RESUMO
Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1-containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM-FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.
Assuntos
Anexinas , Transferência Ressonante de Energia de Fluorescência , Anexinas/genética , Anexinas/metabolismo , Proteínas S100/química , Proteínas S100/metabolismoRESUMO
PURPOSE: To screen proteins/peptides in urine of Renal Cell Carcinoma (RCC) patients by SELDI-TOF (Surface Enhanced Laser Desorption Ionization - Time of Flight) in search of possible biomarkers. MATERIAL AND METHODS: Sixty-one urines samples from Clear Cell RCC and Papillary RCC were compared to 29 samples of control urine on CM10 chip. Mass analysis was performed in a ProteinChip Reader PCS 4,000 (Ciphergen Biosystems, Fremont, CA) with the software Ciphergen Express 3.0. All chips were read at low and at high laser energy. For statistical analysis the urine samples were clustered according to the histological classification (Clear Cell and Papillary Carcinoma). For identification urine was loaded on a SDS PAGE gel and bands of most interest were excised, trypsinized and identified by MS/MS. Databank searches were performed in Swiss-Prot database using the MASCOT search algorithm and in Profound. RESULTS: Proteins that were identified from urine of controls included immunoglobulin light chains, albumin, secreted and transmembrane 1 precursor (protein K12), mannan-binding lectin-associated serine protease-2 (MASP-2) and vitelline membrane outer layer 1 isoform 1. Identification of immunoglobulins and isoforms of albumin are quite common by proteomics and therefore cannot be considered as possible molecular markers. K12 and MASP-2 play important physiological roles, while vitellite membrane outer layer 1 role is unknown since it was never purified in humans. CONCLUSIONS: The down expression of Protein K-12 and MASP-2 make them good candidates for RCC urine marker and should be validated in a bigger cohort including the other less common histological RCC subtypes.
Assuntos
Biomarcadores Tumorais/urina , Carcinoma de Células Renais/urina , Neoplasias Renais/urina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Carcinoma de Células Renais/patologia , Estudos de Coortes , Feminino , Humanos , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteômica/métodosRESUMO
The vertebrate retina harbors rod and cone photoreceptors. Human vision critically depends on cone photoreceptor function. In the phototransduction cascade, cGMP activates distinct rod and cone isoforms of the cyclic nucleotide-gated (CNG) channel. Excessive cGMP levels initiate a pathophysiological rollercoaster, which starts with CNG channel over-activation, typically in rod photoreceptors. This triggers cell death of rods first, and then cones, and is the root cause of many blinding retinal diseases, including Retinitis pigmentosa. While targeting of CNG channels has been proposed for therapeutic purposes, thus far, it has not been possible to inhibit rod CNG channels without compromising cone function. Here, we present a novel strategy, based on cGMP analogues with opposing actions on CNG channels, which enables the selective modulation of either rod or cone photoreceptor activity. The combined treatment with the weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and the cone-selective CNG-channel activator (8-pCPT-cGMP) essentially normalized rod CNG-channel function while preserving cone functionality at physiological and pathological cGMP levels. Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Moreover, this strategy may allow for improvements in visual performance in certain light environments.
RESUMO
Hereditary degeneration of photoreceptors has been linked to over-activation of Ca2+-permeable channels, excessive Ca2+-influx, and downstream activation of Ca2+-dependent calpain-type proteases. Unfortunately, after more than 20 years of pertinent research, unequivocal evidence proving significant and reproducible photoreceptor protection with Ca2+-channel blockers is still lacking. Here, we show that both D- and L-cis enantiomers of the anti-hypertensive drug diltiazem were very effective at blocking photoreceptor Ca2+-influx, most probably by blocking the pore of Ca2+-permeable channels. Yet, unexpectedly, this block neither reduced the activity of calpain-type proteases, nor did it result in photoreceptor protection. Remarkably, application of the L-cis enantiomer of diltiazem even led to a strong increase in photoreceptor cell death. These findings shed doubt on the previously proposed links between Ca2+ and retinal degeneration and are highly relevant for future therapy development as they may serve to refocus research efforts towards alternative, Ca2+-independent degenerative mechanisms.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Diltiazem/farmacologia , Degeneração Retiniana/metabolismo , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Diltiazem/química , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Camundongos , Proteólise , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Anexina A2/fisiologia , Proteínas S100/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Anexina A2/antagonistas & inibidores , Anexina A2/genética , Anexina A2/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células/metabolismo , Células/ultraestrutura , Citoesqueleto/química , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , RNA Interferente Pequeno/farmacologia , Proteínas S100/antagonistas & inibidores , Proteínas S100/genética , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
BACKGROUND: Proteins are able to react in response to distinct stress stimuli by alteration of their subcellular distribution. The stress-responsive protein S100A11 belongs to the family of multifunctional S100 proteins which have been implicated in several key biological processes. Previously, we have shown that S100A11 is directly involved in DNA repair processes at damaged chromatin in the nucleus. To gain further insight into the underlying mechanism subcellular trafficking of S100A11 in response to DNA damage was analyzed. RESULTS: We show that DNA damage induces a nucleolin-mediated translocation of S100A11 from the cytoplasm into the nucleus. This translocation is impeded by inhibition of the phosphorylation activity of PKCα. Translocation of S100A11 into the nucleus correlates with an increased cellular p21 protein level. Depletion of nucleolin by siRNA severely impairs translocation of S100A11 into the nucleus resulting in a decreased p21 protein level. Additionally, cells lacking nucleolin showed a reduced colony forming capacity. CONCLUSIONS: These observations suggest that regulation of the subcellular distribution of S100A11 plays an important role in the DNA damage response and p21-mediated cell cycle control.
Assuntos
Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas S100/metabolismo , Transporte Ativo do Núcleo Celular , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Linhagem Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C-alfa , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas S100/análise , Proteínas S100/genética , NucleolinaRESUMO
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein and its interactor synphilin-1 are major components of these inclusions. Rare mutations in the alpha-synuclein and synphilin-1 genes have been implicated in the pathogenesis of PD; however, the normal function of these proteins is far from being completely elucidated. We, thus, searched for novel synphilin-1-interacting proteins and deciphered periphilin as new interactor. Periphilin isoforms are involved in multiple cellular functions in vivo, and the protein is broadly expressed during embryogenesis and in the adult brain. We show that periphilin displays an overlapping expression pattern with synphilin-1 in cellular and animal models and in Lewy bodies of PD patients. Functional studies demonstrate that periphilin, as previously shown for synphilin-1, displays an antiapoptotic function by reducing caspase-3 activity. Searching for mutations in the periphilin gene, we detected a K69E substitution in two patients of a PD family. Taken together, these findings support for the first time an involvement of periphilin in PD.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Doença de Parkinson/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Proteínas de Transporte/genética , Linhagem Celular , Análise Mutacional de DNA , Humanos , Corpos de Lewy/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Doença de Parkinson/genética , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Human papillomavirus (HPV) infection has been identified as an etiologic agent for a subset of oral squamous cell carcinoma (OSCC) with increasing incidence. HPV DNA-positivity may confer better prognosis but the related oncogenic mechanisms are unknown. For the identification of HPV relevant proteins, we analyzed microdissected cells from HPV DNA-positive (n = 17) and HPV DNA-negative (n = 7) OSCC tissue samples. We identified 18 proteins from tumor tissues by peptide fingerprint mapping and SELDI MS that were separated using 2-DE. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified thioredoxin (TRX) and epidermal-fatty acid binding protein as upregulated in HPV related tumor tissue. This study, investigating for the first time proteomic changes in microdissected HPV infected tumor tissue, provides an indication on the oncogenic potential of viruses.
Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma de Células Escamosas/metabolismo , Proteínas de Ligação a Ácido Graxo/biossíntese , Neoplasias Bucais/metabolismo , Infecções por Papillomavirus/complicações , Proteoma/metabolismo , Tiorredoxinas/biossíntese , Idoso , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/etiologia , DNA Viral/análise , Feminino , Humanos , Masculino , Microdissecção , Pessoa de Meia-Idade , Neoplasias Bucais/química , Neoplasias Bucais/etiologia , Infecções por Papillomavirus/metabolismo , Análise Serial de Proteínas , Proteoma/química , Regulação para CimaRESUMO
Melanoma inhibitory activity (MIA) has been identified as a small protein secreted from malignant melanoma cells, which strongly enhances melanoma cell migration and invasion. Detailed analyses performed by our group showed interaction of MIA with extracellular matrix proteins and integrin alpha4beta1 and alpha5beta1 leading to cellular detachment. In this study, we identified cadherin-7 as a new MIA-binding protein using surface-enhanced laser desorption/ionization-mass spectrometry technology and co-immunoprecipitation. Cadherin-7 is a classical cell-cell adhesion molecule which was shown to be upregulated in malignant melanoma. We demonstrated enhanced expression of cadherin-7 in primary tumor cells compared to metastatic cells. Upregulation of cadherin-7 expression in metastatic cell lines but also downregulation of expression in cells derived from primary melanomas resulted in reduced cell migration. In addition, we speculate that MIA/cadherin-7 interaction may regulate cell-cell adhesion of malignant melanoma cells influencing the migration of the cells. Interestingly, overexpression of cadherin-7 resulted in a decreased MIA mRNA expression. In addition, MIA effects on cell migration were abrogated in cell clones overexpressing cadherin-7. In conclusion, these findings suggest that cadherin-7 regulates the expression and activity of MIA and the migration of melanoma cells playing a role in tumor development of malignant melanoma.
Assuntos
Caderinas/metabolismo , Movimento Celular , Proteínas da Matriz Extracelular/metabolismo , Melanoma/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/patologia , Western Blotting , Caderinas/genética , Adesão Celular , Proliferação de Células , Proteínas da Matriz Extracelular/genética , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoprecipitação , Melanoma/genética , Melanoma/metabolismo , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
DNA damage possesses the capacity to threaten the genomic integrity of an organism. A multitude of proteins are involved in the detection and repair of DNA double-strand breaks (DSBs), a severe kind of DNA damage. The function of DNA repair proteins can be examined by biochemical assays in vitro as well as in cell-based studies. The Ca2+-binding protein S100A11 shows functional interactions with factors involved in the repair of DSBs by homologous recombination (HR), a high-fidelity DNA repair pathway, such as RAD51 and RAD54B. The key enzyme of the homologous recombination repair is RAD51 that catalyzes the invasion of single-stranded DNA (ssDNA) into double-stranded DNA (dsDNA) containing homologous regions and the exchange of these DNA molecules generating heteroduplex DNA (hDNA). In this chapter, we describe a protocol for the purification of S100A11 to near homogeneity. Using purified proteins, we show the ability of S100A11 to stimulate RAD51 in a DNA strand exchange assay. Additionally, we describe a protocol how S100A11 can be localized in sites of DNA repair by immunofluorescence staining. Furthermore, we present a protocol for assessment of chromosomal aberrations after depletion of S100A11 that illustrate the apparent involvement of S100A11 in genome integrity.
Assuntos
DNA/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteínas S100/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular Tumoral , Aberrações Cromossômicas , DNA/química , Dano ao DNA , Imunofluorescência , Humanos , Microscopia ConfocalRESUMO
Knowledge about precise numbers of specific molecules is necessary for understanding and verification of biological pathways. The RAD51 protein is central in the repair of DNA double-strand breaks (DSBs) by homologous recombination repair and understanding its role in cellular pathways is crucial to design mechanistic DNA repair models. Here, we determined the number of RAD51 molecules in several human cell lines including primary fibroblasts. We showed that between 20000 to 100000 of RAD51 molecules are available per human cell that theoretically can be used for simultaneously loading at least 7 DSBs. Interestingly, the amount of RAD51 molecules does not significantly change after the induction of DNA damage using bleomycin or γ-irradiation in cells but an accumulation of RAD51 on the chromatin occurs. Furthermore, we generated an EGFP-RAD51 fusion under the control of HSV thymidine kinase promoter sequences yielding moderate protein expression levels comparable to endogenously expressed RAD51. Initial characterizations suggest that these low levels of ectopically expressed RAD51 are compatible with cell cycle progression of human cells. Hence, we provide parameters for the quantitative understanding and modeling of RAD51-involving processes.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Rad51 Recombinase/genética , Reparo de DNA por Recombinação/genética , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Cromatina/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica/genética , Humanos , Cultura Primária de Células , Timidina Quinase/genéticaRESUMO
Recently, using a proteomic approach we have identified the corepressor Alien as a novel interacting factor of the cell cycle regulator E2F1. Unclear was whether this interaction influences cell proliferation and endogenous E2F1 target gene expression. Here, we show by chromatin immunoprecipitation (ChIP) that Alien is recruited in vivo to the E2F binding sites present in the E2F1 gene promoter, inhibits the transactivation of E2F1 and represses endogenous E2F1 gene expression. Interestingly, using synchronized cells to assess the expression of Alien profile during cell cycle the levels of endogenous Alien are increased during G1, G1/S and G2 phase. Furthermore, stable transfection of Alien leads to reduction of cell proliferation. Thus, the data suggest that Alien acts as a corepressor for E2F1 and is involved in cell cycle regulation.
Assuntos
Proliferação de Células , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/fisiologia , Expressão Gênica , Proteínas Repressoras/fisiologia , Sítios de Ligação , Complexo do Signalossomo COP9 , Ciclo Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Fator de Transcrição E2F1/genética , Fase G1/fisiologia , Fase G2/fisiologia , Células HeLa , Humanos , Masculino , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Ligação Proteica , Fase S/fisiologia , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
Expression of Human Neutrophil Peptides (HNP) 1-3 was recently found to be associated with development of colorectal cancer. Raised defensin-expression in tumours is believed to stem from increased infiltration of neutrophils into tumour environment. To further specify the role of alpha-defensins in tumourigenesis and progression, HNP1-3 were analyzed in colorectal adenomas and carcinomas of 87 patients and quantified in relation to cancer stage and grading. Using the ProteinChip arrays, HNP1-3 were found upregulated in both colorectal adenomas and carcinomas. By combining the array with Laser capture microscopy we were able to confirm that HNP1-3 are expressed by tumour cells but not by neutrophils or other tumour invading cells. These findings suggest that alpha-defensins are more likely to contribute to tumour growth than they are to mount an effective host anti-tumour response. However, the amount of HNP-expression was not found to be related to tumour stage, grading, and serological tumour markers.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , alfa-Defensinas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Adenoma/metabolismo , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Neutrófilos/metabolismo , Prognóstico , Análise Serial de TecidosRESUMO
Synphilin-1 is linked to Parkinson's disease (PD), based on its role as an alpha-synuclein (PARK1)-interacting protein and substrate of the ubiquitin E3 ligase Parkin (PARK2) and because of its presence in Lewy bodies (LB) in brains of PD patients. We found that overexpression of synphilin-1 in cells leads to the formation of ubiquitinated cytoplasmic inclusions supporting a derangement of the ubiquitin-proteasome system in PD. We report here a novel specific interaction of synphilin-1 with the regulatory proteasomal protein S6 ATPase (tbp7). Functional characterization of this interaction on a cellular level revealed colocalization of S6 and synphilin-1 in aggresome-like intracytoplasmic inclusions. Overexpression of synphilin-1 and S6 in cells caused reduced proteasomal activity associated with a significant increase in inclusion formation compared to cells expressing synphilin-1 alone. Steady-state levels of synphilin-1 in cells were not altered after cotransfection of S6 and colocalization of synphilin-1-positive inclusions with lysosomal markers suggests the presence of an alternative lysosomal degradation pathway. Subsequent immunohistochemical studies in brains of PD patients identified S6 ATPase as a component of LB. This is the first study investigating the physiological role of synphilin-1 in the ubiquitin proteasome system. Our data suggest a direct interaction of synphilin-1 with the regulatory complex of the proteasome modulating proteasomal function.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/etiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Encéfalo/patologia , Humanos , Corpos de Inclusão/metabolismo , Corpos de Lewy , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologiaRESUMO
The TANGO gene was originally identified as a new member of the MIA gene family. It codes for a protein of yet unknown function. TANGO revealed a very broad expression pattern in contrast to the highly restricted expression pattern determined for the other family members. The only cells lacking TANGO expression are cells of the hematopoietic system. One of the major differences between mature hematopoietic cells and other tissue cells is the lack of adhesion until these cells leave the bloodstream. In this study, we observed that TANGO expression was induced after adhesion of human monocytic cells to substrate. To understand the mechanism of TANGO function during monocyte adhesion we isolated interacting proteins and found an interaction between TANGO and the leukocyte-specific integrin CD11c. In functional assays, we observed reduced attachment of human monocytic cells to fibrinogen, ICAM-1 and to human microvascular endothelial cells (HMECs) after stimulation with recombinant TANGO protein. Additionally, the migrating capacity of premonocytic cells through fibrinogen or HMECs was increased after stimulation of these cells with recombinant TANGO. Therefore, we suggest that TANGO reduced the attachment to fibrinogen or other cell adhesion molecules. As TANGO does not compete for CD11c ligand binding directly, we hypothesize TANGO function by modulation of integrin activity. Taken together, the results from this study present TANGO as a novel ligand for CD11c, regulating migratory processes of hematopoietic cells.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Antígeno CD11c/metabolismo , Antígenos CD18/metabolismo , Movimento Celular , Monócitos/citologia , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Antígeno CD11c/química , Adesão Celular , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Imunoprecipitação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
FLT3 activating mutations can be detected in about 35% of acute myeloid leukemia (AML). FLT3 internal tandem duplications (FLT3-ITD) represent the majority of FLT3 mutations (25 - 30%) while FLT3-TKD (tyrosine kinase domain) mutations can be found in about 7% of AML patients. In this study, we addressed the question whether especially primary AML cells carrying FLT3-ITD mutations show differences in terms of their protein expression pattern compared to FLT3 wild-type blasts. We investigated bone marrow samples that were isolated at diagnosis from 36 AML patients expressing either FLT3 wild-type (n = 16) or an activating FLT3 mutation (FLT3-ITD, n = 15; FLT3-TKD, n = 5). Proteomic analysis was performed by means of surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry which has shown its high efficiency in finding biomarkers in solid tumors. Here, we demonstrate that a large series of proteins is differently expressed in primary AML blasts harboring FLT3-ITD mutations. Furthermore, there are also significant differences of the protein expression profile between FLT3-ITD and FLT3-TKD mutations. Interestingly, further analysis of FLT3-ITD positive AML according to its response to the induction chemotherapy demonstrates putative prognostic markers for this subgroup of AML. We suggest that SELDI-TOF mass spectrometry represents a promising tool of proteomic analysis of AML that might help to establish new prognostic markers in AML.
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Proteínas de Neoplasias/análise , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , Idoso , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cutaneous T-cell lymphomas (CTCL) are characterized by malignant proliferation of skin homing T-cells. Although prognosis is generally good, reliable markers are needed to identify patients at risk for a more aggressive course. ProteinChip (SELDI) technology was used as a tool for the discovery of protein patterns in lymphocytes from patients with CTCL (n=25) and unaffected controls (n=25). Lymphocytes were separated in CD4+ and CD4- fractions by magnetic cell sorting (MACS). Each whole protein extract was analysed by ProteinChip technology. The resulting protein profiles were submitted for bioinformatic analysis including a clustering algorithm, a rule extraction, a rating and a rule-base construction step. For the generated combined rule base for the CD4- cell fraction, both the sensitivity and specificity for the prediction of CTCL reached 96%, while for the CD4+ fraction they were 92% and 84%, respectively, for sensitivity and specificity. The most significant peak at 3489Da could be identified as HNP3, an alpha-defensin, by immunocapturing. These results open up both the possibility for the use of this protein signature, especially HNP3, to more effectively monitor and screen CTCL, and the avenue to identify the other relevant peaks for a better understanding of the development of this tumour.
Assuntos
Biomarcadores Tumorais/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfoma Cutâneo de Células T/diagnóstico , Neoplasias Cutâneas/diagnóstico , alfa-Defensinas/metabolismo , Antígenos CD4/metabolismo , Estudos de Casos e Controles , HumanosRESUMO
On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.
Assuntos
Análise Serial de Proteínas/métodos , Proteoma/análise , Carcinoma Hepatocelular/química , Colo/química , Neoplasias do Colo/química , Mucosa Gástrica/química , Humanos , Mucosa Intestinal/química , Fígado/química , Neoplasias Hepáticas/química , Sensibilidade e Especificidade , Estômago/química , Neoplasias Gástricas/químicaRESUMO
The aim of the study was to detect proteomic markers usable to distinguish colorectal carcinoma from colon adenoma for a better understanding of the molecular mechanisms in the process of tumourigenesis. Therefore, we microdissected colon carcinoma tissue, epithelial colon adenoma tissue as well as normal adjacent colon epithelium and determined protein profiles by SELDI-TOF MS. A multitude of significantly different signals was detected. For their identification colon biopsis were lysed and subjected to a two-dimensional gel electrophoresis for separation. Subsequently, we identified nearly 100 proteins by tryptic digestion, peptide fingerprint mapping and database search. Calgizzarin (S100A11; S100C) identified by peptide fingerprint mapping correlated very well with a significantly differentially expressed signal found in prior protein profiling. Using an immunodepletion assay we confirmed the identity of this signal as calgizzarin. To localise calgizzarin in tissues we performed immunohistochemistry. For further confirmation of the identity of calgizzarin we re-analysed IHC-positive as well as IHC-negative tissue sections on ProteinChip arrays. This work demonstrates that biomarkers in colorectal cancer can be detected, identified and assessed by a proteomic approach comprising tissue-microdissection, protein profiling and immunological techniques.
Assuntos
Adenoma/genética , Carcinoma/genética , Neoplasias Colorretais/genética , Perfilação da Expressão Gênica/métodos , Proteínas S100/biossíntese , Adenoma/diagnóstico , Adenoma/patologia , Carcinoma/diagnóstico , Carcinoma/patologia , Transformação Celular Neoplásica , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Impressões Digitais de DNA , Diagnóstico Diferencial , Marcadores Genéticos , Humanos , Mucosa Intestinal , Análise Serial de Proteínas , Proteômica , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We applied the novel ProteinChip technology (SELDI-MS) to investigate and identify differentially regulated proteins upon myocardial remodelling in different heart regions. Tissue samples were isolated from the atria, the interventricular septum, and the right and left ventricles three months after surgically-induced myocardial infarction (MI) in rats. Corresponding protein extracts from control versus MI hearts were analysed on two different ProteinChip surfaces. In each of the functionally distinct cardiac regions, we obtained specific protein profile alterations upon myocardial remodelling. Most alterations were observed in the non-infarcted right ventricle, where down-regulation occurred more frequently than up-regulation of protein expression. Three of the differentially regulated proteins were identified: the metabolic enzyme triosephosphate isomerase (TIM), the cell signalling protein Raf-1 kinase inhibitory protein (RKIP), also known as phosphatidylethanolamine binding protein (PEBP), and the small heat shock protein alphaB-crystallin. These proteins showed a pronounced tissue-dependent regulation. TIM was down-regulated only in the atrium and in the left ventricle, RKIP/PEBP was down-regulated only in the right ventricle and in the interventricular septum, and alphaB-crystallin was up-regulated only in the right and in the left ventricle. A simple correlation of peak intensity changes using two of the identified peaks demonstrated the diagnostic potential of SELDI-MS. We conclude that this novel proteomic method is a powerful high-throughput tool for the fast detection of region-specific cardiac protein profiles in small biopsy samples, and that SELDI-MS may become a useful complementary technique for the diagnosis and prognosis of cardiac diseases.