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Perry syndrome (PS) is a rare autosomal dominant disease characterized by parkinsonism, central hypoventilation, weight loss and depression and is caused by pathogenic mutations in the dynactin subunit 1 (DCTN1) gene (encoding p150glued protein). To date, only two cases have been reported in Latin America, specifically in Colombia and Argentina. The present study, to the best of our knowledge, reports the first recorded Mexican family with PS. The clinical features of the proband and a family history of early parkinsonism led to the suspicion of PS. The pathogenic variant NM_004082:c.212G>A, causing a (p.Gly71Glu) mutation in the p150glued protein, was identified in exon 2 of the DCTN1 gene by exome sequencing, confirming the diagnosis of PS. (p.Gly71Glu) has been previously identified in at least 4 cases of PS from different ethnic backgrounds. Genetic counseling was provided to the available family members. To clarify the impact of the (p.Gly71Glu) variant on the structure and function of the cytoskeleton-associated protein Gly rich (CAP-Gly) domain of p150glued, Glu71 mutated CAP-Gly domains were modeled and compared with the wild-type. It was hypothesized that the larger and more charged side chain of Glu may induce conformational and electrostatic changes, imposing a conformational restriction on the peptide backbone that would affect interaction with the p150glued protein partners, causing dysfunction in the dynactin protein complex.
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The adaptive immune system of jawed vertebrates generates a highly diverse repertoire of antibodies to meet the antigenic challenges of a constantly evolving biological ecosystem. Most of the diversity is generated by two mechanisms: V(D)J gene recombination and somatic hypermutation (SHM). SHM introduces changes in the variable domain of antibodies, mostly in the regions that form the paratope, yielding antibodies with higher antigen binding affinity. However, antigen recognition is only possible if the antibody folds into a stable functional conformation. Therefore, a key force determining the survival of B cell clones undergoing somatic hypermutation is the ability of the mutated heavy and light chains to efficiently fold and assemble into a functional antibody. The antibody is the structural context where the selection of the somatic mutations occurs, and where both the heavy and light chains benefit from protective mechanisms that counteract the potentially deleterious impact of the changes. However, in patients with monoclonal gammopathies, the proliferating plasma cell clone may overproduce the light chain, which is then secreted into the bloodstream. This places the light chain out of the protective context provided by the quaternary structure of the antibody, increasing the risk of misfolding and aggregation due to destabilizing somatic mutations. Light chain-derived (AL) amyloidosis, light chain deposition disease (LCDD), Fanconi syndrome, and myeloma (cast) nephropathy are a diverse group of diseases derived from the pathologic aggregation of light chains, in which somatic mutations are recognized to play a role. In this review, we address the mechanisms by which somatic mutations promote the misfolding and pathological aggregation of the light chains, with an emphasis on AL amyloidosis. We also analyze the contribution of the variable domain (VL) gene segments and somatic mutations on light chain cytotoxicity, organ tropism, and structure of the AL fibrils. Finally, we analyze the most recent advances in the development of computational algorithms to predict the role of somatic mutations in the cardiotoxicity of amyloidogenic light chains and discuss the challenges and perspectives that this approach faces.
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Mieloma Múltiplo , Paraproteinemias , Animais , Humanos , Amigos , Ecossistema , Linfócitos B , Paraproteinemias/genéticaRESUMO
Hepatocellular carcinoma (HCC) is a highly lethal liver cancer with late diagnosis; therefore, the identification of new early biomarkers could help reduce mortality. We determine the tissue and plasma status of five annexins during hepatocarcinogenesis by diethylnitrosamine-induced cirrhosis-HCC. We found that Anxa5 was the earliest upregulated gene at week 12 after HCC initiation, while Anxa1 and Anxa2 were upregulated in advanced HCC stages (weeks 18 and 22). Furthermore, the protein level of Annexin A1, A2, A5 and A10 was increased from the early stages. Immunofluorescence and subcellular fractionation revealed Annexin A1, A2, and A5 in the cytoplasm and nuclei of tumor cells. Notably, increased plasma levels of Annexin A5 significantly (r2 = 0.8203) correlated with Annexin A5 levels in liver tissue from week 12 and gradually increased until week 22. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p = 0.0046) and ANXA5 (HR = 1.8, p = 0.00077) was associated with poor survival in HCC patients. In conclusion, we have identified Annexin A1 and A5 as potentially useful early biomarkers for poor prognosis in HCC patients.
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Anexina A1 , Anexina A2 , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Anexina A1/genética , Anexina A1/metabolismo , Anexina A5/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Anexinas/genética , Anexinas/metabolismo , Biomarcadores Tumorais/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide, often preceded by cirrhosis and usually diagnosed at advanced stages; therefore, identifying molecular changes at early stages is an attractive strategy for detection and timely treatment. Here, we investigated the progressive transcriptomic changes during experimental hepatocarcinogenesis to identify novel early tumor markers in an HCC model induced by chronic administration of sublethal doses of diethylnitrosamine. An analysis of differentially expressed genes showed that four processes associated with oxidation-reduction and detoxification were significantly over-represented during hepatocarcinogenesis progression, of which the Nuclear Factor, Erythroid 2 Like 2 pathway showed several dysregulated genes. Interestingly, we also identified 91 genes dysregulated at early HCC stages, but the expression of the indolethylamine N-methyltransferase gene (INMT), as well as the level of its encoding protein, were strongly downregulated. INMT was increased in perivenular hepatocytes of normal livers but decreased in livers of experimental HCC. Furthermore, a gene expression and survival analysis performed using data from the liver hepatocellular carcinoma project of The Cancer Genome Atlas Program revealed that INMT is also significantly downregulated in human HCC and is associated with poor overall survival. In conclusion, by performing a transcriptome analysis of the HCC progression, we identified that INMT is early downregulated in the rat hepatocarcinogenesis and is associated with poor prognosis in human HCC, suggesting that INMT downregulation may be a promising prognostic marker for HCC in high-risk populations.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Regulação para Baixo , Humanos , Neoplasias Hepáticas/patologia , Metiltransferases/genética , RatosRESUMO
The activation of Nuclear Factor, Erythroid 2 Like 2 - Kelch Like ECH Associated Protein 1 (NRF2-KEAP1) signaling pathway plays a critical dual role by either protecting or promoting the carcinogenesis process. However, its activation or nuclear translocation during hepatocellular carcinoma (HCC) progression has not been addressed yet. This study characterizes the subcellular localization of both NRF2 and KEAP1 during diethylnitrosamine-induced hepatocarcinogenesis in the rat. NRF2-KEAP1 pathway was continuously activated along with the increased expression of its target genes, namely Nqo1, Hmox1, Gclc, and Ptgr1. Similarly, the nuclear translocation of NRF2, MAF, and KEAP1 increased in HCC cells from weeks 12 to 22 during HCC progression. Likewise, colocalization of NRF2 with KEAP1 was higher in the cell nuclei of HCC neoplastic nodules than in surrounding cells. Moreover, immunofluorescence analyses revealed that the interaction of KEAP1 with filamentous Actin was disrupted in HCC cells. This disruption may be contributing to the release and nuclear translocation of NRF2 since the cortical actin cytoskeleton serves as anchoring of KEAP1. In conclusion, this evidence indicates that NRF2 is progressively activated and promotes the progression of experimental HCC.
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Carcinoma Hepatocelular/patologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Hepáticas/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/veterinária , Núcleo Celular/metabolismo , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Dietilnitrosamina/toxicidade , Progressão da Doença , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/veterinária , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/genética , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
Hepatocellular carcinoma expressing hepatobiliary progenitor markers, is considered of poor prognosis. By using a hepatocarcinogenesis model, laser capture microdissection, and RNA-Sequencing analysis, we identified an expression profile in GGT/KRT19-positive experimental tumors; 438 differentially expressed genes were found in early and late nodules along with increased collagen deposition. Dysregulated genes were involved in Fatty Acid Metabolism, RXR function, and Hepatic Stellate Cells Activation. Downregulation of Slc27a5, Acsl1, and Cyp2e1, demonstrated that Retinoid X Receptor α (RXRα) function is compromised in GGT/KRT19-positive nodules. Since RXRα controls NRF2 pathway activation, we determined the expression of NRF2 targeted genes; Akr1b8, Akr7a3, Gstp1, Abcc3, Ptgr1, and Txnrd1 were upregulated, indicating NRF2 pathway activation. A comparative analysis in human HCC showed that SLC27A5, ACSL1, CYP2E1, and RXRα gene expression is mutually exclusive with KRT19 gene expression. Our results indicate that the downregulation of Slc27a5, Acsl1, Rxrα, and Cyp2e1 genes is an early event within GGT/KRT19-positive HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/metabolismo , Ácidos Graxos , Humanos , Neoplasias Hepáticas/metabolismo , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , TranscriptomaRESUMO
INTRODUCTION: Deciphering the intricacies of the interactions of glomerulopathic Ig light chains with mesangial cells is key to delineate signaling events responsible for the mesangial pathologic alterations that ensue. METHODS: Human mesangial cells, caveolin 1 (CAV1), wild type (WT) ,and knockout (KO), were incubated with glomerulopathic light chains purified from the urine of patients with light chain-associated (AL) amyloidosis or light chain deposition disease. Associated signaling events induced by surface interactions of glomerulopathic light chains with caveolins and other membrane proteins, as well as the effect of epigallocatechin-3-gallate (EGCG) on the capacity of mesangial cells to intracellularly process AL light chains were investigated using a variety of techniques, including chemical crosslinking with mass spectroscopy, immunofluorescence, and ultrastructural immunolabeling. RESULTS: Crosslinking experiments provide evidence suggesting that sortilin-related receptor (SORL1), a transmembrane sorting receptor that regulates cellular trafficking of proteins, is a component of the receptor on mesangial cells for glomerulopathic light chains. Colocalization of glomerulopathic light chains with SORL1 in caveolae and also in lysosomes when light chain internalization occurred, was documented using double immunofluorescence and immunogold labeling ultrastructural techniques. It was found that EGCG directly blocks c-Fos cytoplasmic to nuclei signal translocation after interactions of AL light chains with mesangial cells, resulting in a decrease in amyloid formation. CONCLUSION: Our findings document for the first time a role for SORL1 linked to glomerular pathology and signaling events that take place when certain monoclonal light chains interact with mesangial cells. This finding may lead to novel therapies for treating renal injury caused by glomerulopathic light chains.
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Patients with plasma cell dyscrasias produce free abnormal monoclonal Ig light chains that circulate in the blood stream. Some of them, termed glomerulopathic light chains, interact with the mesangial cells and trigger, in a manner dependent of their structural and physicochemical properties, a sequence of pathological events that results in either light chain-derived (AL) amyloidosis (AL-Am) or light chain deposition disease (LCDD). The mesangial cells play a key role in the pathogenesis of both diseases. The interaction with the pathogenic light chain elicits specific cellular processes, which include apoptosis, phenotype transformation, and secretion of extracellular matrix components and metalloproteinases. Monoclonal light chains associated with AL-Am but not those producing LCDD are avidly endocytosed by mesangial cells and delivered to the mature lysosomal compartment where amyloid fibrils are formed. Light chains from patients with LCDD exert their pathogenic signaling effect at the cell surface of mesangial cells. These events are generic mesangial responses to a variety of adverse stimuli, and they are similar to those characterizing other more frequent glomerulopathies responsible for many cases of end-stage renal disease. The pathophysiologic events that have been elucidated allow to propose future therapeutic approaches aimed at preventing, stopping, ameliorating, or reversing the adverse effects resulting from the interactions between glomerulopathic light chains and mesangium.
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Immunoglobulin light chain-derived (AL) amyloidosis is a debilitating disease without known cure. Almost nothing is known about the structural factors driving the amyloidogenesis of the light chains. This study aimed to identify the fibrillogenic hotspots of the model protein 6aJL2 and in pursuing this goal, two complementary approaches were applied. One of them was based on several web-based computational tools optimized to predict fibrillogenic/aggregation-prone sequences based on different structural and biophysical properties of the polypeptide chain. Then, the predictions were confirmed with an ad-hoc synthetic peptide library. In the second approach, 6aJL2 protein was proteolyzed with trypsin, and the products incubated in aggregation-promoting conditions. Then, the aggregation-prone fragments were identified by combining standard proteomic methods, and the results validated with a set of synthetic peptides with the sequence of the tryptic fragments. Both strategies coincided to identify a fibrillogenic hotspot located at the CDR1 and ß-strand C of the protein, which was confirmed by scanning proline mutagenesis analysis. However, only the proteolysis-based strategy revealed additional fibrillogenic hotspots in two other regions of the protein. It was shown that a fibrillogenic hotspot associated to the CDR1 is also encoded by several κ and λ germline variable domain gene segments. Some parts of this study have been included in the chapter "The Structural Determinants of the Immunoglobulin Light Chain Amyloid Aggregation", published in Physical Biology of Proteins and Peptides, Springer 2015 (ISBN 978-3-319-21687-4).
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Amiloide/metabolismo , Regiões Determinantes de Complementaridade , Cadeias Leves de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Agregação Patológica de Proteínas/metabolismo , Sequência de Aminoácidos , Amiloide/química , Humanos , Cadeias Leves de Imunoglobulina/química , Modelos Moleculares , Conformação Proteica em Folha beta , Multimerização ProteicaRESUMO
Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show ß-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially 15 N:13 C-labeled samples, that the ß-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.
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Amiloide/metabolismo , Amiloidose/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Amiloide/química , Humanos , Cadeias Leves de Imunoglobulina/química , Espectroscopia de Ressonância Magnética/métodos , Conformação Proteica em Folha beta , Dobramento de ProteínaRESUMO
BACKGROUND: The intrinsic heterogeneity of hepatocellular carcinoma (HCC) represents a great challenge for its molecular classification and for detecting predictive biomarkers. Aldo-keto reductase (Akr) family members have shown differential expression in human HCC, while AKR1B10 overexpression is considered a biomarker; AKR7A3 expression is frequently reduced in HCC. AIMS: To investigate the time-course expression of Akr members in the experimental hepatocarcinogenesis. METHODS: Using DNA-microarray data, we analyzed the time-course gene expression profile from nodules to tumors (4-17 months) of 17 Akr members induced by the resistant hepatocyte carcinogenesis model in the rat. RESULTS: The expression of six members (Akr1c19, Akr1b10, Akr7a3, Akr1b1, Akr1cl1, and Akr1b8) was increased, comparable to that of Ggt and Gstp1, two well-known liver cancer markers. In particular, Akr7a3 and Akr1b10 expression also showed a time-dependent increment at mRNA and protein levels in a second hepatocarcinogenesis model induced with diethylnitrosamine. We confirmed that aldo-keto reductases 7A3 and 1B10 were co-expressed in nine biopsies of human HCC, independently from the presence of glypican-3 and cytokeratin-19, two well-known HCC biomarkers. Because it has been suggested that expression of Akr members is regulated through NRF2 activity at the antioxidant response element (ARE) sequences, we searched and identified at least two ARE sites in Akr1b1, Akr1b10, and Akr7a3 from rat and human gene sequences. Moreover, we observed higher NRF2 nuclear translocation in tumors as compared with non-tumor tissues. CONCLUSIONS: Our results demonstrate that Akr7a3 mRNA and protein levels are consistently co-expressed along with Akr1b10, in both experimental liver carcinogenesis and some human HCC samples. These results highlight the presence of AKR7A3 and AKR1B10 from early stages of the experimental HCC and introduce them as a potential application for early diagnosis, staging, and prognosis in human cancer.
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Aldeído Redutase/metabolismo , Membro B10 da Família 1 de alfa-Ceto Redutase/metabolismo , Aldo-Ceto Redutases/metabolismo , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Aldeído Redutase/genética , Membro B10 da Família 1 de alfa-Ceto Redutase/genética , Aldo-Ceto Redutases/genética , Animais , Biomarcadores/metabolismo , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , RNA Mensageiro/metabolismo , Ratos Endogâmicos F344RESUMO
The Excreted/Secreted (ES) proteins play important roles during Mycobacterium tuberculosis invasion, virulence, and survival inside the host and they are a major source of immunogenic proteins. However, the molecular complexity of the bacillus cell wall has made difficult the experimental isolation of the total bacterial ES proteins. Here, we reported the genomes of two Beijing genotype M. tuberculosis clinical isolates obtained from patients from Vietnam (isolate 46) and South Africa (isolate 48). We developed a bioinformatics pipeline to predict their secretomes and observed that ~12% of the genome-encoded proteins are ES, being PE, PE-PGRS, and PPE the most abundant protein domains. Additionally, the Gene Ontology, KEGG pathways and Enzyme Classes annotations supported the expected functions for the secretomes. The ~70% of an experimental secretome compiled from literature was contained in our predicted secretomes, while only the 34-41% of the experimental secretome was contained in the two previously reported secretomes for H37Rv. These results suggest that our bioinformatics pipeline is better to predict a more complete set of ES proteins in M. tuberculosis genomes. The predicted ES proteins showed a significant higher antigenic density measured by Abundance of Antigenic Regions (AAR) value than the non-ES proteins and also compared to random constructed secretomes. Additionally, we predicted the secretomes for H37Rv, H37Ra, and two M. bovis BCG genomes. The antigenic density for BGG and for isolates 46 and 48 was higher than the observed for H37Rv and H37Ra secretomes. In addition, two sets of immunogenic proteins previously reported in patients with tuberculosis also showed a high antigenic density. Interestingly, mice infected with isolate 46 showed a significant lower survival rate than the ones infected with isolate 48 and both survival rates were lower than the one previously reported for the H37Rv in the same murine model. Finally, after a druggability analysis of the secretomes, we found potential drug targets such as cytochrome P450, thiol peroxidase, the Ag85C, and Ribonucleoside Reductase in the secreted proteins that could be used as drug targets for novel treatments against Tuberculosis.
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Prostaglandin reductase-1 (Ptgr1) is an alkenal/one oxidoreductase that is involved in the catabolism of eicosanoids and lipid peroxidation such as 4-hydroxynonenal (4-HNE). Recently, we reported that Ptgr1 is overexpressed in human clinical and experimentally induced samples of hepatocellular carcinoma (HCC). However, how the expression of this gene is regulated and its role in carcinogenesis are not yet known. Here, we studied parameters associated with antioxidant responses and the mechanisms underlying the induction of Ptgr1 expression by the activation of Nuclear Factor (erythroid-derived-2)-like-2 (NRF2). For these experiments, we used two protocols of induced hepatocarcinogenesis in rats. Furthermore, we determined the effect of PTGR1 on cell proliferation and resistance to oxidative stress in cell cultures of the epithelial liver cell line, C9. Ptgr1 was overexpressed during the early phase in altered hepatocyte foci, and this high level of expression was maintained in persistent nodules until tumors developed. Ptgr1 expression was regulated by NRF2, which bound to an antioxidant response element at -653bp in the rat Ptgr1 gene. The activation of NRF2 induced the activation of an antioxidant response that included effects on proteins such as glutamate-cysteine ligase, catalytic subunit, NAD(P)H dehydrogenase quinone-1 (NQO1) and glutathione-S-transferase-P (GSTP1). These effects may have produced a reduced status that was associated with a high proliferation rate in experimental tumors. Indeed, when Ptgr1 was stably expressed, we observed a reduction in the time required for proliferation and a protective effect against hydrogen peroxide- and 4-HNE-induced cell death. These data were consistent with data showing colocalization between PTGR1 and 4-HNE protein adducts in liver nodules. These findings suggest that Ptgr1 and antioxidant responses act as a metabolic adaptation and could contribute to proliferation and cell-death evasion in liver tumor cells. Furthermore, these data indicate that Ptgr1 could be used to design early diagnostic tools or targeted therapies for HCC.
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Oxirredutases do Álcool/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fator 2 Relacionado a NF-E2/genética , Animais , Antioxidantes/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Peroxidação de Lipídeos/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Estresse Oxidativo/genética , Ratos , Transdução de Sinais/genéticaRESUMO
Variable domain (VL) gene segments exhibit variable tendencies to be associated with light chain amyloidosis (AL). While few of them are very frequent in AL and give rise to most of the amyloidogenic light chains compiled at the sequence databases, other are rarely found among the AL cases. To analyze to which extent these tendencies depend on folding stability and aggregation propensity of the germline VL protein, we characterized VL proteins encoded by four AL-associated germline gene segments and one not associated to AL. We found that the AL-associated germline rVL proteins differ widely in conformational stability and propensity to in vitro amyloid aggregation. While in vitro the amyloid formation kinetics of these proteins correlate well with their folding stabilities, the folding stability does not clearly correlate with their germline's frequencies in AL. We conclude that the association of the VL genes segments to amyloidosis is not determined solely by the folding stability and aggregation propensity of the germline VL protein. Other factors, such as the frequencies of destabilizing mutations and susceptibility to proteolysis, must play a role in determining the light chain amyloidogenicity.
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Amiloide/genética , Amiloidose/genética , Região Variável de Imunoglobulina/genética , Agregação Patológica de Proteínas/genética , Sequência de Aminoácidos , Mutação em Linhagem Germinativa , Humanos , Microscopia Eletrônica de Transmissão , Domínios Proteicos , Estabilidade Proteica , Alinhamento de Sequência , Espectrometria de FluorescênciaRESUMO
In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca(2+)-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the "open" and "closed" conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM's inhibitors correlated well with available experimental data as the r(2) obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca(2+)-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca(2+)-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca(2+)-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs.
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Cálcio/química , Calmodulina/química , Ligantes , Conformação Proteica/efeitos dos fármacos , Benzoxazóis , Sítios de Ligação , Cálcio/metabolismo , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Maleimidas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , TermodinâmicaRESUMO
The advances in experimental methods and the development of high performance bioinformatic tools have substantially improved our understanding of microbial communities associated with human niches. Many studies have documented that changes in microbial abundance and composition of the human microbiome is associated with human health and diseased state. The majority of research on human microbiome is typically focused in the analysis of one level of biological information, i.e., metagenomics or metatranscriptomics. In this review, we describe some of the different experimental and bioinformatic strategies applied to analyze the 16S rRNA gene profiling and shotgun sequencing data of the human microbiome. We also discuss how some of the recent insights in the combination of metagenomics, metatranscriptomics and viromics can provide more detailed description on the interactions between microorganisms and viruses in oral and gut microbiomes. Recent studies on viromics have begun to gain importance due to the potential involvement of viruses in microbial dysbiosis. In addition, metatranscriptomic combined with metagenomic analysis have shown that a substantial fraction of microbial transcripts can be differentially regulated relative to their microbial genomic abundances. Thus, understanding the molecular interactions in the microbiome using the combination of metagenomics, metatranscriptomics and viromics is one of the main challenges towards a system level understanding of human microbiome.
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The high-quality draft genomes of two Vibrio parahaemolyticus strains, one that causes the acute hepatopancreatic necrosis disease (AHPND) in cultured shrimps (FIM-S1708(+)), and another that does not (FIM-S1392(-)) are reported. A chromosome-scale assembly for the FIM-S1392(-) genome is reported here. The analysis of the two genomes gives some clues regarding the genomic differences between the strains.
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To identify novel tumor-associated proteins, we analyzed the protein expression patterns from experimental hepatocellular carcinoma (HCC) that were induced using hepatocarcinogenesis models in rats. Rats were subjected to two previously described protocols of hepatocarcinogenesis using diethylnitrosamine as a carcinogen: the alternative Solt-Farber (aS&F) protocol, which induces HCC within 9 months, and Schiffer's model, which induces cirrhosis and multifocal HCC within 18 weeks. The patterns of protein expression from tumors and normal liver tissue were examined by SDS-PAGE and the bands identified at 33-34 kDa were analyzed by mass spectrometry. The prostaglandin reductase 1 (PTGR1) showed the highest number of peptides, with a confidence of level >99%. The increased expression of PTGR1 in tumors was confirmed in these two models by Western blotting and by increase in alkenal/one oxidoreductase activity (25-fold higher than normal liver). In addition, the gene expression level of Ptgr1, as measured by qRT-PCR, was increased during cancer development in a time-dependent manner (200-fold higher than normal liver). Furthermore, PTGR1 was detected in the cytoplasm of neoplastic cells in rat tumors and in 12 human HCC cases by immunohistochemistry. These analyses were performed by comparing the expression of PTGR1 to that of two well-known markers of hepatocarcinoma, Glutathione S-transferase pi 1 (GSTP1) in rats and glypican-3 in humans. The increased expression and activity of PTGR1 in liver carcinogenesis encourage further research aimed at understanding the metabolic role of PTGR1 in HCC and its potential application for human cancer diagnosis and treatment.
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Oxirredutases do Álcool/biossíntese , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/biossíntese , Oxirredutases do Álcool/genética , Animais , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , RatosRESUMO
It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.
Assuntos
Amiloide/química , Cadeias Leves de Imunoglobulina/química , Cadeias lambda de Imunoglobulina/química , Amiloide/genética , Sítios de Ligação , Cadeias Leves de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
γ-Glutamyl transferase (GGT) is useful as a marker in pathological conditions, including several types of cancer. We optimized the histochemical detection of GGT to assay the gene expression profiles of phenotype-specific cells selected by laser capture microdissection (LCM). For optimization, we used the livers of rats subjected to hepatocarcinogenesis. This model induced nodules of hepatocytes and tumors with GGT activity. To obtain sufficient high-quality RNA after histochemistry and LCM, we included an RNase inhibitor and air-dried the tissue sections. This optimization allowed the visualization of GGT activity in situ and a yield of 1.4 to 2.0 µg of total RNA from 15 to 18 mm² of microdissected tissue (20 µm thickness). The average RNA integrity number in GGT-positive tissue, determined by chip-capillary electrophoresis, was 6.9, and the 28S/18S ribosomal RNA (rRNA) ratio was 1.4. The RNAs were processed for the Rat Gene 1.0 ST Array (Affymetrix). Comparable quality control metrics, such as signal intensity and RNA degradation plots, were found between the LCM samples and non-LCM tissue. The increased expression of Ggt1 expected in GGT-positive tissue was confirmed by microarrays and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). This optimization provided a suitable method for whole-transcript analysis of GGT-positive tissue isolated using LCM.