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1.
J Proteomics ; 176: 13-23, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29331515

RESUMO

To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer cell lines. MHC class I binding probability was used to plot the distribution of the eluted peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. SIGNIFICANCE: By employing proteomic analyses of eluted peptides from breast cancer cells, the current study has built an initial HLA-I-typed antigen collection for breast cancer research. It was also determined that immunogenic epitopes can be identified using established cell lines and that shared immunogenic peptides can be found in different cancer types such as breast cancer and leukemia. Importantly, out of 3196 eluted peptides that included duplicate peptides in different cells 89 peptides either contained mutation in their sequence or were derived from aberrant translation suggesting that mutation-containing epitopes are on the order of 2-3% in breast cancer cells. Finally, our results suggest that interfering with MHC class I function is one of the mechanisms of how tumor cells escape immune system attack.


Assuntos
Neoplasias da Mama/imunologia , Antígenos de Histocompatibilidade Classe I/análise , Sequência de Aminoácidos , Apresentação do Antígeno , Antígenos de Neoplasias , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Epitopos/genética , Antígenos HLA , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Mutação , Proteômica/métodos
2.
Proc Natl Acad Sci U S A ; 113(29): 8272-7, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27307436

RESUMO

Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.


Assuntos
Neoplasias da Mama/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismo , Sequência de Bases , Linhagem Celular , Emulsões , Feminino , Humanos , Reação em Cadeia da Polimerase/métodos
3.
J Biol Chem ; 286(23): 21002-12, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21518756

RESUMO

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.


Assuntos
Colagenases/química , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Inibidores Teciduais de Metaloproteinases/química , Animais , Células CHO , Colagenases/genética , Colagenases/metabolismo , Cricetinae , Cricetulus , Humanos , Camundongos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo
4.
J Biol Chem ; 285(36): 27726-36, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20605791

RESUMO

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.


Assuntos
Furina/metabolismo , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinase 14 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína
5.
Int J Cancer ; 126(5): 1067-78, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19551841

RESUMO

Both invasion-promoting MT1-MMP and its physiological inhibitor TIMP-2 play a significant role in tumorigenesis and are identified in the most aggressive cancers. Despite its antiproteolytic effects in vitro, clinical data suggest that TIMP-2 expression is positively associated with tumor recurrence, thus emphasizing the wide-ranging role of TIMP-2 in malignancies. To shed light on this role of TIMP-2, we report that low concentrations of TIMP-2, by interacting with MT1-MMP (a specific membrane receptor of TIMP-2), induce the MEK/ERK signaling cascade in fibrosarcoma HT1080 cells which express MT1-MMP naturally. TIMP-2 binding with cell surface-associated MT1-MMP stimulates phosphorylation of MEK1/2, which is upstream of ERK1/2, and the ERK1/2 substrate p90RSK. Consistent with volumes of literature, we confirmed that the activation of ERK stimulated cell migration. Both the transcriptional silencing of MT1-MMP and the inhibition of MEK1/2 reversed the signaling effects of TIMP-2/MT1-MMP while the active site-targeting MMP inhibitor GM6001 did not. Our data suggest that both the interactions of TIMP-2 with MT1-MMP, which activate the pro-migratory ERK signaling cascade,and the conventional inhibition of MT1-MMP's catalytic activity by TIMP-2, play a role in the invasion-promoting function of MT1-MMP. The TIMP-2-induced stimulation of ERK signaling in cancer cells explains the direct, as opposed to the inverse, association of TIMP-2 expression with poor prognosis in cancer.


Assuntos
Movimento Celular/fisiologia , Metaloproteinase 14 da Matriz/metabolismo , Invasividade Neoplásica , Transdução de Sinais/fisiologia , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , MAP Quinase Quinase Quinases/metabolismo , Fosforilação , RNA Interferente Pequeno
6.
Mol Cancer Ther ; 8(6): 1515-25, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19509255

RESUMO

Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. The current suboptimal efficiency and selectivity drugs have therapeutic limitations and induce concomitant side effects. Recently, novel cancer therapies based on the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have emerged. TRAIL, a key component of the natural antitumor immune response, selectively kills many tumor cell types. Earlier studies with recombinant TRAIL, however, revealed its many shortcomings including a short half-life, off-target toxicity, and existence of TRAIL-resistant tumor cells. We improved the efficacy of recombinant TRAIL by redesigning its structure and the expression and purification procedures. The result is a highly stable leucine zipper (LZ)-TRAIL chimera that is simple to produce and purify. This chimera functions as a trimer in a manner that is similar to natural TRAIL. The formulation of the recombinant LZ-TRAIL we have developed has displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. Our results have shown that the half-life of LZ-TRAIL is improved and now exceeds 1 h in mice compared with a half-life of only minutes reported earlier for recombinant TRAIL. We have concluded that our LZ-TRAIL construct will serve as a foundation for a new generation of fully human LZ-TRAIL proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.


Assuntos
Zíper de Leucina/genética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Engenharia de Proteínas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Biochem J ; 420(1): 37-47, 2009 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-19232058

RESUMO

Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; DeltaCT) MT1-MMP-FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and DeltaCT constructs. The WT and E240A constructs also interacted with alpha-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the DeltaCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2-FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.


Assuntos
Neoplasias da Mama/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Metabolismo Energético , Feminino , Humanos , Peptídeo Hidrolases/metabolismo , Mapeamento de Interação de Proteínas
8.
Cancer Res ; 68(11): 4086-96, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18519667

RESUMO

Invasion-promoting MT1-MMP is directly linked to tumorigenesis and metastasis. Our studies led us to identify those genes, the expression of which is universally linked to MT1-MMP in multiple tumor types. Genome-wide expression profiling of MT1-MMP-overexpressing versus MT1-MMP-silenced cancer cells and a further data mining analysis of the preexisting expression database of 190 human tumors of 14 cancer types led us to identify 11 genes, the expression of which correlated firmly and universally with that of MT1-MMP (P < 0.00001). These genes included regulators of energy metabolism (NNT), trafficking and membrane fusion (SLCO2A1 and ANXA7), signaling and transcription (NR3C1, JAG1, PI3K delta, and CK2 alpha), chromatin rearrangement (SMARCA1), cell division (STK38/NDR1), apoptosis (DAPK1), and mRNA splicing (SNRPB2). Our subsequent extensive analysis of cultured cells, tumor xenografts, and cancer patient biopsies supported our data mining. Our results suggest that transcriptional reprogramming of the specific downstream genes, which themselves are associated with tumorigenesis, represents a distinctive "molecular signature" of the proteolytically active MT1-MMP. We suggest that the transactivation activity of MT1-MMP contributes to the promigratory cell phenotype, which is induced by this tumorigenic proteinase. The activated downstream gene network then begins functioning in unison with MT1-MMP to rework the signaling, transport, cell division, energy metabolism, and other critical cell functions and to commit the cell to migration, invasion, and, consequently, tumorigenesis.


Assuntos
Metaloproteinase 14 da Matriz/genética , Neoplasias/genética , Ativação Transcricional , Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Metabolismo Energético , Perfilação da Expressão Gênica , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
J Biol Chem ; 283(1): 87-99, 2008 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-17991754

RESUMO

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.


Assuntos
Proliferação de Células , Metaloproteinase 14 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Alanina/metabolismo , Alanina/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Ativação Enzimática , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Proteínas ras/metabolismo
10.
J Biol Chem ; 282(44): 32106-11, 2007 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-17761671

RESUMO

Human diabetes mellitus (IDDM; type I diabetes) is a T cell-mediated disease that is closely modeled in non-obese diabetic (NOD) mice. The pathogenesis of IDDM involves the transmigration of autoimmune T cells into the pancreatic islets and the subsequent destruction of insulin-producing beta cells. Therapeutic interventions leading to beta cell regeneration and the reversal of established IDDM are exceedingly limited. We report here that specific inhibition of T cell intra-islet transmigration by using a small molecule proteinase inhibitor restores beta cell functionality, increases insulin-producing beta cell mass, and alleviates the severity of IDDM in acutely diabetic NOD mice. As a result, acutely diabetic NOD mice do not require insulin injections for survival for a significant time period, thus providing a promising clue to effect IDDM reversal in humans. The extensive morphometric analyses and the measurements of both the C-peptide blood levels and the proinsulin mRNA levels in the islets support our conclusions. Diabetes transfer experiments suggest that the inhibitor specifically represses the T cell transmigration and homing processes as opposed to causing immunosuppression. Overall, our data provide a rationale for the pharmacological control of the T cell transmigration step in human IDDM.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Linfócitos T/metabolismo , Animais , Peptídeo C/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Glucose/metabolismo , Imunossupressores/uso terapêutico , Insulina/genética , Metaloproteinase 14 da Matriz , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Compostos Orgânicos/uso terapêutico , RNA Mensageiro/metabolismo
11.
Cancer Res ; 66(21): 10460-5, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17079467

RESUMO

An elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with multiple malignancies. Recently, we discovered that recycled MT1-MMP was trafficked along the tubulin cytoskeleton into the centrosomal compartment and cleaved the integral centrosomal protein pericentrin-2. These events correlated with the induction of chromosome instability and aneuploidy in nonmalignant Madine-Darby canine kidney cells. Accordingly, we hypothesized that MT1-MMP is an oncogene that promotes malignant transformation of normal cells rather than just an enzyme that supports growth of preexisting tumors. To prove our hypothesis, we transfected normal 184B5 human mammary epithelial cells with MT1-MMP (184B5-MT1 cells). MT1-MMP was colocalized with pericentrin in the centrosomal compartment and especially in the midbody of dividing cells. 184B5-MT1 cells acquired the ability to activate MMP-2, to cleave pericentrin, and to invade the Matrigel matrix. 184B5-MT1 cells exhibited aneuploidy, and they were efficient in generating tumors in the orthotopic xenograft model in immunodeficient mice. Because of the absence of tumor angiogenesis and the resulting insufficient blood supply, the tumors then regressed with significant accompanying necrosis. Gene array studies confirmed a significant up-regulation of oncogenes and tumorigenic genes but not the angiogenesis-promoting genes in 184B5-MT1 cells. We believe that our data point to a novel function of MT1-MMP in the initial stages of malignant transformation and to new and hitherto unknown transition mechanism from normalcy to malignancy.


Assuntos
Aneuploidia , Mama/enzimologia , Neoplasias Mamárias Experimentais/etiologia , Metaloproteinase 14 da Matriz/fisiologia , Animais , Mama/ultraestrutura , Linhagem Celular , Instabilidade Cromossômica , Células Epiteliais/enzimologia , Células Epiteliais/ultraestrutura , Feminino , Humanos , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos
12.
FASEB J ; 20(11): 1793-801, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16940151

RESUMO

It has been well established that invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP), a multifunctional membrane-tethered enzyme, functions in cancer cells as a mediator of pericellular proteolysis and directly cleaves several cell surface receptors, including CD44. In this report, we confirm that adhesion of diabetogenic T cells promotes the activation of endogenous MT1-MMP. Activated protease then cleaves CD44 in adherent T cells. We have validated that the T cell CD44 receptor is critical for the adhesion of diabetogenic insulin-specific, CD8-positive, K(d)-restricted cells to the matrix as well as for the subsequent transmigration of the adherent T cells through the endothelium and homing of the transmigrated T cells into the pancreatic islets. We have determined that the inhibition of MT1-MMP by low dosages of AG3340 (a subnanomolar range hydroxamate inhibitor of MMPs that has been widely tested in cancer patients) inhibited both T cell MT1-MMP activity and MT1-MMP-dependent shedding of CD44, immobilized T cells on the endothelium, repressed the homing of diabetogenic T cells into the pancreatic islets, reduced insulitis and mononuclear cell infiltration, and promoted either the recovery or the rejuvenation of the functional insulin-producing beta cells in diabetic NOD mice with freshly developed type I diabetes (IDDM). We believe our data constitute a mechanistic and substantive rationale for clinical trials of selected MT1-MMP inhibitors in the therapy of IDDM in humans.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Animais , Adesão Celular , Sobrevivência Celular , Ativação Enzimática , Citometria de Fluxo , Receptores de Hialuronatos/fisiologia , Camundongos , Camundongos Endogâmicos NOD
13.
Cancer Res ; 66(12): 6258-63, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16778201

RESUMO

Neoplasms have developed strategies to protect themselves against the complement-mediated host immunity. Invasion- and metastasis-promoting membrane type-1 (MT1) matrix metalloproteinase (MMP) is strongly associated with many metastatic cancer types. The relative importance of the individual functions of MT1-MMP in metastasis was, however, unknown. We have now determined that the expression of murine MT1-MMP in murine melanoma B16F1 cells strongly increased the number of metastatic loci in the lungs of syngeneic C57BL/6 mice. In contrast, MT1-MMP did not affect the number of metastatic loci in complement-deficient C57BL/6-C3-/- mice. Our results indicated, for the first time, that the anticomplement activity of MT1-MMP played a significant role in promoting metastasis in vivo and determined the relative importance of the anticomplement activity in the total metastatic effect of this multifunctional proteolytic enzyme. We believe that our results shed additional light on the functions of MT1-MMP in cancer and clearly make this protease a promising drug target in metastatic malignancies.


Assuntos
Complemento C3/imunologia , Proteínas Inativadoras do Complemento/imunologia , Metaloproteinases da Matriz/imunologia , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/imunologia , Animais , Complemento C3/deficiência , Complemento C3/genética , Proteínas Inativadoras do Complemento/genética , Proteínas Inativadoras do Complemento/metabolismo , Fibrossarcoma/enzimologia , Fibrossarcoma/genética , Fibrossarcoma/imunologia , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Metaloproteinase 14 da Matriz , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Transfecção
14.
J Biol Chem ; 281(25): 16897-905, 2006 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-16627478

RESUMO

MT1-MMP is a key enzyme in cancer cell invasion and metastasis. The activity of cellular MT1-MMP is regulated by furin-like proprotein convertases, TIMPs, shedding, autoproteolysis, dimerization, exocytosis, endocytosis, and recycling. Our data demonstrate that, in addition to these already known mechanisms, MT1-MMP is regulated by O-glycosylation of its hinge region. Insignificant autolytic degradation is characteristic for naturally expressed, glycosylated, MT1-MMP. In turn, extensive autolytic degradation, which leads to the inactivation of the protease and the generation of its C-terminal membrane-tethered degraded species, is a feature of overexpressed MT1-MMP. We have determined that incomplete glycosylation stimulates extensive autocatalytic degradation and self-inactivation of MT1-MMP. Self-proteolysis commences during the secretory process of MT1-MMP through the cell compartment to the plasma membrane. The strongly negatively charged sialic acid is the most important functional moiety of the glycopart of MT1-MMP. We hypothesize that sialic acid of the O-glycosylation cassette restricts the access of the catalytic domain to the hinge region and to the autolytic cleavage site and protects MT1-MMP from autolysis. Overall, our results point out that there is a delicate balance between glycosylation and self-proteolysis of MT1-MMP in cancer cells and that when this balance is upset the catalytically potent MT1-MMP pool is self-proteolyzed.


Assuntos
Metaloproteinases da Matriz/química , Animais , Células CHO , Domínio Catalítico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cricetinae , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicosilação , Humanos , Metaloproteinase 14 da Matriz , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Transplante de Neoplasias
15.
Cancer Res ; 66(5): 2716-24, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16510592

RESUMO

Estrogens have many cellular functions, including their interactions with estrogen receptors alpha and beta (ERalpha and ERbeta). Earlier, we determined that the estrogen-ER complex stimulates the transcriptional activity of the matrix metalloproteinase 26 (MMP-26) gene promoter. We then determined that ERbeta is susceptible to MMP-26 proteolysis whereas ERalpha is resistant to the protease. MMP-26 targets the NH(2)-terminal region of ERbeta coding for the divergent NH(2)-terminal A/B domain that is responsible for the ligand-independent transactivation function. As a result, MMP-26 proteolysis generates the COOH-terminal fragments of ERbeta. Immunohistochemical analysis of tissue microarrays derived from 121 cancer patients corroborated these data and revealed an inverse correlation between the ERalpha-dependent expression of MMP-26 and the levels of the intact ERbeta in breast carcinomas. MMP-26 is not expressed in normal mammary epithelium. The levels of MMP-26 are strongly up-regulated in ductal carcinoma in situ (DCIS). In the course of further disease progression through stages I to III, the expression of MMP-26 decreases. In contrast to many tumor-promoting MMPs, the expression of MMP-26 in DCIS correlated with a longer patient survival. Our data suggest the existence of an MMP-26-mediated intracellular pathway that targets ERbeta and that MMP-26, a novel and valuable cancer marker, contributes favorably to the survival of the ERalpha/beta-positive cohort of breast cancer patients.


Assuntos
Neoplasias da Mama/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Ductal de Mama/metabolismo , Receptor beta de Estrogênio/metabolismo , Metaloproteinases da Matriz/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Carcinoma in Situ/enzimologia , Carcinoma in Situ/patologia , Carcinoma Ductal de Mama/enzimologia , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Humanos , Metaloproteinases da Matriz Secretadas , Estadiamento de Neoplasias , Taxa de Sobrevida
16.
Arch Biochem Biophys ; 446(1): 52-9, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16384550

RESUMO

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.


Assuntos
Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Endocitose , Furina/metabolismo , Animais , Antibacterianos/farmacologia , Bacillus anthracis/química , Sítios de Ligação , Células Cultivadas , Dimerização , Furina/deficiência , Camundongos , Modelos Moleculares , Peso Molecular , Pró-Proteína Convertases/metabolismo , Receptores de Superfície Celular/metabolismo , Venenos de Víboras
17.
Biochem J ; 393(Pt 2): 503-11, 2006 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16229682

RESUMO

Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9-12-mer peptides are potent inhibitors of WNV NS3 with K(i) values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.


Assuntos
Arginina/análise , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Animais , Arginina/química , Células Cultivadas , Sequência Conservada , Furina/química , Furina/metabolismo , Camundongos , Dados de Sequência Molecular , Bainha de Mielina/metabolismo , Neurônios/metabolismo , Peptídeos/química , Peptídeos/farmacologia , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Serpinas/farmacologia , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Vírus do Nilo Ocidental/genética
18.
J Cell Sci ; 118(Pt 21): 4975-84, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16219679

RESUMO

Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades the extracellular matrix, initiates the activation pathway of soluble MMPs and regulates the functionality of cell adhesion signaling receptors, thus playing an important role in many cell functions. Intracellular transport mechanisms, currently incompletely understood, regulate the presentation of MT1-MMP at the cell surface. We have focused our efforts on identifying these mechanisms. To understand the transport of MT1-MMP across the cell, we used substitution and deletion mutants, the trafficking of which was examined using antibody uptake and Chariot delivery experiments. Our experiments have demonstrated that the microtubulin cytoskeleton and the centrosomes (the microtubulin cytoskeleton-organizing centers) are essential for the trafficking and the internalization of MT1-MMP. We determined that after reaching the plasma membrane, MT1-MMP is internalized in the Rab-4-positive recycling endosomes and the Rab-11-positive pericentrosomal recycling endosomes. The microtubular trafficking causes the protease to accumulate in the pericentrosomal region of the cell. We believe that the presence of the transmembrane domain is required for the microtubular vesicular trafficking of MT1-MMP because the soluble mutants are not presented at the cell surface and they are not delivered to the centrosomes. The observed transport mechanisms provide a vehicle for the intracellular targets and, accordingly, for an intracellular cleavage function of MT1-MMP in malignant cells, which routinely overexpress this protease.


Assuntos
Membrana Celular/enzimologia , Metaloendopeptidases/metabolismo , Microtúbulos/enzimologia , Animais , Sítios de Ligação de Anticorpos , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Centrossomo/enzimologia , Centrossomo/metabolismo , Cricetinae , Citoesqueleto/fisiologia , Cães , Humanos , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Microtúbulos/metabolismo , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Deleção de Sequência , Vesículas Transportadoras/enzimologia , Vesículas Transportadoras/genética , Tubulina (Proteína)/fisiologia
19.
Proc Natl Acad Sci U S A ; 102(27): 9499-504, 2005 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-15983377

RESUMO

Inhalation anthrax is a deadly disease for which there is currently no effective treatment. Bacillus anthracis lethal factor (LF) metalloproteinase is an integral component of the tripartite anthrax lethal toxin that is essential for the onset and progression of anthrax. We report here on a fragment-based approach that allowed us to develop inhibitors of LF. The small-molecule inhibitors we have designed, synthesized, and tested are highly potent and selective against LF in both in vitro tests and cell-based assays. These inhibitors do not affect the prototype human metalloproteinases that are structurally similar to LF. Initial in vivo evaluation of postexposure efficacy of our inhibitors combined with antibiotic ciprofloxacin against B. anthracis resulted in significant protection. Our data strongly indicate that the scaffold of inhibitors we have identified is the foundation for the development of novel, safe, and effective emergency therapy of postexposure inhalation anthrax.


Assuntos
Bacillus anthracis/efeitos dos fármacos , Toxinas Bacterianas/antagonistas & inibidores , Ciprofloxacino/farmacologia , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Rodaminas/metabolismo , Animais , Antígenos de Bactérias , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Fluorescência , Espectroscopia de Ressonância Magnética , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
20.
J Biol Chem ; 280(30): 27755-8, 2005 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-15944163

RESUMO

We have discovered that clinically tested inhibitors of matrix metalloproteinases can control the functional activity of T cell membrane type-1 matrix metalloproteinase (MT1-MMP) and the onset of disease in a rodent model of type 1 diabetes in non-obese diabetic mice. We determined that MT1-MMP proteolysis of the T cell surface CD44 adhesion receptor affects the homing of T cells into the pancreas. We also determined that both the induction of the intrinsic T cell MT1-MMP activity and the shedding of cellular CD44 follow the adhesion of insulin-specific, CD8-positive, Kd-restricted T cells to the matrix. Conversely, inhibition of these events by AG3340 (a potent hydroxamate inhibitor that was widely used in clinical trials in cancer patents) impedes the transmigration of diabetogenic T cells into the pancreas and protects non-obese diabetic mice from diabetes onset. Overall, our studies have divulged a previously unknown function of MT1-MMP and identified a promising novel drug target in type I diabetes.


Assuntos
Inibidores Enzimáticos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Pâncreas/metabolismo , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Domínio Catalítico , Separação Celular , Citometria de Fluxo , Receptores de Hialuronatos/biossíntese , Metaloproteinase 14 da Matriz , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinases da Matriz Associadas à Membrana , Camundongos , Camundongos Endogâmicos NOD , Microscopia de Fluorescência , Compostos Orgânicos/farmacologia , Linfócitos T/imunologia , Fatores de Tempo
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