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1.
Bioconjug Chem ; 31(9): 2201-2210, 2020 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-32786505

RESUMEN

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click" ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.


Asunto(s)
Alcadienos/química , Colorantes Fluorescentes/química , Alcadienos/síntesis química , Animales , Bovinos , Química Clic , Reacción de Cicloadición , Ciclooctanos/síntesis química , Ciclooctanos/química , Colorantes Fluorescentes/síntesis química , Albúmina Sérica Bovina/química , Staphylococcus aureus/citología , Staphylococcus aureus/aislamiento & purificación
2.
Proc Natl Acad Sci U S A ; 113(31): E4558-66, 2016 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-27432991

RESUMEN

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-ß receptor expression and signaling, and blocking TGF-ß receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-ß signaling with BET bromodomain inhibition may offer new therapeutic benefits.


Asunto(s)
Azepinas/farmacología , Proteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Triazoles/farmacología , Azepinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Medicamentos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HEK293 , Humanos , Estructura Molecular , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Triazoles/química
3.
Bioconjug Chem ; 27(5): 1332-40, 2016 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-27077642

RESUMEN

Cyclic CNGRC (cCNGRC) peptides are very important targeting ligands for Aminopeptidase N (APN or CD13), which is overexpressed on the surface of many cancer cells. In this work we have (1) developed an efficient solid-phase synthesis and (2) tested on purified porcine APN and APN-expressing human cells two different classes of cCNGRC peptides: the first carrying a biotin affinity tag or a fluorescent tag attached to the carboxyl Arg-Cys-COOH terminus and the second with the tags attached to the amino H2N-Cys-Asn terminus. Carboxyl-terminus functionalized cCNGRC peptides 3, 6, and 8 showed good affinity for porcine APN and very good capacity to target and be internalized into APN-expressing cells. In contrast, amino-terminus functionalized cCNGRC peptides 4, 5, and 7 displayed significantly decreased affinity and targeting capacity. These results, which are in agreement with the recently reported X-ray structure of a cCNGRC peptide bound to APN showing important stabilizing interactions between the unprotected cCNGRC amino terminus and the APN active site, indicate that the carboxyl and not the amino-terminus of cCNGRC peptides should be used as a "handle" for the attachment of toxic payloads for therapy or isotopically labeled functions for imaging and nuclear medicine.


Asunto(s)
Antígenos CD13/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Humanos , Modelos Moleculares , Conformación Proteica , Porcinos
4.
J Biol Chem ; 288(44): 32093-105, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-24052262

RESUMEN

Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of filamins. Here, we show that the calponin homology domain 1 (CH1), within the filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of filamins.


Asunto(s)
Filaminas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Filaminas/genética , Humanos , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Supresoras de la Señalización de Citocinas/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/fisiología
5.
J Cell Sci ; 125(Pt 16): 3858-69, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22595522

RESUMEN

Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered filamin expression or filamin mutations.


Asunto(s)
Proteínas Contráctiles/metabolismo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de Microfilamentos/metabolismo , Actinas/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proteínas Contráctiles/deficiencia , Proteínas Contráctiles/genética , Activación Enzimática , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibrosarcoma/enzimología , Fibrosarcoma/genética , Filaminas , Técnicas de Silenciamiento del Gen , Humanos , Integrinas/metabolismo , Metaloproteinasa 14 de la Matriz , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Invasividad Neoplásica , Fenotipo , Estructura Terciaria de Proteína
6.
J Cell Sci ; 124(Pt 15): 2631-41, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21750192

RESUMEN

Filamins are an important family of actin-binding and crosslinking proteins that mediate remodeling of the actin cytoskeleton and maintain extracellular matrix connections by anchoring transmembrane proteins to actin filaments and linking them to intracellular signaling cascades. We recently found that filamins are targeted for proteasomal degradation by the E3 ubiquitin ligase specificity subunit ASBα and that acute degradation of filamins through this ubiquitin-proteasome pathway correlates with cell differentiation. Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2α triggers filamin degradation and recapitulates early events crucial for cell differentiation. ASB2α is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2α interacts with filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2α to actin-rich structures is dependent on filamin and that the actin-binding domain (ABD) of filamin mediates the interaction with ASB2α. Furthermore, we show that the ABD is necessary and sufficient for ASB2α-mediated filamin degradation. We propose that ASB2α exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting filamins for degradation. These studies provide the molecular basis for ASB2α-mediated filamin degradation and unravel an important mechanism by which filamin levels can be acutely regulated.


Asunto(s)
Proteínas Contráctiles/metabolismo , Proteínas de Microfilamentos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Células Cultivadas , Proteínas Contráctiles/genética , Cricetinae , Cricetulus , Filaminas , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Immunoblotting , Ratones , Proteínas de Microfilamentos/genética , Unión Proteica , Ubiquitina-Proteína Ligasas/genética
7.
J Biol Chem ; 286(35): 30571-30581, 2011 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-21737450

RESUMEN

By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor α oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2α) and a muscle-type (ASB2ß) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2α is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2α structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2α, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of ß1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2α, together with ankyrin repeats 1 to 10, is necessary for association of ASB2α with filamin A. Importantly, the ASB2α N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and ß integrins. Together, these data provide new insights into the molecular mechanisms of ASB2α binding to filamin.


Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Integrinas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Adhesión Celular , Fibronectinas/metabolismo , Células HeLa , Humanos , Ratones , Músculos/metabolismo , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato
8.
Nat Cell Biol ; 6(11): 1071-81, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15502824

RESUMEN

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.


Asunto(s)
Aparato de Golgi/ultraestructura , Transporte Biológico , Compartimento Celular , Línea Celular , Retículo Endoplásmico/ultraestructura , Microscopía Electrónica
9.
Front Cell Infect Microbiol ; 11: 660689, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33898333

RESUMEN

Salmonella Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of S. Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic S. Typhi infection, the mechanisms that macrophages use to control the growth of S. Typhi and the role of these mechanisms in the bacterium's adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of S. Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescent-labeled S. Typhi, infected cells are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells.


Asunto(s)
Salmonella typhi , Fiebre Tifoidea , Animales , Macrófagos/metabolismo , Ratones , ARN Interferente Pequeño , Salmonella typhi/genética , Proteínas de Unión al GTP rab/metabolismo
10.
J Clin Med ; 10(3)2021 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-33498782

RESUMEN

Congenital Generalized Lipodystrophy type 2 (CGL2) is the most severe form of lipodystrophy and is caused by mutations in the BSCL2 gene. Affected patients exhibit a near complete lack of adipose tissue and suffer severe metabolic disease. A recent study identified infection as a major cause of death in CGL2 patients, leading us to examine whether Bscl2 loss could directly affect the innate immune response. We generated a novel mouse model selectively lacking Bscl2 in the myeloid lineage (LysM-B2KO) and also examined the function of bone-marrow-derived macrophages (BMDM) isolated from global Bscl2 knockout (SKO) mice. LysM-B2KO mice failed to develop lipodystrophy and metabolic disease, providing a model to study the direct role of Bscl2 in myeloid lineage cells. Lipopolysaccharide-mediated stimulation of inflammatory cytokines was not impaired in LysM-B2KO mice or in BMDM isolated from either LysM-B2KO or SKO mice. Additionally, intracellular fate and clearance of bacteria in SKO BMDM challenged with Staphylococcus aureus was indistinguishable from that in BMDM isolated from littermate controls. Overall, our findings reveal that selective Bscl2 deficiency in macrophages does not critically impact the innate immune response to infection. Instead, an increased susceptibility to infection in CGL2 patients is likely to result from severe metabolic disease.

11.
Sci Adv ; 7(3)2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33523895

RESUMEN

Macrophages provide a first line of defense against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here, we report that the Rab32 guanosine triphosphatase and its guanine nucleotide exchange factor BLOC-3 (biogenesis of lysosome-related organelles complex-3) are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This host-defense mechanism is active in both human and murine macrophages and is independent of well-known antimicrobial mechanisms such as the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-dependent oxidative burst, production of nitric oxide, and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1-encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defense pathway and protects mammalian species from various pathogens.


Asunto(s)
Salmonella typhi , Proteínas de Unión al GTP rab , Animales , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Lisosomas/metabolismo , Macrófagos/metabolismo , Mamíferos/metabolismo , Ratones , Proteínas de Unión al GTP rab/metabolismo
12.
Blood ; 112(13): 5130-40, 2008 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-18799729

RESUMEN

The ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and filamin degradation; conversely, ASB2 expression in leukemia cells induces filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both filamin A and filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of filamins for degradation.


Asunto(s)
Proteínas Contráctiles/metabolismo , Leucemia/patología , Proteínas de Microfilamentos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Actinas/metabolismo , Adhesión Celular , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proteínas Contráctiles/genética , Filaminas , Humanos , Leucemia/tratamiento farmacológico , Proteínas de Microfilamentos/genética , ARN Interferente Pequeño/farmacología , Proteínas Supresoras de la Señalización de Citocinas/genética , Tretinoina/farmacología
13.
Front Cell Infect Microbiol ; 10: 581024, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33392103

RESUMEN

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localization on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing.


Asunto(s)
Melanosomas , Proteínas de Unión al GTP rab , Animales , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Melanosomas/metabolismo , Ratones , Salmonella typhi/metabolismo , Proteínas de Unión al GTP rab/metabolismo
14.
Nat Struct Mol Biol ; 25(10): 918-927, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30224736

RESUMEN

Actin-cross-linking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion, and motility. Missense mutations in the tandem calponin homology domains of their actin-binding domains (ABDs) underlie numerous genetic diseases, but a molecular understanding of these pathologies is hampered by the lack of high-resolution structures of any actin-cross-linking protein bound to F-actin. Here, taking advantage of a high-affinity, disease-associated mutant of the human filamin A (FLNa) ABD, we combine cryo-electron microscopy and functional studies to reveal at near-atomic resolution how the first calponin homology domain (CH1) and residues immediately N-terminal to it engage actin. We further show that reorientation of CH2 relative to CH1 is required to avoid clashes with actin and to expose F-actin-binding residues on CH1. Our data explain localization of disease-associated loss-of-function mutations to FLNaCH1 and gain-of-function mutations to the regulatory FLNaCH2. Sequence conservation argues that this provides a general model for ABD-F-actin binding.


Asunto(s)
Actinas/química , Filaminas/química , Actinas/metabolismo , Microscopía por Crioelectrón , Filaminas/metabolismo , Humanos , Modelos Moleculares , Mutación Missense , Dominios Proteicos
15.
Mol Biol Cell ; 14(3): 1074-84, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12631724

RESUMEN

The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we show that the dynamin 2 (Dyn2), a GTPase implicated in the control of actin-driven cytoskeletal remodeling events and membrane transport, is necessary for focalized matrix degradation at invadopodia. Dynamin was inhibited by using two approaches: 1) expression of dominant negative GTPase-impaired or proline-rich domain-deleted Dyn2 mutants; and 2) inhibition of the dynamin regulator calcineurin by cyclosporin A. In both cases, the number and extension of ECM degradation foci were drastically reduced. To understand the site and mechanism of dynamin action, the cellular structures devoted to ECM degradation were analyzed by correlative confocal light-electron microscopy. Invadopodia were found to be organized into a previously undescribed ECM-degradation structure consisting of a large invagination of the ventral plasma membrane surface in close spatial relationship with the Golgi complex. Dyn2 seemed to be concentrated at invadopodia.


Asunto(s)
Extensiones de la Superficie Celular/metabolismo , Dinamina II/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Dinamina II/genética , Humanos , Invasividad Neoplásica , Proteínas Recombinantes de Fusión/metabolismo
16.
Eur J Cell Biol ; 85(3-4): 159-64, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16546558

RESUMEN

The controlled degradation of extracellular matrix is crucial in physiological and pathological cell invasion alike. In cultured cells, degradation occurs at specific sites where invasive cells make contact with the extracellular matrix via specialized plasma membrane protrusions termed invadopodia. Considerable progress has been made in recent years towards understanding the basic molecular components and the ultrastructural features of invadopodia. This current knowledge will be reviewed here together with some of the most important open questions in invadopodia biology. Considering the substantial interest and momentum in the field, the need for an operational framework to correctly define and identify invadopodia will also be discussed.


Asunto(s)
Extensiones de la Superficie Celular/fisiología , Animales , Extensiones de la Superficie Celular/ultraestructura , Matriz Extracelular/fisiología , Humanos , Microscopía Confocal , Modelos Biológicos , Células Tumorales Cultivadas
17.
Eur J Cell Biol ; 85(12): 1217-31, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17010475

RESUMEN

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.


Asunto(s)
Actinas/metabolismo , Extensiones de la Superficie Celular/fisiología , Matriz Extracelular/metabolismo , Actinas/ultraestructura , Comunicación Celular/fisiología , Línea Celular Tumoral , Estructuras de la Membrana Celular/fisiología , Estructuras de la Membrana Celular/ultraestructura , Extensiones de la Superficie Celular/ultraestructura , Matriz Extracelular/ultraestructura , Humanos , Melanoma/fisiopatología , Invasividad Neoplásica/fisiopatología , Neoplasias Cutáneas/fisiopatología
18.
Reprod Toxicol ; 21(2): 216-9, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16213123

RESUMEN

Mancozeb is known to alter reproductive performance in exposed animals, but its specific mechanism of action is still unclear. We investigated whether in female mice of the F1 generation, mancozeb could affect oocyte ability to undergo complete meiotic maturation and fertilization. Female mice were treated with 50 and 500 mg/kg of mancozeb (or vehicle in the controls) from gestational day 2 to postnatal day 20. Results demonstrated that only at the highest dose, mancozeb induced a significant decrease in the number of ovulated eggs. Moreover, at this dose mancozeb caused a significant decrease of fertilizability related to a reduction of the formation of male and female pronuclei.


Asunto(s)
Fertilización In Vitro/efectos de los fármacos , Fungicidas Industriales/toxicidad , Maneb/toxicidad , Oocitos/efectos de los fármacos , Zineb/toxicidad , Animales , Femenino , Masculino , Meiosis/efectos de los fármacos , Ratones , Espermatozoides/efectos de los fármacos
19.
Eur J Cancer ; 41(3): 470-6, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15691648

RESUMEN

The naturally occurring phosphoinositide metabolite, glycerophosphoinositol 4-phosphate, has recently been shown to induce rearrangements in the actin cytoskeleton through modulation of the small GTPases, Rac and Rho. Since this is directly linked to cell spreading and remodelling, we have evaluated the potential role of glycerophosphoinositol 4-phosphate and related metabolites in tumour cell invasion. The biological effects of these compounds were tested in a number of cellular activities related to cell spreading, including cell migration and cell invasion. We find that unlike other inositol-containing molecules, such as the inositol phosphates, glycerophosphoinositol and glycerophosphoinositol 4-phosphate prevent the invasion of epithelium-derived MDA-MB-231 breast carcinoma and A375MM melanoma cell lines through the extracellular matrix; this is due to a decreased ability to degrade matrix components. These data identify a specific activity of the glycerophosphoinositols that can be exploited for their development as novel anti-invasive drugs.


Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Matriz Extracelular/patología , Fosfatos de Inositol/uso terapéutico , Melanoma/patología , Actinas/química , Neoplasias de la Mama/patología , Quimiotaxis/efectos de los fármacos , Citoesqueleto/química , Femenino , Humanos , Metaloproteinasas de la Matriz/metabolismo , Invasividad Neoplásica/prevención & control
20.
Front Biosci ; 9: 1944-53, 2004 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-14977600

RESUMEN

Cells make contact with the extracellular matrix (ECM) through extensions of the plasma membrane; these range from irregular dynamic structures, e.g. lamellipodia, ruffles and pseudopodia, to more localized and highly defined protrusions, e.g. podosomes and invadopodia. Both might be instruments through which cells sample the immediate extracellular environment and maintain polarized activities such as chemotaxis and focal degradation of the matrix. Podosomes are expressed in cells of the monocytic lineage, and most studies point to a role for podosomes in adhesion/motility. Invadopodia are prominent in certain aggressive cancer cells (or transformed cells) and appear to be directly responsible for focal ECM degradation. Recent studies have revived interest in these structures in terms of the actin regulation machinery. Within this framework, the atypical GTP-binding protein dynamin, a central modulator of protrusive events, has been associated to podosome and invadopodia structure and function. Here, we specifically discuss the role played by dynamin in controlling the activities and function of these structures.


Asunto(s)
Estructuras de la Membrana Celular/fisiología , Dinaminas/fisiología , Animales , Adhesión Celular , Estructuras de la Membrana Celular/metabolismo , Movimiento Celular , Humanos , Proteínas de Microfilamentos/metabolismo , Monocitos/fisiología , Monocitos/ultraestructura , Invasividad Neoplásica , Neoplasias/ultraestructura , Osteoclastos/fisiología , Osteoclastos/ultraestructura
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