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1.
J Immunol ; 196(12): 5156-65, 2016 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-27183614

RESUMEN

The impact of microRNAs (miRNAs) known to regulate numerous biologic processes on complement-dependent cytotoxicity (CDC) was investigated in K562 cells. The C5b-9 complex is the executioner of CDC. Cells protect themselves from CDC by C5b-9 elimination, a process involving the mitochondrial chaperone mortalin/GRP75. Potential miR-200 (b and c) and miR-217 regulatory sites were identified in mortalin mRNA. Overexpression of miR-200b/c or miR-217 lowered the expression of mortalin mRNA. miRNA inhibitors for miR-200b, miR-200c, or miR-217 enhanced mortalin mRNA level. Unexpectedly, these miRNA modulators had no significant effect on mortalin protein level. Metabolic labeling analysis demonstrated that, to compensate for reduction in mortalin mRNA level, the cells increased the rate of synthesis of mortalin protein. Cells overexpressing miR-200b/c or miR-217 showed reduced sensitivity to CDC, whereas inhibition of miR-200c and miR-217 enhanced cell death. miR-200b/c overexpression reduced C5b-9 binding and enhanced its release from the cells and promoted mortalin relocation to the plasma membrane. Inhibition of miR-200 (b and c) and miR-217 had no effect on the expression level of the membrane complement-regulatory proteins CD46, CD55, and CD59. However, overexpression of miR-200b/c or miR-217 enhanced expression of CD46 and CD55 (not of CD59). Overall, the data demonstrate miRNA regulation of cell sensitivity to CDC. We identified miR-200b, miR-200c, and miR-217 as regulators of mortalin and, perhaps indirectly, of CD46 and CD55. Cell exposure to a sublytic dose of complement was shown to increase expression of miR-200 (b and c), suggesting that complement C5b-9 exerts a feedback-regulatory effect on these miRNAs.


Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Citotoxicidad Inmunológica , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , MicroARNs/genética , Antígenos CD55/genética , Antígenos CD59/genética , Línea Celular Tumoral , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Humanos , Células K562 , Proteína Cofactora de Membrana/genética , ARN Mensajero
2.
Int J Cancer ; 141(11): 2329-2335, 2017 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-28791678

RESUMEN

Mitochondrial mortalin and cytosolic Hsp70 are essential chaperones overexpressed in cancer cells. Our goals were to reproduce our earlier findings of elevated circulating levels of mortalin and Hsp70 in colorectal cancer (CRC) patients with a larger patient cohort, to compare death risk assessment of mortalin, Hsp70, CEA and C19-9 and to assess their prognostic value in various CRC stages. Mortalin, Hsp70, CEA and CA19-9 levels were determined in sera of 235 CRC patients enrolled in the study and followed up 5 years after surgery. Association between their concentrations and patients' survival was analyzed by Kaplan-Meier estimator and subjected to Cox Proportional hazards analysis. Serum level of mortalin was independent of that of Hsp70, CEA and CA19-9, whereas Hsp70 level weakly correlated with CEA and CA19-9 levels. Improved short-term survival was found in early or advanced disease stages associated with lower mortalin and Hsp70 levels. Cox regression analysis showed a high mortality hazard (HR = 3.7, p < 0.001) in patients with both high mortalin and Hsp70 circulating levels. Multivariate analysis showed that high mortalin and Hsp70 significantly enhances risk score over a baseline model of age, number of affected lymph nodes, CEA, CA19-9, disease stage and perioperative therapy. Analysis of mortalin and Hsp70 in CRC patients' sera adds a high prognostic value to TNM stage and to CEA and CA19-9 and identifies patients with lower or higher survival probability in all CRC stages. Determination of mortalin and Hsp70 in blood could be a useful additive prognostic tool in guiding clinical management of patients.


Asunto(s)
Adenocarcinoma/sangre , Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/sangre , Proteínas HSP70 de Choque Térmico/sangre , Proteínas Mitocondriales/sangre , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adulto , Anciano , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Citosol , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Riesgo
3.
Neurobiol Dis ; 96: 84-94, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27544484

RESUMEN

In Alzheimer's disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aß42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aß clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aß uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD.


Asunto(s)
Envejecimiento , Enfermedad de Alzheimer , Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Encéfalo/patología , Regulación de la Expresión Génica/genética , Fármacos Neuroprotectores/uso terapéutico , Fragmentos de Péptidos/metabolismo , Envejecimiento/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Antígeno CD11b/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Receptores de Complemento/metabolismo
4.
J Biol Chem ; 289(21): 15014-22, 2014 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-24719326

RESUMEN

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.


Asunto(s)
Complemento C9/inmunología , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Adenosina Difosfato/inmunología , Adenosina Difosfato/farmacología , Adenosina Trifosfatasas/inmunología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/inmunología , Adenosina Trifosfato/farmacología , Sitios de Unión/genética , Sitios de Unión/inmunología , Western Blotting , Complemento C9/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Células K562 , Microscopía Confocal , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Interferencia de ARN , Cloruro de Sodio/inmunología , Cloruro de Sodio/farmacología
5.
J Immunol ; 189(2): 860-6, 2012 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-22685314

RESUMEN

Nucleated cells are equipped with several mechanisms that support their resistance to complement-dependent cytotoxicity (CDC). The role of the NF-κB pathway in cell protection from CDC was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 subunit of NF-κB or the IκB kinases IKKα or IKKß, and in cells treated with p65 small interfering RNA. Pretreatment with the IKK inhibitor PS-1145 also enhanced CDC of wild-type cells (WT) but not of p65(-/-) cells. Furthermore, reconstitution of p65 into p65(-/-) cells and overexpression of p65 in WT cells lowered their sensitivity to CDC. The postulated effect of p65 on the JNK-mediated death-signaling pathway activated by complement was examined. p65 small interfering RNA enhanced CDC in WT cells but not in cells lacking JNK. JNK phosphorylation induced by complement was more pronounced in p65(-/-) cells than in WT cells. The results indicate that the NF-κB pathway mediates cell resistance to CDC, possibly by suppressing JNK-dependent programmed necrotic cell death.


Asunto(s)
Activación de Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/fisiología , Citotoxicidad Inmunológica/inmunología , Transducción de Señal/inmunología , Factor de Transcripción ReIA/fisiología , Animales , Comunicación Celular/genética , Comunicación Celular/inmunología , Activación de Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/deficiencia , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Citotoxicidad Inmunológica/genética , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/inmunología , Células Madre Embrionarias/metabolismo , Fibroblastos/enzimología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Quinasa I-kappa B/deficiencia , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/metabolismo , MAP Quinasa Quinasa 4/fisiología , Ratones , Ratones Noqueados , Subunidades de Proteína/deficiencia , Transducción de Señal/genética , Factor de Transcripción ReIA/deficiencia , Factor de Transcripción ReIA/metabolismo
6.
Front Immunol ; 15: 1337215, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38715618

RESUMEN

Background: Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70. It protects cells from various types of damages and from senescence. Our goal was to determine whether COVID-19 patients have circulating mortalin in their blood and to assess its prognostic value in anticipating disease severity. Methods: Mortalin was determined by ELISA in the sera of 83 COVID-19 patients enrolled in the study. Patients were categorized into 4 groups: critical patients who died (FATAL) or required intensive care and survived (ICU), patients of mild severity (hospitalized but not critical) who required nasal oxygen support (HOSP+O2), and patients who did not need oxygen therapy (HOSP). Results: The mortalin concentration in the serum of all COVID-19 patients in the cohort was 194-2324 pg/mL. A comparison of the mortalin levels by peak severity among the various patient groups showed a highly significant difference between the HOSP and FATAL groups and a significant difference between the HOSP and the ICU groups. COVID-19 patients who eventually failed to survive had at hospitalization a markedly higher level of mortalin in their sera. Cox regression analysis revealed a high mortality hazard (HR=3.96, p<0.01) in patients with high mortalin circulating levels (above the median, ≥651 pg/mL). This was confirmed in survival curve analysis (Kaplan-Meier; p=0.0032, log-rank test). Mortalin remained an independent predictor of mortality even after adjusting for age and sex or various complement activation products. Complement activation data collected in an earlier study in the same cohort was compared regarding the mortalin levels. Patients with higher circulating mortalin levels also had higher levels of complement C3a but reduced levels of properdin. Discussion: This is the first report on circulating mortalin in COVID-19 patients. Higher mortalin levels were associated with more severe illnesses and a higher risk of death. We claim that quantifying the blood levels of mortalin and activated complement proteins will provide important information on the prognosis of COVID-19 patients and will serve as a useful tool for guiding their clinical management and treatment.


Asunto(s)
COVID-19 , Proteínas HSP70 de Choque Térmico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores/sangre , Activación de Complemento , COVID-19/sangre , COVID-19/diagnóstico , COVID-19/inmunología , Proteínas HSP70 de Choque Térmico/sangre , Pronóstico , SARS-CoV-2/fisiología , Índice de Severidad de la Enfermedad
7.
J Biol Chem ; 287(24): 19904-15, 2012 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-22528500

RESUMEN

The complement system, an important element of both innate and adaptive immunity, is executing complement-dependent cytotoxicity (CDC) with its C5b-9 protein complex that is assembled on cell surfaces and transmits to the cell death signals. In turn, cells, and in particular cancer cells, protect themselves from CDC in various ways. Thus, cells actively remove the C5b-9 complexes from their plasma membrane by endocytosis. Inhibition of clathrin by transfection with shRNA or of EPS-15 with a dominant negative plasmid had no effect on C5b-9 endocytosis and on cell death. In contrast, inhibition of caveolin-1 (Cav-1) by transfection with an shRNA or a dominant negative plasmid sensitized cells to CDC and inhibited C5b-9 endocytosis. Similarly, both inhibition of dynamin-2 by transfection with a dominant negative plasmid or by treatment with Dynasore reduced C5b-9 endocytosis and enhanced CDC. C5b-9 endocytosis was also disrupted by pretreatment of the cells with methyl-ß-cyclodextrin or Filipin III, hence implicating membrane cholesterol in the process. Analyses by confocal microscopy demonstrated co-localization of Cav-1-EGFP with C5b-9 at the plasma membrane, in early endosomes, at the endocytic recycling compartment and in secreted vesicles. Further investigation of the process of C5b-9 removal by exo-vesiculation demonstrated that inhibition of Cav-1 and cholesterol depletion abrogated C5b-9 exo-vesiculation, whereas, over-expression of Cav-1 increased C5b-9 exo-vesiculation. Our results show that Cav-1 and dynamin-2 (but not clathrin) support cell resistance to CDC, probably by facilitating purging of the C5b-9 complexes by endocytosis and exo-vesiculation.


Asunto(s)
Caveolina 1/metabolismo , Membrana Celular/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Dinamina II/metabolismo , Endocitosis/fisiología , Antibacterianos/farmacología , Caveolina 1/genética , Complejo de Ataque a Membrana del Sistema Complemento/genética , Dinamina II/genética , Endocitosis/efectos de los fármacos , Filipina/farmacología , Humanos , Células K562 , beta-Ciclodextrinas/farmacología
8.
Int J Cancer ; 133(2): 514-8, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23319326

RESUMEN

Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70 (HSP70). It protects cells from senescence and apoptosis and is overexpressed in cancer cells. Cell resistance to complement-dependent cytotoxicity depends on mortalin and during complement attack mortalin is released from cells. Our goal was to determine whether cancer patients have circulating mortalin in blood. The significance of mortalin in blood to survival prospects of colorectal cancer patients was evaluated. Occurrence of extracellular soluble HSP70 (sHSP70) is documented. We developed a sensitive ELISA for mortalin. The association between mortalin level and survival was subjected to the Cox proportional hazards analysis (univariate and multivariate analyses). Mortalin concentration in serum of colorectal cancer patients was 10-214 ng/ml. Survival data of the patients were known from an earlier study of sHSP70 in these samples. Cox regression analysis indicated that high mortalin (>60 ng/ml) is a risk factor for shorter survival. Serum levels of sHSP70 and mortalin in patients were independent variables. Concurrence of high sHSP70 and mortalin was associated with rapid disease progression (HR = 4, 2.04-8.45, p < 0.001). Addition of high sHSP70 and mortalin to a baseline model of age, sex and TNM stage, significantly (p < 0.001) enhanced the risk score to 8 (3.26-20.46). This is the first demonstration of circulating mortalin in cancer patients. Analysis of mortalin in blood, and even more so of mortalin and sHSP70, adds a high prognostic value to the TNM stage and will identify colorectal cancer patients at high risk of poor survival.


Asunto(s)
Neoplasias Colorrectales/sangre , Regulación Neoplásica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/sangre , Mitocondrias/metabolismo , Estudios de Cohortes , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Citosol/metabolismo , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Análisis Multivariante , Modelos de Riesgos Proporcionales , Factores de Riesgo
9.
Biomedicines ; 11(7)2023 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-37509610

RESUMEN

Cell-penetrating peptides (CPPs), developed for more than 30 years, are still being extensively studied due to their excellent delivery performance. Compared with other delivery vehicles, CPPs hold promise for delivering different types of drugs. Here, we review the development process of CPPs and summarize the composition and classification of the CPP-based delivery systems, cellular uptake mechanisms, influencing factors, and biological barriers. We also summarize the optimization routes of CPP-based macromolecular drug delivery from stability and targeting perspectives. Strategies for enhanced endosomal escape, which prolong its half-life in blood, improved targeting efficiency and stimuli-responsive design are comprehensively summarized for CPP-based macromolecule delivery. Finally, after concluding the clinical trials of CPP-based drug delivery systems, we extracted the necessary conditions for a successful CPP-based delivery system. This review provides the latest framework for the CPP-based delivery of macromolecular drugs and summarizes the optimized strategies to improve delivery efficiency.

10.
J Immunol ; 182(1): 515-21, 2009 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19109183

RESUMEN

The membrane attack complex (MAC) of the complement system induces a necrotic-type cell death. Earlier findings suggested that Bcl-2 protects cells from MAC-induced necrosis. Here we examined the involvement of Bid, a proapoptotic protein, in MAC-induced cytotoxicity. Bid knockout (Bid-/-) mouse embryonic fibroblasts (MEF) and primary fibroblasts were damaged by complement but to a significantly lower extent than wild-type (WT) fibroblasts. Bid silencing with small interfering RNA duplexes led to elevated resistance of mouse fibroblasts, human K562, and Jurkat cells to lysis by complement. Bid-/- MEF were also resistant to toxic doses of streptolysin O, melittin, and A23187. Analysis of complement protein deposition on fibroblasts demonstrated that less complement C3 and C9 bound to Bid-/- than to WT cells, even though expression of the membrane complement inhibitors Crry and CD59 was relatively reduced on Bid-/- cells. Bid was rapidly cleaved in WT MEF subjected to lytic doses of MAC. Pretreatment of the cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone reduced Bid cleavage and cell lysis. These results indicate that complement MAC activates two cell death pathways, one involving caspases and Bid and one that is Bid-independent.


Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/fisiología , Complejo de Ataque a Membrana del Sistema Complemento/fisiología , Transducción de Señal/inmunología , Animales , Animales Recién Nacidos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/deficiencia , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasas/metabolismo , Muerte Celular/inmunología , Línea Celular Transformada , Células Cultivadas , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunidad Innata/genética , Células Jurkat , Células K562 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Transducción de Señal/genética
11.
Int J Cancer ; 126(6): 1428-35, 2010 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-19739077

RESUMEN

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.


Asunto(s)
Complemento C9/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Interferencia de ARN , Animales , Proteínas Bacterianas/farmacología , Western Blotting , Calcimicina/farmacología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HCT116 , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Hemólisis/efectos de los fármacos , Humanos , Ionóforos/farmacología , Células K562 , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Piridinas/farmacología , Conejos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Estreptolisinas/farmacología , Tiazoles/farmacología
12.
Int J Oncol ; 57(4): 1013-1026, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32700755

RESUMEN

Cancer cells have developed numerous strategies to maintain their proliferative capacity and to withstand different kinds of stress. The mitochondrial stress­70 protein named glucose regulated protein 75 (GRP75), also known as mortalin, is an intriguing cancer pro­survival factor. It is constitutively expressed in normal tissues but is upregulated in many tumors, and was shown to be a cancer prognostic biomarker. Mortalin is an inhibitor of complement­dependent cytotoxicity (CDC) and may therefore protect cells from antibody­based immunotherapy. To target mortalin for cancer therapy, our laboratory designed several mortalin mimetic peptides with sequences predicted to be involved in mortalin binding to its client proteins. The peptides were synthesized with a C­terminal transactivator of transcription sequence. By using cell death methodologies, the mechanism of action of the mortalin mimetic peptides on cancer cells was studied. Two peptides in particular, Mot­P2 and Mot­P7, were found to be highly toxic to lymphoma and ovarian, breast and prostate carcinoma cells. The analysis of their mode of action revealed that they may induce, within minutes, plasma membrane perturbations and mitochondrial stress. Furthermore, Mot­P2 and Mot­P7 activated necrotic cell death, leading to plasma membrane perforation, mitochondrial inner membrane depolarization and decrease in ATP level. In addition, Mot­P7, but not Mot­P2, required extracellular calcium ions to fully mediate cell death and was partially inhibited by plasma membrane cholesterol. At sub­toxic concentrations, the two peptides moderately inhibited cancer cell proliferation and blocked cell cycle at G2/M. Both peptides may bind intracellularly to mortalin and/or a mortalin­binding protein, hence knocking down mortalin expression reduced cell death. Combining treatment with Mot­P2 or Mot­P7 and CDC resulted in increased cell death. This study identified highly cytotoxic mortalin mimetic peptides that may be used as monotherapy or combined with complement­activating antibody therapy to target mortalin for precision cancer therapy.


Asunto(s)
Proteínas del Sistema Complemento/inmunología , Proteínas HSP70 de Choque Térmico/farmacología , Proteínas Mitocondriales/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Péptidos/farmacología , Peptidomiméticos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/inmunología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Péptidos/química , Unión Proteica
13.
Front Immunol ; 10: 752, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31024572

RESUMEN

The interactions of cancer cells with components of the complement system are highly complex, leading to an outcome that is either favorable or detrimental to cancer cells. Currently, we perceive only the "tip of the iceberg" of these interactions. In this review, we focus on the complement terminal C5b-9 complex, known also as the complement membrane attack complex (MAC) and discuss the complexity of its interaction with cancer cells, starting with a discussion of its proposed mode of action in mediating cell death, and continuing with a portrayal of the strategies of evasion exhibited by cancer cells, and closing with a proposal of treatment approaches targeted at evasion strategies. Upon intense complement activation and membrane insertion of sufficient C5b-9 complexes, the afflicted cells undergo regulated necrotic cell death with characteristic damage to intracellular organelles, including mitochondria, and perforation of the plasma membrane. Several pro-lytic factors have been proposed, including elevated intracellular calcium ion concentrations and activated JNK, Bid, RIPK1, RIPK3, and MLKL; however, further research is required to fully characterize the effective cell death signals activated by the C5b-9 complexes. Cancer cells over-express a multitude of protective measures which either block complement activation, thus reducing the number of membrane-inserted C5b-9 complexes, or facilitate the elimination of C5b-9 from the cell surface. Concomitantly, cancer cells activate several protective pathways that counteract the death signals. Blockage of complement activation is mediated by the complement membrane regulatory proteins CD46, CD55, and CD59 and by soluble complement regulators, by proteases that cleave complement proteins and by protein kinases, like CK2, which phosphorylate complement proteins. C5b-9 elimination and inhibition of cell death signals are mediated by caveolin and dynamin, by Hsp70 and Hsp90, by the mitochondrial stress protein mortalin, and by the protein kinases PKC and ERK. It is conceivable that various cancers and cancers at different stages of development will utilize distinct patterns of these and other MAC resistance strategies. In order to enhance the impact of antibody-based therapy on cancer, novel precise reagents that block the most effective protective strategies will have to be designed and applied as adjuvants to the therapeutic antibodies.


Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Neoplasias/inmunología , Animales , Señalización del Calcio , Muerte Celular/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Activación de Complemento , Proteínas Inactivadoras de Complemento/inmunología , Proteínas Inactivadoras de Complemento/metabolismo , Citotoxicidad Inmunológica , Humanos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Modelos Inmunológicos , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral/inmunología
14.
Cancer Immunol Res ; 7(12): 1970-1983, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31537542

RESUMEN

MicroRNAs (miR) are small RNA molecules that shape the cell transcriptome and proteome through regulation of mRNA stability and translation. Here, we examined their function as determinants of cell resistance to complement-dependent cytotoxicity (CDC). To achieve this goal, we compared the expression of microRNAs between complement-resistant and -sensitive K562 leukemia, Raji lymphoma, and HCT-116 colorectal carcinoma cells. Global microRNA array analysis identified miR-150, miR-328, and miR-616 as regulators of CDC resistance. Inhibition of miR-150 reduced resistance, whereas inhibition of miR-328 or miR-616 enhanced cell resistance. Treatment of K562 cells with a sublytic dose of complement was shown to rapidly increase miR-150, miR-328, and miR-616 expression. Protein targets of these microRNAs were analyzed in K562 cells by mass spectrometry-based proteomics. Expression of the complement membrane regulatory proteins CD46 and CD59 was significantly enhanced after inhibition of miR-328 and miR-616. Enrichment of proteins of mitochondria, known target organelles in CDC, was observed after miR-150, miR-328, and miR-616 inhibition. In conclusion, miR-150, miR-328, and miR-616 regulate cell resistance to CDC by modifying the expression of the membrane complement regulators CD46 and CD59 and the response of the mitochondria to complement lytic attack. These microRNAs may be considered targets for intervention in complement-associated diseases and in anticancer, complement-based therapy.


Asunto(s)
Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica/inmunología , MicroARNs/inmunología , Mitocondrias/inmunología , Humanos , Células K562
15.
Front Immunol ; 9: 306, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29527209

RESUMEN

The complement system participates in the pathogenesis of many diseases. Complement activation produces several active protein complexes and peptides, including the terminal C5b-9 complexes. It was reported that C5b-9 complexes insert into the plasma membrane and cause membrane perturbation, intracellular calcium surge, metabolic depletion, and osmotic lysis. Previously, we showed that complement-dependent cytotoxicity (CDC) is regulated by JNK and Bid. Here, we demonstrate that three mediators in TNFα-induced necroptosis (regulated necrosis), the receptor-interacting protein kinases, receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like protein (MLKL), are activated by complement and contribute to CDC. Cell treatment with necrostatin-1 (Nec-1), a RIPK1 inhibitor, GSK'872, a RIPK3 inhibitor, or necrosulfonamide and GW806742X, MLKL inhibitors, restrain CDC. These findings were confirmed by using specific siRNAs targeting the synthesis of these proteins. Mouse fibroblasts lacking RIPK3 or MLKL were found to be less sensitive to C5b-9 than were wild-type (WT) fibroblasts. Enhanced CDC was achieved by RIPK1 or RIPK3 overexpression but not by the overexpression of a RHIM-RIPK1 mutant nor by a kinase-dead RIPK3 mutant. Nec-1 reduces the CDC of WT but not of RIPK3-knockout fibroblasts. Cells treated with a sublytic dose of complement exhibit co-localization of RIPK3 with RIPK1 in the cytoplasm and co-localization of RIPK3 and MLKL with C5b-9 at the plasma membrane. Data supporting cooperation among the RIP kinases, MLKL, JNK, and Bid in CDC are presented. These results provide a deeper insight into the cell death process activated by complement and identify potential points of cross talk between complement and other inducers of inflammation and regulated necrosis.


Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Fibroblastos/inmunología , Proteínas Quinasas/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Animales , Complejo de Ataque a Membrana del Sistema Complemento/genética , Proteínas del Sistema Complemento , Humanos , Células K562 , Ratones , Ratones Noqueados , Necrosis , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética
16.
Cell Death Dis ; 9(2): 150, 2018 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-29396434

RESUMEN

Cancer cells are commonly more resistant to cell death activated by the membranolytic protein complex C5b-9. Several surface-expressed and intracellular proteins that protect cells from complement-dependent cytotoxicity (CDC) have been identified. In this study, we investigated the function of heat shock protein 90 (Hsp90), an essential and ubiquitously expressed chaperone, overexpressed in cancer cells, in C5b-9-induced cell death. As shown, inhibition of Hsp90 with geldanamycin or radicicol is enhancing sensitivity of K562 erythroleukemia cells to CDC. Similarly, Hsp90 inhibition confers in Ramos B cell lymphoma cells elevated sensitivity to treatment with rituximab and complement. C5b-9 deposition is elevated on geldanamycin-treated cells. Purified Hsp90 binds directly to C9 and inhibits zinc-induced C9 polymerization, indicating that Hsp90 may act directly on the C5b-9 complex. Mortalin, also known as stress protein 70 or GRP75, is a mitochondrial chaperone that confers resistance to CDC. The postulated cooperation between Hsp90 and mortalin in protection from CDC was tested. Geldanamycin failed to sensitize toward CDC cells with knocked down mortalin. Direct binding of Hsp90 to mortalin was shown by co-immunoprecipitation in cell extracts after triggering with complement as well as by using purified recombinant proteins. These results provide an insight into the protective mechanisms utilized by cancer cells to evade CDC. They suggest that Hsp90 protects cells from CDC by inhibiting, together with mortalin, C5b-9 assembly and/or stability at the plasma membrane.


Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Muerte Celular , Línea Celular Tumoral , Complemento C9/metabolismo , Citoprotección , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Unión Proteica
17.
Dev Comp Immunol ; 31(9): 889-902, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17287019

RESUMEN

The colonial ascidian Botryllus schlosseri manifests a unique allorecognition system that is controlled by a single histocompatibility haplotype, the Fu/HC locus. When two allogeneic incompatible colonies come into direct contact, they develop inflammatory-like rejection lesions, called points of rejection (POR). While screening for differentially expressed genes during POR formation, we developed and analyzed a cDNA library of expressed sequence tags (ESTs) with 1693 unique ESTs that were clustered and assembled into 217 contigs and 1476 singlets. About 51% of these ESTs showed high similarity (E-value 0.005) to known database sequences, of which 123 matches were identified as immune-relevant genes encoding for stress proteins, pattern recognition receptors and complement proteins, proteases and protease inhibitors, cell adhesion and coagulation proteins, cytokine-related proteins, programmed cell death and proteasome-associated proteins. This first EST wide-screening analysis of the Botryllus allorecognition effector arm reveals a complex innate immune system, hallmarked by a whole genome response to allorecognition challenge.


Asunto(s)
Regulación de la Expresión Génica/genética , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/inmunología , Urocordados/genética , Urocordados/inmunología , Animales , Apoptosis/genética , Secuencia de Bases , Coagulación Sanguínea/genética , Adhesión Celular , Citocinas/genética , ADN Complementario/genética , Bases de Datos de Ácidos Nucleicos , Etiquetas de Secuencia Expresada , Proteínas de Choque Térmico/genética , Lectinas/genética , Proteínas de Neoplasias/genética , Péptido Hidrolasas/genética , Inhibidores de Proteasas/metabolismo , Urocordados/citología
18.
Int J Parasitol ; 37(1): 67-75, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17123534

RESUMEN

Schistosomes are believed to evade complement-mediated damage by expression of complement inhibitory proteins. Our previous results [Deng, J., Gold, D., LoVerde, P.T., Fishelson, Z., 2003. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect. Immun. 71, 6402-6410.] have demonstrated that paramyosin (Pmy) of the blood fluke S. mansoni binds to the human complement proteins C8 and C9, inhibits complement activation at the terminal stage and protects the parasite from complement-mediated damage. In order to locate the Pmy binding site to C8 and C9, various fragments of Pmy cDNA were PCR-cloned into a pET28a bacterial expression vector. Recombinant His-tagged Pmy fragments were expressed in BL21 Escherichia coli and purified over a nickel-nitrilotriacetic acid column. Binding assays by Western blotting with monoclonal anti-His antibody demonstrated that PmyCC (Pmy amino acids (744)Asp-(866)Met) was the only Pmy fragment that bound to human C8 and C9. Functional analyses demonstrated that PmyCC inhibited hemolysis of rabbit erythrocytes and of antibody-sensitized sheep erythrocytes by human complement. Importantly, PmyCC inhibited in vitro killing of trypsin-sensitized schistosomula of S. mansoni by human complement. In the presence of PmyCC, Zn(2+)-induced C9 polymerization was inhibited. Most of the immunodominant B-cell antigenic epitopes of Pmy are present in the PmyCC region, as antibodies collected from mice immunized with recombinant Pmy bound primarily to PmyCC. Taken together, this study has mapped the complement regulatory domain in Pmy, capable of binding to C8 and C9 and preventing polyC9 formation, to its C-terminal region.


Asunto(s)
Complemento C9/metabolismo , Schistosoma mansoni/metabolismo , Tropomiosina/metabolismo , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/metabolismo , Sitios de Unión , Activación de Complemento/inmunología , Complemento C8/inmunología , Complemento C8/metabolismo , Complemento C9/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Hemólisis/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Conejos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/inmunología , Ovinos , Tropomiosina/inmunología
20.
Immunobiology ; 221(12): 1395-1406, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27475989

RESUMEN

Mortalin/GRP75 is a ubiquitously expressed mitochondrial chaperon that is overexpressed in cancer. Mortalin protects cells from complement-dependent cytotoxicity (CDC) and facilitates elimination of the complement C5b-9 complexes from the cell surface. We performed a nanoscopical study aimed at imaging the distribution of the C5b-9 complexes in the plasma membrane and the postulated relocation of mortalin from the mitochondria to the plasma membrane. To gain a resolution of 35nm, the locations of the C5b-9 complex and mortalin were imaged with a STED (Stimulated Emission Depletion) microscope at sub-diffraction resolution. Early changes in the spatial distribution of the C5b-9 on the cell surface are described. Juxtaposition of the labeled mortalin and C5b-9 at the plasma membrane region within minutes after complement attack is evident. Microscopical analysis of the distribution of mortalin in the vicinity of the mitochondria of complement-treated cells shows a more diffused pattern relative to control cells, proposing exit of mortalin from the mitochondria in response to complement-induced stress. In support, analysis of cytoplasmic mortalin by immunoblotting shows enhanced level of mortalin in the cytoplasm in complement-treated cells. Our data demonstrates that cells can sense complement activation at the plasma membrane and in response, swiftly send mortalin to this region in order to deactivate it.


Asunto(s)
Membrana Celular/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Mitocondrias/metabolismo , Neoplasias/metabolismo , Activación de Complemento , Citoplasma/metabolismo , Citotoxicidad Inmunológica , Daño del ADN , Humanos , Células K562 , Microscopía Electrónica de Transmisión , Nanotecnología , Transporte de Proteínas
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