RESUMO
Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.
Assuntos
Apoptose/genética , Plaquetas/citologia , Linfócitos T/citologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Linhagem Celular , Sobrevivência Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
Bisphosphonates are the first-choice treatment of osteoporosis and Paget's disease of bone. Among the bisphosphonates, the non-amino-bisphosphonates, such as clodronic acid, are intracellular converted into toxic analogues of ATP and induce cellular apoptosis whereas the amino-bisphosphonates, such as zoledronic acid, inhibit the farnesyl-diphosphate-synthase, an enzyme of the mevalonate pathway. This pathway regulates cholesterol and glucose homeostasis and is a target for statins. In this retrospective cohort study, we evaluated the effects of an intravenous infusion of zoledronic acid (5 mg) or clodronic acid (1500 mg) on blood lipid (i.e. total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol and triglycerides) and glucose levels in patients with osteoporosis and Paget's disease of bone. All patients were evaluated before, 1 and 6 months after bisphosphonate treatment. Pagetic and osteoporotic patients treated with zoledronic acid showed a significant reduction in glucose and atherogenic lipids during follow-up whereas these phenomena were not observed after clodronic treatment. The effect on circulating lipid levels was similar in naïve and re-treated Pagetic patients. Zoledronic acid treatment was associated with a reduction in blood glucose and atherogenic lipids in patients with metabolic bone disorders. The extent of change was similar to that obtained with the regular assumption of a low-intensity statin. Further studies are warranted to better evaluate the clinical implications of these observations.
Assuntos
Conservadores da Densidade Óssea , Osteíte Deformante , Difosfonatos , Glucose , Humanos , Lipídeos , Estudos Retrospectivos , Ácido ZoledrônicoRESUMO
Osteoporosis and nephrolithiasis are common multifactorial disorders with high incidence and prevalence in the adult population worldwide. Both are associated with high morbidity and mortality if not correctly diagnosed and accurately treated. Nephrolithiasis is considered a risk factor for reduced bone mineral density. Aim of this retrospective longitudinal study was to evaluate if osteoporosis is a predictive factor for the nephrolithiasis occurrence. Free-living subjects referring to "COMEGEN" general practitioners cooperative operating in Naples, Southern Italy. Twelve thousand seven hundred ninety-four Caucasian subjects (12,165 female) who performed bone mineral density by dual-energy X-ray absorptiometry and have a negative personal history for nephrolithiasis. Subjects aged less than 40 years or with signs or symptoms suggestive of secondary osteoporosis were excluded from the study. In a mean lapse of time of 19.5 months, 516 subjects had an incident episode of nephrolithiasis. Subjects with osteoporosis had an increased risk of nephrolithiasis than subjects without osteoporosis (Hazard Ratio = 1.33, 95% Confidence Interval 1.01-1.74, p = 0.04). Free-living adult subjects over the age of 40 with idiopathic osteoporosis have an increased risk of incident nephrolithiasis, suggesting the advisability of appropriate investigation and treatment of the metabolic alterations predisposing to nephrolithiasis in patients with osteoporosis. The study protocol was approved by the ASL Napoli 1 Ethical Committee, protocol number 0018508/2018.
Assuntos
Nefrolitíase/epidemiologia , Osteoporose/epidemiologia , Absorciometria de Fóton , Adulto , Densidade Óssea , Feminino , Medicina Geral , Humanos , Itália/epidemiologia , Estudos Longitudinais , Masculino , Estudos RetrospectivosRESUMO
Beclin 1 is a 450 amino acid protein that plays critical roles in the early stages of autophagosome formation. We recently reported the successful expression, purification and structural characterisation of the entire N-terminal region of Beclin 1 (residues 1-150), including its backbone NMR chemical shift assignments. Based on assigned backbone NMR chemical shifts, it has been established that the N-terminal region of Beclin 1 (1-150), including the BH3 domain (112-123), is intrinsically disordered in the absence of its interaction partners. Here, a detailed study of its conformational preference and backbone dynamics obtained from an analysis of its secondary structure populations using the δ2D method, and the measurements of effective hydrodynamic radius as well as (1)H temperature coefficients, (1)H solvent exchange rates, and (15)N relaxation parameters of backbone amides using NMR spectroscopy is reported. These data provide further evidence for the intrinsically disordered nature of the N-terminal region of Beclin 1 and support the view that the helical conformation adopted by the Beclin 1 BH3 domain upon interaction with binding partners such as BCL-2 pro-survival proteins is likely induced rather than pre-existing.
Assuntos
Proteína Beclina-1/química , Proteínas Intrinsicamente Desordenadas/química , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Isótopos de Nitrogênio , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Coloração e Rotulagem/métodos , TermodinâmicaRESUMO
Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity "BH3-in-groove" complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteína bcl-X/química , Sequência de Aminoácidos , Animais , Apoptose , Proteínas Reguladoras de Apoptose/fisiologia , Sobrevivência Celular , Células Cultivadas , Cristalografia por Raios X , Citocromos c/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteína bcl-X/fisiologiaRESUMO
Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and ß-amino acid residues ("α/ß-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/ß-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/ß-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/ß-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/ß-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.
Assuntos
Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteína 11 Semelhante a Bcl-2 , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/metabolismo , Citocromos c/metabolismo , Células HCT116 , Humanos , Proteínas de Membrana/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteólise , Proteínas Proto-Oncogênicas/químicaRESUMO
Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2-regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2-like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with "BH3 mimetic" drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.
Assuntos
Apoptose/fisiologia , Proteínas de Helminto/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Schistosoma japonicum/metabolismo , Schistosoma mansoni/metabolismo , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Cristalografia por Raios X , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Nitrofenóis/farmacologia , Fragmentos de Peptídeos/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Schistosoma japonicum/genética , Schistosoma mansoni/genética , Esquistossomose Japônica/tratamento farmacológico , Esquistossomose Japônica/genética , Esquistossomose Japônica/metabolismo , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/genética , Esquistossomose mansoni/metabolismo , Sulfonamidas/farmacologiaRESUMO
Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.
Assuntos
Apoptose , Células Epiteliais , Camundongos , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Apoptose/genética , Células Epiteliais/metabolismo , Autofagia/genética , Homeostase , MamíferosRESUMO
We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/ß-peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl-x(L) but weakly to Mcl-1. The crystal structure of the Puma-derived α/ß-peptide complexed to Bcl-x(L) was used as the basis for computational design of variants intended to display improved binding to Mcl-1. Molecular modelling suggested modification of three α residues of the original α/ß backbone. Individually, each substitution caused only a modest (4- to 15-fold) gain in affinity; however, together the three substitutions led to a 250-fold increase in binding to Mcl-1. These modifications had very little effect on affinity for Bcl-x(L). Crystal structures of a number of the new α/ß-peptides bound to either Mcl-1 or Bcl-x(L) validated the selection of each substitution. Overall, our findings demonstrate that structure-guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.
Assuntos
Peptídeos/química , Peptídeos/metabolismo , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/químicaRESUMO
BACKGROUND: Monepantel is an anti-helminthic drug that also has anti-cancer properties. Despite several studies over the years, the molecular target of monepantel in mammalian cells is still unknown, and its mechanism-of-action is not fully understood, though effects on cell cycle, mTOR signalling and autophagy have been implicated. METHODS: Viability assays were performed on >20 solid cancer cell cells, and apoptosis assays were performed on a subset of these, including 3D cultures. Genetic deletion of BAX/BAK and ATG were used to establish roles of apoptosis and autophagy in killing activity. RNA-sequencing was performed on four cell lines after monepantel treatment, and differentially regulated genes were confirmed by Western blotting. RESULTS: We showed that monepantel has anti-proliferative activity on a broad range of cancer cell lines. In some, this was associated with induction of apoptosis which was confirmed using a BAX/BAK-deficient cell line. However, proliferation is still inhibited in these cells following monepantel treatment, indicating cell-cycle disruption as the major anti-cancer effect. Previous studies have also indicated autophagic cell death occurs following monepantel treatment. We showed autophagy induction in multiple cell lines; however, deletion of a key autophagy regulator ATG7 had minimal impact on monepantel's anti-proliferative activity, suggesting autophagy is associated with, but not required for its anti-tumour effects. Transcriptomic analysis of four cell lines treated with monepantel revealed downregulation of many genes involved in the cell cycle, and upregulation of genes linked to ATF4-mediated ER stress responses, especially those involved in amino-acid metabolism and protein synthesis. CONCLUSIONS: As these outcomes are all associated with mTOR signalling, cell cycle and autophagy, we now provide a likely triggering mechanism for the anti-cancer activity of monepantel.
Assuntos
Estresse do Retículo Endoplasmático , Neoplasias , Animais , Humanos , Proteína X Associada a bcl-2 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Apoptose , Serina-Treonina Quinases TOR/metabolismo , Autofagia , Linhagem Celular Tumoral , Mamíferos/metabolismoRESUMO
Background: HyperDoppler is a new echocardiographic color Doppler-based technique that can assess intracardiac flow dynamics. The aim of this study was to verify the feasibility and reproducibility of this technique in unselected patients and its capability to differentiate measures of vortex flow within the left ventricle (LV) in normal sedentary subjects, athletes, and patients with heart failure. Methods: Two hundred unselected, consecutive patients presenting at the echocardiographic laboratory, 50 normal subjects, 30 athletes, and 50 patients with chronic heart failure and LV ejection fraction <50% were enrolled. Images were acquired using a MyLab X8 echo-scanner. Area, intensity, depth, length, and kinetic energy dissipation (KED) of vortex flow were measured. Results: The HyperDoppler technique feasibility was 94.5%. According to the intraclass correlation coefficient evaluations, repeatability and reproducibility of vortex flow measures were good for vortex area (0.82, 0.85), length (0.83, 0.82), and depth (0.87, 0.84) and excellent for intensity (0.92, 0.90) and KED (0.98, 0.98). Combining different vortex flow measures, the LV flow profile of healthy sedentary individuals, athletes, and heart failure patients could be differentiated. Conclusions: HyperDoppler is a feasible, reliable, and practical technique for the assessment of LV flow dynamics and may distinguish normal subjects and patients with heart failure.
RESUMO
The crystal structure of a complex between the prosurvival protein Bcl-x(L) and an α/ß-peptide 21-mer is described. The α/ß-peptide contains six ß-amino acid residues distributed periodically throughout the sequence and adopts an α-helix-like conformation that mimics the bioactive shape of the Puma BH3 domain. The α/ß-peptide forms all of the noncovalent contacts that have previously been identified as necessary for recognition of the prosurvival protein by an authentic BH3 domain. Comparison of our α/ß-peptide:Bcl-x(L) structure with structures of complexes between native BH3 domains and Bcl-2 family proteins reveals how subtle adjustments, including variations in helix radius and helix bowing, allow the α/ß-peptide to engage Bcl-x(L) with high affinity. Geometric comparisons of the BH3-mimetic α/ß-peptide with α/ß-peptides in helix-bundle assemblies provide insight on the conformational plasticity of backbones that contain combinations of α- and ß-amino acid residues. The BH3-mimetic α/ß-peptide displays prosurvival protein-binding preferences distinct from those of Puma BH3 itself, even though these two oligomers have identical side-chain sequences. Our results suggest origins for this backbone-dependent change in selectivity.
Assuntos
Fragmentos de Peptídeos/química , Peptídeos/química , Proteínas Proto-Oncogênicas/química , Proteína bcl-X/química , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Peptidomiméticos/química , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteína bcl-X/metabolismoRESUMO
Malignant pleural mesothelioma (MPM) is an aggressive cancer with treatment limited to Cisplatin and Pemetrexed chemotherapy. Recently, we showed that drugs targeting the BCL-2-regulated apoptosis pathway could kill MPM cell lines in vitro, and control tumor growth in vivo. These studies showed BCL-XL was the dominant pro-survival BCL-2 family member correlating with its high-level expression in cells and patient tumor samples. In this study we show another inhibitor, AZD4320 that targets BCL-XL (and BCL-2), can also potently kill MPM tumor cells in vitro (EC50 values in the 200 nM range) and this effect is enhanced by co-inhibition of MCL-1 using AZD5991. Moreover, we show that a novel nanoparticle, AZD0466, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer, was as effective as standard-of-care chemotherapy, Cisplatin, at inhibiting tumor growth in mouse xenograft studies, and this effect was enhanced when both drugs were combined. Critically, the degree of thrombocytopenia, an on-target toxicity associated with BCL-XL inhibition, was significantly reduced throughout the treatment period compared to other BCL-XL-targeting BH3-mimetics. These pre-clinical findings provide a rationale for the future clinical evaluation for novel BH3-mimetic formulations in MPM, and indeed, other solid tumor types dependent on BCL-XL.
RESUMO
Limited therapeutic options are available for the treatment of human schistosomiasis caused by the parasitic Schistosoma flatworm. The B cell lymphoma-2 (BCL-2)-regulated apoptotic cell death pathway in schistosomes was recently characterized and shown to share similarities with the intrinsic apoptosis pathway in humans. Here, we exploit structural differences in the human and schistosome BCL-2 (sBCL-2) pro-survival proteins toward a novel treatment strategy for schistosomiasis. The benzothiazole hydrazone scaffold previously employed to target human BCL-XL was repurposed as a starting point to target sBCL-2. We utilized X-ray structural data to inform optimization and then applied a scaffold-hop strategy to identify the 5-carboxamide thiazole hydrazone scaffold (43) with potent sBCL-2 activity (IC50 30 nM). Human BCL-XL potency (IC50 13 nM) was inadvertently preserved during the optimization process. The lead analogues from this study exhibit on-target activity in model fibroblast cell lines dependent on either sBCL-2 or human BCL-XL for survival. Further optimization of the thiazole hydrazone class is required to exhibit activity in schistosomes and enhance the potential of this strategy for treating schistosomiasis.
Assuntos
Hidrazonas , Schistosoma , Animais , Apoptose , Benzotiazóis , Humanos , Hidrazonas/farmacologia , Proteína bcl-X/genéticaRESUMO
CONTEXT: Nephrolithiasis (NL) and primary hyperparathyroidism (HPTH) are metabolic complications of Paget disease of bone (PDB), but recent data regarding their prevalence in PDB patients are lacking. OBJECTIVES: Study 1: To compare the prevalence of primary HPTH and NL in 708 patients with PDB and in 1803 controls. Study 2: To evaluate the prevalence of NL-metabolic risk factors in 97 patients with PDB and NL, 219 PDB patients without NL, 364 NL patients without PDB, and 219 controls, all of them without HPTH. DESIGN: Cross-sectional multicentric study. SETTING: Italian referral centers for metabolic bone disorders. PARTICIPANTS: Patients with PDB from the Associazione Italiana malati di osteodistrofia di Paget registry. Participants in the Olivetti Heart and the Siena Osteoporosis studies. MAIN OUTCOME MEASURES: HPTH; NL; NL-metabolic risk factors. RESULTS: Patients with PDB showed higher prevalence of primary HPTH and NL compared with controls (P < 0.01). The NL recurrence occurs more frequently in patients with polyostotic PDB. About one-half of patients with PDB but without NL showed 1 or more NL-related metabolic risk factors. The hyperoxaluria (HyperOx) prevalence was higher in patients with PDB and NL compared with patients with NL but without PDB and in patients with PDB without NL compared with controls (P = 0.01). Patients with PDB and HyperOx showed a longer lapse of time from the last aminobisphosphonate treatment. CONCLUSIONS: NL and HPTH are frequent metabolic complication of PDB. The NL occurrence should be evaluated in patients with PDB, particularly in those with polyostotic disease and/or after aminobisphosphonate treatment to apply an adequate prevention strategy.
Assuntos
Hiperoxalúria/epidemiologia , Hiperparatireoidismo/epidemiologia , Nefrolitíase/epidemiologia , Osteíte Deformante/epidemiologia , Idoso , Estudos Transversais , Feminino , Humanos , Hiperoxalúria/complicações , Hiperparatireoidismo/complicações , Masculino , Pessoa de Meia-Idade , Nefrolitíase/complicações , Nefrolitíase/metabolismo , Osteíte Deformante/complicações , Osteíte Deformante/metabolismo , Prevalência , Fatores de RiscoRESUMO
Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans apoptosis has several unique features compared with human apoptosis. To date, there has been no detailed analysis of apoptosis regulators in nematodes other than C. elegans. Here, we discovered BCL-2 orthologues in 89 free-living and parasitic nematode taxa representing four evolutionary clades (I, III, IV and V). Unlike in C. elegans, 15 species possess multiple (two to five) BCL-2-like proteins, and some do not have any recognisable BCL-2 sequences. Functional studies provided no evidence that BAX/BAK proteins have evolved in nematodes, and structural studies of a BCL-2 protein from the basal clade I revealed it lacks a functionally important feature of the C. elegans orthologue. Clade I CED-4/APAF-1 proteins also possess WD40-repeat sequences associated with apoptosome assembly, not present in C. elegans, or other nematode taxa studied.
Assuntos
Apoptose , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Transdução de Sinais , Animais , Apoptose/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Genes de Helmintos , Camundongos , Filogenia , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Despite having one of the lowest survival rates of all cancers, there have been no new approved treatments for malignant pleural mesothelioma (MPM) in over a decade. Standard-of-care treatment relies on Cisplatin plus Pemetrexed chemotherapy. Here, we tested a suite of BH3-mimetic drugs targeting BCL-2 pro-survival proteins of the intrinsic apoptotic pathway. We found BCL-XL is the dominant pro-survival protein in a panel of cell lines in vitro, though potent, synergistic cell killing occurred with MCL-1 co-targeting. This correlates with high-level expression of BCL-XL and MCL-1 in cell lines and a large cohort of patient tumour samples. BCL-XL inhibition combined with Cisplatin also enhanced cell killing. In vivo BCL-XL inhibition was as effective as Cisplatin, and the combination enhanced tumour growth control and survival. Genetic ablation of MCL-1 also enhanced the effects of BCL-XL inhibitors, in vivo. Combined, these data provide a compelling rationale for the clinical investigation of BH3-mimetics targeting BCL-XL in MPM.
RESUMO
Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.
Assuntos
Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Placenta/citologia , Âmnio/citologia , Âmnio/imunologia , Animais , Antígenos de Superfície/metabolismo , Adesão Celular , Diferenciação Celular , Córion/citologia , Córion/imunologia , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/transplante , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Tolerância Imunológica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Placenta/imunologia , Gravidez , Transplante de Células-Tronco , Células Estromais/citologia , Células Estromais/imunologia , Bancos de Tecidos , Trofoblastos/citologia , Trofoblastos/imunologiaRESUMO
The paper deals with the peculiarities of a firearm wound regarding the dynamic of the accident and particularly considering the path followed by the bullet. The patient's firearm wound is described on the basis of the instrumental examinations done. These last give us a precious help to confirm the thesis, already guessed on the clinical examination of an introduction and path followed through the digestive tract, and to show the dynamics and the still positions of the bullet inside the patient's body. The bullet was expelled spontaneously by defecation. After control of no internal damages this firearm wound has been treated simply as a normal tip wound.
Assuntos
Lesões do Pescoço/complicações , Faringe/lesões , Ferimentos por Arma de Fogo/complicações , Acidentes , Adulto , Fezes , Migração de Corpo Estranho/complicações , Balística Forense , Humanos , Masculino , Lesões do Pescoço/diagnóstico , Lesões do Pescoço/cirurgia , Ferimentos por Arma de Fogo/diagnóstico , Ferimentos por Arma de Fogo/cirurgiaRESUMO
BECN1/Beclin 1 is a critical protein in the initiation of autophagosome formation. Recent studies have shown that phosphorylation of BECN1 by STK4/MST1 at threonine 108 (T108) within its BH3 domain blocks macroautophagy/autophagy by increasing BECN1 affinity for its negative regulators, the anti-apoptotic proteins BCL2/Bcl-2 and BCL2L1/Bcl-xL. It was proposed that this increased binding is due to formation of an electrostatic interaction with a conserved histidine residue on the anti-apoptotic molecules. Here, we performed biophysical studies which demonstrated that a peptide corresponding to the BECN1 BH3 domain in which T108 is phosphorylated (p-T108) does show increased affinity for anti-apoptotic proteins that is significant, though only minor (<2-fold). We also determined X-ray crystal structures of BCL2 and BCL2L1 with T108-modified BECN1 BH3 peptides, but only showed evidence of an interaction between the BH3 peptide and the conserved histidine residue when the histidine flexibility was restrained due to crystal contacts. These data, together with molecular dynamics studies, indicate that the histidine is highly flexible, even when complexed with BECN1 BH3. Binding studies also showed that detergent can increase the affinity of the interaction. Although this increase was similar for both the phosphorylated and non-phosphorylated peptides, it suggests factors such as membranes could impact on the interaction between BECN1 and BCL2 proteins, and therefore, on the regulation of autophagy. Hence, we propose that phosphorylation of BECN1 by STK4/MST1 can increase the affinity of the interaction between BECN1 and anti-apoptotic proteins and this interaction can be stabilized by local environmental factors. Abbreviations: asu: asymmetric unit; BH3: BCL2/Bcl-2 homology 3; DAPK: death associated protein kinase; MD: molecular dynamics; MST: microscale thermophoresis; NMR: nuclear magnetic resonance; PDB: protein data bank; p-T: phosphothreonine; SPR: surface plasmon resonance; STK4/MST1: serine/threonine kinase 4.