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We recently reported the benefit of the IV transferring of active exogenous mitochondria in a short-term pharmacological AD (Alzheimer's disease) model. We have now explored the efficacy of mitochondrial transfer in 5XFAD transgenic mice, aiming to explore the underlying mechanism by which the IV-injected mitochondria affect the diseased brain. Mitochondrial transfer in 5XFAD ameliorated cognitive impairment, amyloid burden, and mitochondrial dysfunction. Exogenously injected mitochondria were detected in the liver but not in the brain. We detected alterations in brain proteome, implicating synapse-related processes, ubiquitination/proteasome-related processes, phagocytosis, and mitochondria-related factors, which may lead to the amelioration of disease. These changes were accompanied by proteome/metabolome alterations in the liver, including pathways of glucose, glutathione, amino acids, biogenic amines, and sphingolipids. Altered liver metabolites were also detected in the serum of the treated mice, particularly metabolites that are known to affect neurodegenerative processes, such as carnosine, putrescine, C24:1-OH sphingomyelin, and amino acids, which serve as neurotransmitters or their precursors. Our results suggest that the beneficial effect of mitochondrial transfer in the 5XFAD mice is mediated by metabolic signaling from the liver via the serum to the brain, where it induces protective effects. The high efficacy of the mitochondrial transfer may offer a novel AD therapy.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Ratones , Animales , Péptidos beta-Amiloides/metabolismo , Proteoma/metabolismo , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Mitocondrias/metabolismo , Ratones Transgénicos , Hígado/metabolismoRESUMEN
To overcome the lack of specificity of cancer therapeutics and thus create a more potent and effective treatment, we developed a novel chimeric protein, IL2-Smurf2. Here, we describe the production of this chimeric IL2-Smurf2 protein and its variants, with inactive or over-active killing components. Using Western blots, we demonstrated the chimeric protein's ability to specifically enter target cells alone. After entering the cells, the protein showed biological activity, causing cell death that was not seen with an inactive variant, and that was shown to be apoptotic. The chimeric protein also proved to be active as an E3 ligase, as demonstrated by testing total ubiquitination levels along with targeted ubiquitination for degradation. Finally, we tested IL2-Smurf2 and its variants in an in vivo mouse model of leukemia and demonstrated its potential as a drug for the targeted treatment of cancer cells. In the course of this work, we established for the first time the feasibility of the use of Smurf2 as a killing component in chimeric targeting proteins. Utilizing the IL2 cytokine to target cells overexpressing IL-2R and Smurf2 to cause protein degradation, we were able to produce a chimeric protein with dual functionality which causes targeted cell death.
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Much effort has been dedicated in the recent decades to find novel protein/enzyme-based therapies for human diseases, the major challenge of such therapies being the intracellular delivery and reaching sub-cellular organelles. One promising approach is the use of cell-penetrating peptides (CPPs) for delivering enzymes/proteins into cells. In this review, we describe the potential therapeutic usages of CPPs (mainly trans-activator of transcription protein, TAT) in enabling the uptake of biologically active proteins/enzymes needed in cases of protein/enzyme deficiency, concentrating on mitochondrial diseases and on the import of enzymes or peptides in order to destroy pathogenic cells, focusing on cancer cells.
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Pathogenesis of neurodegenerative diseases involves dysfunction of mitochondria, one of the most important cell organelles in the brain, with its most prominent roles in producing energy and regulating cellular metabolism. Here we investigated the effect of transferring active intact mitochondria as a potential therapy for Alzheimer's disease (AD), in order to correct as many mitochondrial functions as possible, rather than a mono-drug related therapy. For this purpose, AD-mice (amyloid-ß intracerebroventricularly injected) were treated intravenously (IV) with fresh human isolated mitochondria. One to two weeks later, a significantly better cognitive performance was noticed in the mitochondria treated AD-mice relative to vehicle treated AD-mice, approaching the performance of non-AD mice. We also detected a significant decrease in neuronal loss and reduced gliosis in the hippocampus of treated mice relative to untreated AD-mice. An amelioration of the mitochondrial dysfunction in brain was noticed by the increase of citrate-synthase and cytochrome c oxidase activities relative to untreated AD-mice, reaching activity levels of non-AD-mice. Increased mitochondrial activity was also detected in the liver of mitochondria treated mice. No treatment-related toxicity was noted. Thus, IV mitochondrial transfer may possibly offer a novel therapeutic approach for AD.
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Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Trastornos del Conocimiento/patología , Trastornos del Conocimiento/terapia , Gliosis/patología , Mitocondrias/trasplante , Neuronas/patología , Enfermedad de Alzheimer/inducido químicamente , Péptidos beta-Amiloides/administración & dosificación , Animales , Conducta Animal , Citrato (si)-Sintasa/metabolismo , Cognición , Trastornos del Conocimiento/inducido químicamente , Complejo IV de Transporte de Electrones/metabolismo , Células HeLa , Humanos , Inyecciones Intraventriculares , Masculino , Aprendizaje por Laberinto , Ratones , Mitocondrias Hepáticas/metabolismo , Desempeño PsicomotorRESUMEN
Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA.
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Adenosina Trifosfato/biosíntesis , Fibroblastos/enzimología , Productos del Gen tat/genética , Metilmalonil-CoA Mutasa/genética , Mitocondrias/enzimología , Proteínas Recombinantes de Fusión/genética , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/patología , Errores Innatos del Metabolismo de los Aminoácidos/terapia , Sistemas CRISPR-Cas , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/patología , Expresión Génica , Productos del Gen tat/metabolismo , Terapia Genética/métodos , Células Hep G2 , Humanos , Hígado/enzimología , Hígado/patología , Potencial de la Membrana Mitocondrial , Ácido Metilmalónico/metabolismo , Metilmalonil-CoA Mutasa/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Enfermedades Mitocondriales/terapia , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , Ingeniería de Proteínas/métodos , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders.
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Proteínas de Unión a Hierro/metabolismo , Mitocondrias/metabolismo , Aconitato Hidratasa/metabolismo , Ataxia de Friedreich/metabolismo , Humanos , Estrés Oxidativo , Transporte de Proteínas , FrataxinaRESUMEN
Plants have been used for medical purposes since the beginning of human history and are the basis of modern medicine. Most chemotherapeutic drugs for cancer treatment are molecules identified and isolated from plants or their synthetic derivatives. Our hypothesis was that whole plant extracts selected according to ethnobotanical sources of historical use might contain multiple molecules with antitumor activities that could be very effective in killing human cancer cells. This study examined the effects of three whole plant extracts (ethanol extraction) on human tumor cells. The extracts were from Urtica membranacea (Urticaceae), Artemesia monosperma (Asteraceae), and Origanum dayi post (Labiatae). All three plant extracts exhibited dose- and time-dependent killing capabilities in various human derived tumor cell lines and primary cultures established from patients' biopsies. The killing activity was specific toward tumor cells, as the plant extracts had no effect on primary cultures of healthy human cells. Cell death caused by the whole plant extracts is via apoptosis. Plant extract 5 (Urtica membranacea) showed particularly strong anticancer capabilities since it inhibited actual tumor progression in a breast adenocarcinoma mouse model. Our results suggest that whole plant extracts are promising anticancer reagents.
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Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias/tratamiento farmacológico , Extractos Vegetales/administración & dosificación , Plantas Medicinales/química , Animales , Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Asteraceae/química , Línea Celular Tumoral , Humanos , Lamiaceae/química , Ratones , Extractos Vegetales/química , Urticaceae/químicaRESUMEN
The discovery and therapeutic use of antibiotics made a major contribution to the reduction of human morbidity and mortality. However, the growing resistance to antibiotics has become a matter of huge concern. In this study we aimed to develop an innovative approach to treat bacterial infections utilizing two components: the human antibacterial peptide ß-defensin-3 (BD3) and the bacterial protein flagellin (F). This combination was designed to provide an efficient weapon against bacterial infections with the peptide killing the bacteria directly, while the flagellin protein triggers the immune system and acts against bacteria escaping from the peptide's action. We designed, expressed and purified the fusion protein flagellin BD3 (FBD3) and its two components, the F protein and the native BD3 peptide. FBD3 fusion protein and native BD3 peptide had antibacterial activity in vitro against various bacterial strains. FBD3 and F proteins could also recognize their receptor expressed on target cells and stimulated secretion of IL-8. In addition, F and FBD3 proteins had a partial protective effect in mice infected by pathogenic Escherichia coli bacteria that cause a lethal disease. Moreover, we were able to show partial protection of mice infected with E. coli using a flagellin sequence from Salmonella. We also explored flagellin's basic mechanisms of action, focusing on its effects on CD4+ T cells from healthy donors. We found that F stimulation caused an increase in the mRNA levels of the Th1 response cytokines IL12A and IFNγ. In addition, F stimulation affected its own receptor.
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Disorders of the oxidative phosphorylation (OXPHOS) system frequently result in a severe multisystem disease with the consequence of early childhood death. Among these disorders, isolated complex I deficiency is the most frequently diagnosed, accounting for one-third of all cases of respiratory chain deficiency. We chose to focus on complex I deficiency, caused by mutation in the assembly factor chromosome 6, open reading frame 66 (C6ORF66; NADH dehydrogenase [ubiquinone] complex I assembly factor 4 [NDUFAF4]) protein. We used the approach of cell- and organelle-directed protein/enzyme replacement therapy, with the transactivator of transcription (TAT) peptide as the moiety delivery system. This step will enable us to deliver the wild-type assembly factor C6ORF66 into patient cells and their mitochondria, leading to the proper assembly and function of complex I and, as a result, to a functional OXPHOS system. We designed and constructed the TAT-ORF fusion protein by gene fusion techniques, expressed the protein in an Escherichia coli expression system and highly purified it. Our results indicate that TAT-ORF enters patients' cells and their mitochondria rapidly and efficiently. TAT-ORF is biologically active and led to an increase in complex I activity. TAT-ORF also increased the number of patient cells and improved the activity of their mitochondria. Moreover, we observed an increase in ATP production, a decrease in the content of mitochondria and a decrease in the level of reactive oxygen species. Our results suggest that this approach of protein replacement therapy for the treatment of mitochondrial disorders is a promising one.
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Proteínas de Unión a Calmodulina/farmacología , Complejo I de Transporte de Electrón/metabolismo , Productos del Gen tat/farmacología , Enfermedades Mitocondriales/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas de Unión a Calmodulina/genética , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Productos del Gen tat/genética , Humanos , Enfermedades Mitocondriales/genéticaRESUMEN
BACKGROUND: A wide range of hematologic malignancies arises from numerous cell types. In an attempt to offer a new target for treating B-cell malignancies, in this study, the authors tested the possibility of using the CD40/CD40L system as a common targeting system for the various malignancies in this group. METHODS: Two chimeric proteins, soluble CD40 ligand (sCD40L)-caspase 3 (sCD40L-l-Caspase3) and sCD40L-pseudomonas exotoxin 38 (PE38) (sCD40L-l-PE38), were constructed, expressed, and partially purified. The ability of the chimeric proteins to kill tumor cells that expressed CD40 was tested by using proliferation assays. In addition, the induction of apoptosis in treated cells was followed by measuring expression levels of apoptotic proteins using real-time polymerases chain reaction analysis, caspase 3 enzymatic activity, and tracking changes in the cell cycle with fluorescence-activated cell-sorting analysis. RESULTS: The chimeric proteins exhibited concentration-dependent and time-dependent killing ability. The new chimeric proteins had no effect in several carcinoma cell lines that did not express the CD40 receptor. Treating tumor cells with sCD40L-based chimeric proteins led to internalization of the fusion proteins into the cell cytoplasm of B cells. Shortly after treatment, a sharp rise in B-cell chronic lymphocytic leukemia/lymphoma 2 (Bcl2) expression levels occurred. Approximately 36 hours after the initiation of treatment, Bcl2 levels dropped, whereas Bcl2-associated X protein (Bax) expression levels rose, pushing the cells toward apoptosis. Concomitantly, caspase 3 RNA levels rose. CONCLUSIONS: sCD40L-based chimeric proteins were able to bind and internalize into B cells that expressed the CD40 receptor and specifically and efficiently induced apoptotic death. Moreover, the current results validated for the first time the ability of sCD40L to serve as a direct delivery system for targeted molecules. sCD40L-based chimeric cytotoxic proteins offer a new weapon in the everlasting war against cancer.
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Apoptosis , Linfocitos B/patología , Ligando de CD40/metabolismo , Caspasa 3/metabolismo , Leucemia de Células B/terapia , Linfoma de Células B/terapia , Proteínas Recombinantes de Fusión/uso terapéutico , Western Blotting , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligando de CD40/genética , Caspasa 3/genética , Ciclo Celular , Exotoxinas/metabolismo , Humanos , Leucemia de Células B/metabolismo , Leucemia de Células B/patología , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pseudomonas aeruginosa , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Designing a chimeric protein and developing a procedure for its stable production as a biologically active protein, are key steps in its potential application to clinical trails. IL2-Caspase3 chimeric protein designed to target activated T lymphocytes was found to be a promising molecule for targeted treatment, however was found to be difficult to produce as a biological active molecule. Thus, we designed a new version of the molecule, IL2-Caspase3s, in which six amino acids (aa 29-34) from the N-terminus of the large subunit of caspase 3 were excluded. Repeated expressions, productions, and partial purifications of the IL2-Caspase3s yielded reproducible batches with consistent results. We found that IL2-Caspase3s causes cell death in a specific, dose-, and time-dependent manner. Cell death due to IL2-Caspase3s is caused by apoptosis. This improved and biologically stable IL2-Caspase3s chimeric protein may be developed in the future for clinical trails as a promising therapy for several pathologies involving activated T-cells. Moreover, this truncated caspase 3 sequence, lacking the N-terminal six amino acids of its large subunit, may be used in other caspase 3-based chimeric proteins targeted against various human diseases, using the appropriate targeting moiety.
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Caspasa 3/química , Caspasa 3/metabolismo , Interleucina-2/farmacología , Secuencias de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn-dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over-expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer.
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Modelos Animales de Enfermedad , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Lesiones Precancerosas/patología , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Carcinoma Pulmonar de Lewis , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Lesiones Precancerosas/metabolismoRESUMEN
INTRODUCTION: The development of specific immunosuppressive reagents remains the major goal in the treatment of human diseases. One such approach is the use of recombinant immunotoxins/chimeric proteins, composed of targeting and killing moieties, fused at the cDNA level. Most of these 'magic bullets' use bacterial or plant toxins to induce cell death. These toxins are extremely potent, but they also cause severe toxicity and systemic side effects that limit the maximal doses given to patients. Moreover, being of non-human origin, they are highly immunogenic, and the resulting neutralizing antibody production impairs their efficacy. AREAS COVERED: This review describes recombinant immunotoxins/chimeric proteins composed of the classical delivering, cell-targeting molecules, fused to highly cytotoxic human proteins capable of generating an intense apoptotic response within the target cell. This review focuses on the new 'Human Killing Moieties' of these targeted proteins and describes recent progress in the development of these promising molecules. EXPERT OPINION: Human toxin-based immunotoxins/chimeric proteins for the targeted delivery of drugs are still in their early stages of development. However, they are certain to advance in the very near future to become an extra weapon in the everlasting war against human diseases, mainly cancer.
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Inmunotoxinas/uso terapéutico , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Sistemas de Liberación de Medicamentos , Humanos , Inmunotoxinas/administración & dosificación , Inmunotoxinas/efectos adversos , Inmunotoxinas/inmunología , Neoplasias/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
One of the main problems of conventional anticancer therapy is multidrug resistance (MDR), whereby cells acquire resistance to structurally and functionally unrelated drugs following chemotherapeutic treatment. One of the main causes of MDR is overexpression of the P-glycoprotein transporter. In addition to extruding the chemotherapeutic drugs, it also inhibits apoptosis through the inhibition of caspases. To overcome MDR, we constructed a novel chimeric protein, interleukin (IL)-2 granzyme A (IGA), using IL-2 as a targeting moiety and granzyme A as a killing moiety, fused at the cDNA level. IL-2 binds to the high-affinity IL-2 receptor that is expressed in an array of abnormal cells, including malignant cells. Granzyme A is known to cause caspase 3-independent cell death. We show here that the IGA chimeric protein enters the target sensitive and MDR cancer cells overexpressing IL-2 receptor and induces caspase 3-independent cell death. Specifically, after its entry, IGA causes a decrease in the mitochondrial potential, triggers translocation of nm23-H1, a granzyme A-dependent DNase, from the cytoplasm to the nucleus, where it causes single-strand DNA nicks, thus causing cell death. Moreover, IGA is able to overcome MDR and kill cells resistant to chemotherapeutic drugs. We believe that overcoming MDR with targeted molecules such as IGA chimeric protein that causes caspase-independent apoptotic cell death could be applied to many other resistant types of tumors using the appropriate targeting moiety. Thus, this novel class of targeted molecules could open up new vistas in the fight against human cancer.
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Apoptosis , Caspasa 3/metabolismo , Resistencia a Múltiples Medicamentos , Granzimas/metabolismo , Interleucina-2/metabolismo , Linfoma/tratamiento farmacológico , Proteínas Recombinantes de Fusión/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Western Blotting , Núcleo Celular/metabolismo , Proliferación Celular , Citoplasma/metabolismo , Daño del ADN , Resistencia a Antineoplásicos , Granzimas/genética , Humanos , Interleucina-2/genética , Linfoma/metabolismo , Linfoma/patología , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Transporte de Proteínas , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Células Tumorales CultivadasRESUMEN
Medicine today offers no cure for patients suffering from mitochondrial disorders, such as lipoamide dehydrogenase (LAD; also known as E3) deficiency, and treatment is limited to symptomatic care. LAD is one of the components of the α-ketoacid dehydrogenase complexes, which are mitochondrial multienzyme complexes crucial for the metabolism of carbohydrates and amino acids. Recently, we tested the therapeutic approach for treating mitochondrial disorders whereby the activity of multicomponent complexes in the mitochondria is restored by TAT-mediated enzyme replacement therapy (ERT). The LAD deficiency disease was used before as a proof-of-principle in vitro, in patients' cells, utilizing the TAT-LAD fusion protein. In this report, we present successful TAT-mediated ERT in an in vivo mouse model using E3-deficient mice. We demonstrate the delivery of TAT-LAD into E3-deficient mice tissues and that a single administration of TAT-LAD results in a significant increase in the enzymatic activity of the mitochondrial multienzyme complex pyruvate dehydrogenase complex within the liver, heart and, most importantly, the brain of TAT-LAD-treated E3-deficient mice. We believe that this TAT-mediated ERT approach could change the management of mitochondrial disorders and of other metabolic diseases in modern medicine.
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Dihidrolipoamida Deshidrogenasa/deficiencia , Terapia de Reemplazo Enzimático , Productos del Gen tat/metabolismo , Mitocondrias/enzimología , Proteínas Recombinantes de Fusión , Animales , Dihidrolipoamida Deshidrogenasa/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Especificidad de Órganos/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Targeted depletion of immune cells expressing the interleukin-2 (IL-2) receptor can exacerbate inflammatory bowel disease (IBD) through elimination of regulatory T (Treg) cells, or ameliorate its course by depletion of cytotoxic cells. To answer this question we used a fusion protein composed of IL-2 and caspase-3 (IL2-cas) in an experimental model of DSS-induced toxic colitis. In a preventive setting, co-administration of DSS with a daily therapeutic dose of IL2-cas for seven days improved all disease parameters. Although CD4(+)CD25(+) T cells were depleted in the mesenteric lymph nodes, a fractional increase in CD4(+)FoxP3(+) T cells was observed in the spleen. Likewise, IL2-cas therapy improved the outcome of established disease in a chronic model of colitis. These data demonstrate that therapies that use IL-2 as a targeting moiety exert a protective effect over the colon under conditions of inflammation. The efficacy of IL-2-targeted therapy is attributed to reduced activity of reactive T cells, which ameliorates the secondary inflammatory infiltration. IL2-cas evolves as a potential therapeutic tool in IBD.
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Caspasa 3/metabolismo , Colitis/prevención & control , Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Animales , Peso Corporal/efectos de los fármacos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Caspasa 3/genética , Colitis/inducido químicamente , Sulfato de Dextran , Factores de Transcripción Forkhead/metabolismo , Inyecciones Intravenosas , Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
IL2-caspase3 chimeric protein was designed to target and kill cells expressing the high affinity IL-2 receptor. Its effects on lymphocyte reactivity and on experimental autoimmune encephalomyelitis (EAE), a T-cell mediated disease, were tested in this study. Our data show that IL2-caspase3 promoted cell specific apoptosis both in vitro and in vivo. Cell lines preferentially expressing the IL-2R alpha chain and encephalitogenic lymphocytes derived from EAE-induced mice were highly sensitive to the chimeras' activity. This was demonstrated by increased DNA fragmentation and annexin labeling together with reduced specific T-cell proliferation in response to IL2-casepase3 treatment. Furthermore, IL2-caspase3 treatment of EAE-induced mice caused a significant delay in disease onset together with a reduction in disease burden. The efficacy of IL2-caspase3 treatment was dependent on the time at which treatment begun, with the chimera ameliorating EAE only when administered at maximal activation of peripheral lymphocytes. According to our findings we suggest that the chimeric protein IL2-caspase3 may provide a novel approach for the treatment of a variety of autoimmune disorders, such as multiple sclerosis, as well as for other pathological conditions that involve uncontrolled expansion of activated T cells.
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Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Linfocitos T/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Caspasa 3/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Progresión de la Enfermedad , Sistemas de Liberación de Medicamentos , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Interleucina-2/genética , Ratones , Ingeniería de Proteínas , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
There has been great progress in the use of TAT-based drug delivery systems for the delivery of different macromolecules into cells in vitro and in vivo, thus circumventing the bioavailability barrier that is a problem for so many drugs. There are many advantages to using this system, such as the ability to deliver these cargoes into all types of cells in culture and into all organs in vivo. This system can even deliver cargoes into the brain across the blood-brain barrier. In addition, the ability to target specific intracellular sub-localizations such as the nuclei, the mitochondria and lysosomes further expands the possibilities of this drug delivery system to the development of sub-cellular organelle-targeted therapy. The therapeutic applications seem almost unlimited, and the use of the TAT-based delivery system has extended from proteins to a large variety of cargoes such as oligonucleotides, imaging agents, low molecular mass drugs, nanoparticles, micelles and liposomes. In this review the most recent advances in the use of the TAT-based drug delivery system will be described, mainly discussing TAT-mediated protein delivery and the use of the TAT system for enzyme replacement therapy.
Asunto(s)
Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Productos del Gen tat/administración & dosificación , Productos del Gen tat/metabolismo , Animales , Humanos , Modelos Biológicos , Nanopartículas/administración & dosificación , Ácidos Nucleicos/administración & dosificación , Ácidos Nucleicos de Péptidos/administración & dosificación , Proteínas/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificaciónRESUMEN
Modern medicine offers no cure for mitochondrial disorders such as lipoamide dehydrogenase (LAD) deficiency. LAD is the E3 subunit shared by alpha-ketoacid dehydrogenase complexes in the mitochondrial matrix, and these complexes are crucial for the metabolism of carbohydrates and amino acids. We propose a novel concept for restoring the activity of an immense multicomponent enzymatic complex by replacing one mutated component, the LAD subunit. Our approach entails the fusing of LAD with the transactivator of transcription (TAT) peptide, which is capable of rapidly crossing biological membranes, thereby allowing TAT-LAD to be delivered into cells and their mitochondria where it can replace the mutated endogenous enzyme. Our results show that TAT-LAD is indeed delivered into the cells and their mitochondria, where it is processed, restoring LAD activity to normal values and, most importantly, increasing the activity of pyruvate dehydrogenase complex. We report here, for the first time, that TAT-mediated replacement of one mutated component restores the activity of an essential mitochondrial multicomponent enzymatic complex in cells of patients with enzyme deficiencies. We believe that this approach involving TAT-mediated enzyme replacement therapy (ERT) can be applied to the treatment of LAD deficiency as well as to other mitochondrial and metabolic disorders.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/deficiencia , Dihidrolipoamida Deshidrogenasa/genética , Mitocondrias/efectos de los fármacos , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Células Cultivadas , Dihidrolipoamida Deshidrogenasa/metabolismo , Activación Enzimática , Humanos , Mitocondrias/enzimología , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Homozygosity mapping was performed in five patients from a consanguineous family who presented with infantile mitochondrial encephalomyopathy attributed to isolated NADH:ubiquinone oxidoreductase (complex I) deficiency. This resulted in the identification of a missense mutation in a conserved residue of the C6ORF66 gene, which encodes a 20.2 kDa mitochondrial protein. The mutation was also detected in a patient who presented with antenatal cardiomyopathy. In muscle of two patients, the levels of the C6ORF66 protein and of the fully assembled complex I were markedly reduced. Transfection of the patients' fibroblasts with wild-type C6ORF66 cDNA restored complex I activity. These data suggest that C6ORF66 is an assembly factor of complex I. Interestingly, the C6ORF66 gene product was previously shown to promote breast cancer cell invasiveness.