are compounds widely found in products considered healthy, such as fruits, vegetables, and herbs. Many studies indicated not only the neuroprotective effects of these compounds but also their ability to reverse changes occurring during the progression of diseases such as Alzheimer's, Parkinson's and amyotrophic lateral sclerosis. Here, we present the main groups of flavonoids, discussing their characteristics and mechanisms of action. The most widely described mechanisms point to neuroprotective functions due to strong antioxidant and anti-inflammatory effects, accompanied with their ability to penetrate the blood-brain barrier, as well as the ability to inhibit the formation of protein aggregates. The latter feature, together with promoting removal of the aggregates is especially important in neurodegenerative diseases. We discuss a therapeutic potential of selected flavonoids in the fight against neurodegenerative diseases, based on in vitro studies, and their impact when included in the diet of animals (laboratory research) and humans (population studies). Thus, this review summarizes flavonoids' actions and impacts on neurodegenerative diseases. Therapeutic use of these compounds in the future is potentially possible but depends on overcoming key challenges such as low bioavailability, determining the therapeutic dose, and defining what a flavonoid-rich diet is and determining its potential negative effects. This review also suggests further research directions to address these challenges.
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Estrogen is a group of hormones that collaborate with the nervous system to impact the overall well-being of all genders. It influences many processes, including those occurring in the central nervous system, affecting learning and memory, and playing roles in neurodegenerative diseases and mental disorders. The hormone's action is mediated by specific receptors. Significant roles of classical estrogen receptors, ERα and ERß, in various diseases were known since many years, but after identifying a structurally and locationally distinct receptor, the G protein-coupled estrogen receptor (GPER), its role in human physiology and pathophysiology was investigated. This review compiles GPER-related information, highlighting its impact on homeostasis and diseases, while putting special attention on functions and dysfunctions of this receptor in neurobiology and biobehavioral processes. Understanding the receptor modulation possibilities is essential for therapy, as disruptions in receptors can lead to diseases or disorders, irrespective of correct estrogen levels. We conclude that studies on the GPER receptor have the potential to develop therapies that regulate estrogen and positively impact human health.
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Estrogênios , Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/fisiologia , Estrogênios/metabolismo , Doenças Neurodegenerativas/metabolismo , Transtornos Mentais/metabolismo , Transtornos Mentais/fisiopatologia , AnimaisRESUMO
Glucocerebrosidase (GCase), encoded by the GBA1 gene, is one of the lysosomal enzymes responsible for hydrolyzing the glycosphingolipids. Deficiency in GCase activity (in patients with two defective alleles of GBA1) leads to glucosylceramide storage in lysosomes which in turn results in the development of the Gaucher diseases, a lysosomal storage disorder, while a heterozygous state may be correlated with the GBA1 mutation-associated Parkinson disease. One of the proposed forms of therapy for these two conditions is the use of pharmacological chaperones which work by facilitating the achievement of the correct conformation of abnormally folded enzymes. Several compounds with chaperone activities against GCase have already been tested, one of which turned out to be ambroxol. Studies conducted on the action of this compound have indeed indicated its effectiveness in increasing GCase levels and activity. However, some data have begun to question its activity as a chaperone against certain GCase variants. Then, a number of articles appeared pointing to other mechanisms of action of ambroxol, which may also contribute to the improvement of patients' condition. This paper summarizes the biological mechanisms of action of ambroxol in Gaucher disease and GBA1 mutation-associated Parkinson disease, focused on its activity as a chaperone, modulator of ERAD pathways, inducer of autophagy, and pain reliever in cellular and animal models as well as in patients. The effects of these activities on the reduction of disease markers and symptoms in patients are also discussed. Consideration of all the properties of ambroxol can help in the appropriate choice of therapy and the determination of the effective drug dose.
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Ambroxol , Doença de Gaucher , Glucosilceramidase , Mutação , Doença de Parkinson , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Ambroxol/farmacologia , Ambroxol/uso terapêutico , Humanos , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , AnimaisRESUMO
Mucopolysaccharidoses (MPS) are a group of diseases caused by mutations in genes encoding lysosomal enzymes that catalyze reactions of glycosaminoglycan (GAG) degradation. As a result, GAGs accumulate in lysosomes, impairing the proper functioning of entire cells and tissues. There are 14 types/subtypes of MPS, which are differentiated by the kind(s) of accumulated GAG(s) and the type of a non-functional lysosomal enzyme. Some of these types (severe forms of MPS types I and II, MPS III, and MPS VII) are characterized by extensive central nervous system disorders. The aim of this work was to identify, using transcriptomic methods, organelle-related genes whose expression levels are changed in neuronopathic types of MPS compared to healthy cells while remaining unchanged in non-neuronopathic types of MPS. The study was conducted with fibroblast lines derived from patients with neuronopathic and non-neuronopathic types of MPS and control (healthy) fibroblasts. Transcriptomic analysis has identified genes related to cellular organelles whose expression is altered. Then, using fluorescence and electron microscopy, we assessed the morphology of selected structures. Our analyses indicated that the genes whose expression is affected in neuronopathic MPS are often associated with the structures or functions of the cell nucleus, endoplasmic reticulum, or Golgi apparatus. Electron microscopic studies confirmed disruptions in the structures of these organelles. Special attention was paid to up-regulated genes, such as PDIA3 and MFGE8, and down-regulated genes, such as ARL6IP6, ABHD5, PDE4DIP, YIPF5, and CLDN11. Of particular interest is also the GM130 (GOLGA2) gene, which encodes golgin A2, which revealed an increased expression in neuronopathic MPS types. We propose to consider the levels of mRNAs of these genes as candidates for biomarkers of neurodegeneration in MPS. These genes may also become potential targets for therapies under development for neurological disorders associated with MPS and candidates for markers of the effectiveness of these therapies. Although fibroblasts rather than nerve cells were used in this study, it is worth noting that potential genetic markers characteristic solely of neurons would be impractical in testing patients, contrary to somatic cells that can be relatively easily obtained from assessed persons.
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One of the hopes for overcoming the antibiotic resistance crisis is the use of bacteriophages to combat bacterial infections, the so-called phage therapy. This therapeutic approach is generally believed to be safe for humans and animals as phages should infect only prokaryotic cells. Nevertheless, recent studies suggested that bacteriophages might be recognized by eukaryotic cells, inducing specific cellular responses. Here we show that in chickens infected with Salmonella enterica and treated with a phage cocktail, bacteriophages are initially recognized by animal cells as viruses, however, the cGAS-STING pathway (one of two major pathways of the innate antiviral response) is blocked at the stage of the IRF3 transcription factor phosphorylation. This inhibition is due to the inability of RNA polymerase III to recognize phage DNA and to produce dsRNA molecules which are necessary to stimulate a large protein complex indispensable for IRF3 phosphorylation, indicating the mechanism of the antiviral response impairment.
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Bacteriófagos , Terapia por Fagos , Humanos , Animais , Bacteriófagos/fisiologia , Galinhas , Imunidade , AntiviraisRESUMO
The overuse of antibiotics in both humans and livestock has led to the antibiotic resistance phenomenon which is now considered one of the biggest problems in the modern world. Some antibiotics used to control or prevent infections in livestock poultry were registered a long time ago, and as a result, data on the possible side effects of their use, both for birds and humans, are incomplete and should be updated. An example of such an antibiotic is enrofloxacin which has been widely used in poultry since 1989. Data in recent years have begun to indicate that this antibiotic induces the process of apoptosis in diverse types of eukaryotic cells. Unfortunately, such studies have never been conducted on chicken models even though it is in poultry that this antibiotic is most commonly used. Therefore, the purpose of this work was to investigate whether enrofloxacin induces apoptosis in chicken cells of the UMNSAH/DF-1 line and to study the molecular mechanism of its action. The results of these experiments indicated that enrofloxacin induces apoptosis in chicken cells but not in human HEK-293 and PC3 cells. This induction was accompanied by changes in the morphology and size of mitochondria, the process of apoptosome formation and activation of executive caspases, which clearly indicates the role of the mitochondrial pathway in the induction of apoptosis by enrofloxacin. This study is the first to show the toxicity of enrofloxacin against chicken cells and to demonstrate the exact mechanism of its action. The results presented in this work show the need to monitor the concentration of antibiotic residues in poultry foods as well as to study their impact on public health to guarantee consumer safety and prevent the phenomenon of antibiotic resistance in bacteria.
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Antibacterianos , Apoptose , Galinhas , Enrofloxacina , Fluoroquinolonas , Mitocôndrias , Enrofloxacina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Antibacterianos/farmacologia , Antibacterianos/efeitos adversos , Antibacterianos/toxicidade , Fluoroquinolonas/farmacologia , Fluoroquinolonas/toxicidade , Fluoroquinolonas/efeitos adversos , Aves Domésticas , Células HEK293 , Caspases/metabolismo , Linhagem CelularRESUMO
Huntington disease (HD) is a neurodegenerative disorder caused by a mutation in the HTT gene. The expansion of CAG triplets leads to the appearance of misfolded HTT (huntingtin) forming aggregates and leading to impairment of neuronal functions. Here we demonstrate that stimulation of macroautophagy/autophagy by genistein (4',5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4 H-1-benzopyran-4-one) caused a reduction of levels of mutated HTT in brains of HD mice and correction of their behavior as assessed in a battery of cognitive, anxiety and motor tests, even if the compound was administered after symptoms had developed in the animals. Biochemical and immunological parameters were also improved in HD mice. Studies on molecular mechanisms of genistein-mediated stimulation of autophagy in HD cells indicated the involvement of the FOXO3-related pathway. In conclusion, treatment with genistein stimulates the autophagy process in the brains of HD mice, leading to correction of symptoms of HD, suggesting that it might be considered as a potential drug for this disease. Combined with a very recently published report indicating that impaired autophagy may be a major cause of neurodegenerative changes, these results may indicate the way to the development of effective therapeutic approaches for different neurodegenerative diseases by testing compounds (or possibly combinations of compounds) capable of stimulating autophagy and/or unblocking this process.Abbreviations: CNS: central nervous system; EPM: elevated plus-maze; GOT1/ASPAT: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALAT/ALT: glutamic pyruvic transaminase, soluble; HD: Huntington disease; HTT: huntingtin; IL: interleukin; mHTT: mutant huntingtin; NOR: novel object recognition; MWM: Morris water maze; OF: open field; ROS: reactive oxygen species; TNF: tumor necrosis factor.
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Mucopolysaccharidoses (MPS) are a group of inherited disorders, caused by mutations in the genes coding for proteins involved (directly or indirectly) in glycosaminoglycan (GAG) degradation. A lack or drastically decreased residual activity of a GAG-degrading enzyme leads to the storage of these compounds, thus damaging proper functions of different cells, including neurons. The disease leads to serious psycho-motor dysfunctions and death before reaching the adulthood. Until now, induction of the autophagy process was considered as one of the therapeutic strategies for treatment of diseases caused by protein aggregation (Alzheimer's, Parkinson's, and Huntington's diseases). However, this strategy has only been recently suggested as a potential therapy for MPS. In this work, we show that the pharmacological stimulation of autophagy, by using valproic acid and lithium chloride, led to accelerated degradation of accumulated GAGs. Cytotoxicity tests indicated the safety of the use of the investigated compounds. We observed an increased number of lysosomes and enhanced degradation of heparan sulfate (one of GAGs). Induction of the autophagy process was confirmed by measuring abundance of the marker proteins, including LC3-II. Moreover, inhibition of this process resulted in abolition of the valproic acid- and LiCl-mediated reduction in GAG levels. This is the first report on the possibility of using valproic acid and lithium chloride for reducing levels of GAGs in neuronopathic forms of MPS.
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Morquio disease, also called mucopolysaccharidosis IV (MPS IV), belongs to the group of lysosomal storage diseases (LSD). Due to deficiencies in the activities of galactose-6-sulfate sulfatase (in type A) or ß-galactosidase (in type B), arising from mutations in GALNS or GLB1, respectively, keratan sulfate (one of glycosaminoglycans, GAGs) cannot be degraded efficiently and accumulates in lysosomes. This primary defect leads to many cellular dysfunctions which then cause specific disease symptoms. Recent works have indicated that different secondary effects of GAG accumulation might significantly contribute to the pathomechanisms of MPS. Apoptosis is among the cellular processes that were discovered to be affected in MPS cells on the basis of transcriptomic studies and some cell biology experiments. However, Morquio disease is the MPS type which is the least studied in light of apoptosis dysregulation, while RNA-seq analyses suggested considerable changes in the expression of genes involved in apoptosis in MPS IVA and IVB fibroblasts. Here we demonstrate that cytochrome c release from mitochondria is more efficient in MPS IVA and IVB fibroblasts relative to control cells, both under the standard cultivation conditions and after treatment with staurosporine, an apoptosis inducer. This indication of apoptosis stimulation was corroborated by measurements of the levels of caspases 9, 3, 6, and 7, as well as PARP, cleaved at specific sites, in Morquio disease and control fibroblasts. The more detailed analyses of the transcriptomic data revealed which genes related to apoptosis are down- and up-regulated in MPS IVA and IVB fibroblasts. We conclude that apoptosis is stimulated in Morquio disease under both standard cell culture conditions and after induction with staurosporine which may contribute to the pathomechanism of this disorder. Dysregulation of apoptosis in other MPS types is discussed.
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Condroitina Sulfatases , Mucopolissacaridose IV , Humanos , Mucopolissacaridose IV/terapia , Estaurosporina/farmacologia , Sulfato de Queratano/metabolismo , Fibroblastos/metabolismo , Apoptose/genética , Condroitina Sulfatases/genéticaRESUMO
Until now, only a few studies have focused on the early onset of symptoms of alkaptonuria (AKU) in the pediatric population. This prospective, longitudinal study is a comprehensive approach to the assessment of children with recognized AKU during childhood. The study includes data from 32 visits of 13 patients (five males, eight females; age 4-17 years) with AKU. A clinical evaluation was performed with particular attention to eye, ear, and skin pigmentation, musculoskeletal complaints, magnetic resonance imaging (MRI), and ultrasound (US) imaging abnormalities. The cognitive functioning and adaptive abilities were examined. Molecular genetic analyses were performed. The most common symptoms observed were dark urine (13/13), followed by joint pain (6/13), and dark ear wax (6/13). In 4 of 13 patients the values obtained in the KOOS-child questionnaire were below the reference values. MRI and US did not show degenerative changes in knee cartilages. One child had nephrolithiasis. Almost half of the children with AKU (5/13) presented deficits in cognitive functioning and/or adaptive abilities. The most frequent HGD variants observed in the patients were c.481G>A (p.Gly161Arg) mutation and the c.240A>T (p.His80Gln) polymorphism. The newly described allele of the HGD gene (c.948G>T, p.Val316Phe) which is potentially pathogenic was identified.
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Alcaptonúria , Criança , Masculino , Feminino , Humanos , Pré-Escolar , Adolescente , Alcaptonúria/diagnóstico , Alcaptonúria/genética , Alcaptonúria/patologia , Homogentisato 1,2-Dioxigenase/genética , Estudos Prospectivos , Estudos Longitudinais , MutaçãoRESUMO
The main approach used in the current therapy of mucopolysaccharidosis (MPS) is to reduce the levels of glycosaminoglycans (GAGs) in cells, the deposits considered to be the main cause of the disease. Previous studies have revealed significant differences in the expression of genes encoding proteins involved in many processes, like those related to actin filaments, in MPS cells. Since the regulation of actin filaments is essential for the intracellular transport of specific molecules, the process which may affect the course of MPSs, the aim of this study was to evaluate the changes that occur in the actin cytoskeleton and focal adhesion in cells derived from patients with this disease, as well as in the MPS I mouse model, and to assess whether they could be potential therapeutic targets for different MPS types. Western-blotting, flow cytometry and transcriptomic analyses were employed to address these issues. The levels of the key proteins involved in the studied processes, before and after specific treatment, were assessed. We have also analyzed transcripts whose levels were significantly altered in MPS cells. We identified genes whose expressions were changed in the majority of MPS types and those with particularly highly altered expression. For the first time, significant changes in the expression of genes involved in the actin cytoskeleton structure/functions were revealed which may be considered as an additional element in the pathogenesis of MPSs. Our results suggest the possibility of using the actin cytoskeleton as a potential target in therapeutic approaches for this disease.
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Mucopolissacaridoses , Mucopolissacaridose I , Animais , Camundongos , Adesões Focais/metabolismo , Polimerização , Mucopolissacaridoses/terapia , Mucopolissacaridose I/terapia , Mucopolissacaridose I/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
The phage display technology is based on the presentation of peptide sequences on the surface of virions of bacteriophages. Its development led to creation of sophisticated systems based on the possibility of the presentation of a huge variability of peptides, attached to one of proteins of bacteriophage capsids. The use of such systems allowed for achieving enormous advantages in the processes of selection of bioactive molecules. In fact, the phage display technology has been employed in numerous fields of biotechnology, as diverse as immunological and biomedical applications (in both diagnostics and therapy), the formation of novel materials, and many others. In this paper, contrary to many other review articles which were focussed on either specific display systems or the use of phage display in selected fields, we present a comprehensive overview of various possibilities of applications of this technology. We discuss an usefulness of the phage display technology in various fields of science, medicine and the broad sense of biotechnology. This overview indicates the spread and importance of applications of microbial systems (exemplified by the phage display technology), pointing to the possibility of developing such sophisticated tools when advanced molecular methods are used in microbiological studies, accompanied with understanding of details of structures and functions of microbial entities (bacteriophages in this case).
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Introduction: The problem of antibiotic resistance is a global one, involving many industries and entailing huge financial outlays. Therefore, the search for alternative methods to combat drug-resistant bacteria has a priority status. Great potential is seen in bacteriophages which have the natural ability to kill bacterial cells. Bacteriophages also have several advantages over antibiotics. Firstly, they are considered ecologically safe (harmless to humans, plants and animals). Secondly, bacteriophages preparations are readily producible and easy to apply. However, before bacteriophages can be authorized for medical and veterinary use, they must be accurately characterized in vitro and in vivo to determinate safety. Methods: Therefore, the aim of this study was to verify for the first time the behavioral and immunological responses of both male and female mice (C57BL/6J) to bacteriophage cocktail, composed of two bacteriophages, and to two commonly used antibiotics, enrofloxacin and tetracycline. Animal behavior, the percentage of lymphocyte populations and subpopulations, cytokine concentrations, blood hematological parameters, gastrointestinal microbiome analysis and the size of internal organs, were evaluated. Results: Unexpectedly, we observed a sex-dependent, negative effect of antibiotic therapy, which not only involved the functioning of the immune system, but could also significantly impaired the activity of the central nervous system, as manifested by disruption of the behavioral pattern, especially exacerbated in females. In contrast to antibiotics, complex behavioral and immunological analyses confirmed the lack of adverse effects during the bacteriophage cocktail administration. Discussion: The mechanism of the differences between males and females in appearance of adverse effects, related to the behavioral and immune functions, in the response to antibiotic treatment remains to be elucidated. One might imagine that differences in hormones and/or different permeability of the blood-brain barrier can be important factors, however, extensive studies are required to find the real reason(s).
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Antibacterianos , Bacteriófagos , Feminino , Humanos , Masculino , Animais , Camundongos , Camundongos Endogâmicos C57BL , Antibacterianos/farmacologia , Tetraciclina , EnrofloxacinaRESUMO
Although mucopolysaccharidoses (MPS) are monogenic diseases, caused by mutations in genes coding for enzymes involved in degradation of glycosaminoglycans (GAGs), recent studies suggested that changes in expressions of various genes might cause secondary and tertiary cellular dysfunctions modulating the course of these diseases. In this report, we demonstrate that vesicle trafficking regulation is affected in fibroblasts derived from patients suffering from 11 different types of MPS due to changes in levels of crucial proteins (estimated by automated Western-blotting) involved in this process, including caveolin, clathrin, huntingtin (Htt), APPL1, EEA1, GOPC, Rab5, and Rab7. Microscopic studies confirmed these results, while investigations of tissue samples derived from the MPS I mouse model indicated differences between various organs in this matter. Moreover, transcriptomic analyses provided a global picture for changes in expressions of genes related to vesicle trafficking in MPS cells. We conclude that vesicle trafficking is dysregulated in MPS cells and changes in this process might contribute to the molecular mechanisms of this disease. Most probably, primary GAG storage might cause a cellular stress response leading to dysregulation of expression of many genes which, in turn, results in changes in cellular processes like vesicle trafficking. This can significantly modulate the course of the disease due to enhancing accumulation of GAGs and altering crucial cellular processes. This hypothesis has been supported by normalization of levels of clathrin in MPS cells treated with either an active form of the deficient GAG-degrading enzyme or a compound (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) indirectly reducing the efficiency of GAG synthesis.
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Mucopolissacaridoses , Camundongos , Animais , Linhagem Celular , Mucopolissacaridoses/genética , Mucopolissacaridoses/tratamento farmacológico , Mucopolissacaridoses/metabolismo , Glicosaminoglicanos/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Chimeric antigen receptor T (CAR-T) cells are specifically modified T cells which bear recombinant receptors, present at the cell surface and devoted to detect selected antigens of cancer cells, and due to the presence of transmembrane and activation domains, able to eliminate the latter ones. The use of CAR-T cells in anti-cancer therapies is a relatively novel approach, providing a powerful tool in the fight against cancer and bringing new hope for patients. However, despite huge possibilities and promising results of preclinical studies and clinical efficacy, there are various drawbacks to this therapy, including toxicity, possible relapses, restrictions to specific kinds of cancers, and others. Studies desiring to overcome these problems include various modern and advanced methods. One of them is transcriptomics, a set of techniques that analyze the abundance of all RNA transcripts present in the cell at certain moment and under certain conditions. The use of this method gives a global picture of the efficiency of expression of all genes, thus revealing the physiological state and regulatory processes occurring in the investigated cells. In this review, we summarize and discuss the use of transcriptomics in studies on and applications of CAR-T cells, especially in approaches focused on improved efficacy, reduced toxicity, new target cancers (like solid tumors), monitoring the treatment efficacy, developing novel analytical methods, and others.
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Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases (LSD) caused by mutations in genes coding for enzymes responsible for degradation of glycosaminoglycans (GAGs). Most types of these severe disorders are characterized by neuronopathic phenotypes. Although lysosomal accumulation of GAGs is the primary metabolic defect in MPS, secondary alterations in biochemical processes are considerable and influence the course of the disease. Early hypothesis suggested that these secondary changes might be due to lysosomal storage-mediated impairment of activities of other enzymes, and subsequent accumulation of various compounds in cells. However, recent studies indicated that expression of hundreds of genes is changed in MPS cells. Therefore, we asked whether metabolic effects observed in MPS are caused primarily by GAG-mediated inhibition of specific biochemical reactions or appear as results of dysregulation of expression of genes coding for proteins involved in metabolic processes. Transcriptomic analyses of 11 types of MPS (using RNA isolated from patient-derived fibroblasts), performed in this study, showed that a battery of the above mentioned genes is dysregulated in MPS cells. Some biochemical pathways might be especially affected by changes in expression of many genes, including GAG metabolism and sphingolipid metabolism which is especially interesting as secondary accumulation of various sphingolipids is one of the best known additional (while significantly enhancing neuropathological effects) metabolic defects in MPS. We conclude that severe metabolic disturbances, observed in MPS cells, can partially arise from changes in the expression of many genes coding for proteins involved in metabolic processes.
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Mucopolissacaridoses , Transcriptoma , Humanos , Transcriptoma/genética , Mucopolissacaridoses/genética , Mucopolissacaridoses/patologia , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Linhagem Celular , Lisossomos/metabolismoRESUMO
The methylotrophic yeast Komagataella phaffii is considered one of the most effective producers of recombinant proteins of industrial importance. Effective producers should be characterized by the maximal reduction of degradation of the cytosolic recombinant proteins. The mechanisms of degradation of cytosolic proteins in K. phaffii have not been elucidated; however, data suggest that they are partially degraded in the autophagic pathway. To identify factors that influence this process, a developed system for the selection of recombinant strains of K. phaffii with impaired autophagic degradation of the heterologous model cytosolic protein (yeast ß-galactosidase) was used for insertional tagging of the genes involved in cytosolic proteins degradation. In one of the obtained strains, the insertion cassette disrupted the open reading frame of the gene encoding ß-1,6-N-acetylglucosaminyltransferase. A recombinant strain with deletion of this gene was also obtained. The rate of degradation of the ß-galactosidase enzyme was two times slower in the insertion mutant and 1.5 times slower in the deletion strain as compared to the parental strain with native ß-1,6-N-acetylglucosaminyltransferase. The rate of degradation of native K. phaffii cytosolic and peroxisomal enzymes, formaldehyde dehydrogenase, formate dehydrogenase, and alcohol oxidase, respectively, showed similar trends to that of ß-galactosidase-slower degradation in the deletion and insertional mutants as compared to the wild-type strain, but faster protein degradation relative to the strain completely defective in autophagy. We conclude that K. phaffii gene designated ACG1, encoding ß-1,6-N-acetylglucosaminyltransferase, is involved in autophagy of the cytosolic and peroxisomal proteins.
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N-Acetilglucosaminiltransferases , Saccharomycetales , Saccharomycetales/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase , Autofagia/genéticaRESUMO
Mucopolysaccharidoses (MPS) are rare genetic disorders belonging to the lysosomal storage diseases. They are caused by mutations in genes encoding lysosomal enzymes responsible for degrading glycosaminoglycans (GAGs). As a result, GAGs accumulate in lysosomes, leading to impairment of cells, organs and, consequently, the entire body. Many of the therapies proposed thus far require the participation of chaperone proteins, regardless of whether they are therapies in common use (enzyme replacement therapy) or remain in the experimental phase (gene therapy, STOP-codon-readthrough therapy). Chaperones, which include heat shock proteins, are responsible for the correct folding of other proteins to the most energetically favorable conformation. Without their appropriate levels and activities, the correct folding of the lysosomal enzyme, whether supplied from outside or synthesized in the cell, would be impossible. However, the baseline level of nonspecific chaperone proteins in MPS has never been studied. Therefore, the purpose of this work was to determine the basal levels of nonspecific chaperone proteins of the Hsp family in MPS cells and to study the effect of normalizing GAG concentrations on these levels. Results of experiments with fibroblasts taken from patients with MPS types I, II, IIIA, IIIB, IIIC, IID, IVA, IVB, VI, VII, and IX, as well as from the brains of MPS I mice (Idua-/-), indicated significantly reduced levels of the two chaperones, Hsp70 and Hsp40. Interestingly, the reduction in GAG levels in the aforementioned cells did not lead to normalization of the levels of these chaperones but caused only a slight increase in the levels of Hsp40. An additional transcriptomic analysis of MPS cells indicated that the expression of other genes involved in protein folding processes and the cell response to endoplasmic reticulum stress, resulting from the appearance of abnormally folded proteins, was also modulated. To summarize, reduced levels of chaperones may be an additional cause of the low activity or inactivity of lysosomal enzymes in MPS. Moreover, this may point to causes of treatment failure where the correct structure of the enzyme supplied or synthesized in the cell is crucial to lower GAG levels.