ABSTRACT
Immune cells are especially dependent on the proper functioning of the actin cytoskeleton, and both innate and adaptive responses rely on it. Leukocytes need to adhere not only to substrates but also to cells in order to form synapses that pass on instructions or kill infected cells. Neutrophils literally squeeze their cell body during blood extravasation and efficiently migrate to the inflammatory focus. Moreover, the development of immune cells requires the remodeling of their cytoskeleton as it depends on, among other processes, adhesive contacts and migration. In recent years, the number of reports describing cytoskeletal defects that compromise the immune system has increased immensely. Furthermore, a new emerging paradigm points toward a role for the cellular actin content as an essential component of the so-called homeostasis-altering molecular processes that induce the activation of innate immune signaling pathways. Here, we review the role of critical actin-cytoskeleton-remodeling proteins, including the Arp2/3 complex, cofilin, coronin and WD40-repeat containing protein 1 (WDR1), in immune pathophysiology, with a special focus on autoimmune and autoinflammatory traits.
Subject(s)
Cytoskeletal Proteins , Immune System Diseases , Actin Cytoskeleton , Actin Depolymerizing Factors , Actins , HumansABSTRACT
Diarrheic diseases account for the annual death of approximately 1.9 million children under the age of 5 years, and it is a major cause of work absenteeism in developed countries. As diarrheagenic bacteria, enteropathogenic Escherichia coli (EPEC) attach to cells in the small intestine, causing local disappearance of microvilli and inducing the formation of actin-rich pedestals that disrupt the intestinal barrier and help EPEC adhere to and infect intestinal cells. Antibiotics and other bioactive compounds can often be found by analyzing traditional medicines. Here a crude aqueous extract of Hibiscus sabdariffa, which typically grows in subtropical and tropical areas and is a popular medicinal tisane in many countries, was analyzed for antibacterial activity against EPEC. In standard microdilution assays, the extract showed a minimum inhibitory concentration of 6.5 mg/ml against EPEC growth. Time-kill kinetics assays demonstrated significant 24 h bactericidal activity at 25 mg/ml. The extract is able to impede pedestal induction. Not only did the extract inhibit preformed pedestals but it prevented pedestal induction as well. Remarkably, it also promoted the formation of EPEC filaments, as observed with other antibiotics. Our results in vitro support the potential of Hibiscus sabdariffa as an antimicrobial agent against EPEC.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enterotoxigenic Escherichia coli/growth & development , Enterotoxigenic Escherichia coli/ultrastructure , Hibiscus/chemistry , Plant Extracts/pharmacology , Anti-Bacterial Agents/chemistry , Plant Extracts/chemistryABSTRACT
Autoinflammatory diseases are hyperinflammatory, immune dysregulatory diseases caused by innate immune cells dysregulation that present typically in the perinatal period with systemic and organ-targeted inflammation, but with improved genetic testing and the development of diagnostic criteria, milder and later-onset forms are being detected in adulthood. While the discovery of gain-of-function mutations in innate sensors linked to the production of proinflammatory cytokines provided the bases for anti-cytokine therapies that changed disease and patient outcomes, the field is expanding with the increasing discovery of disease-causing loss-of-function mutations in genes with cellular house-keeping functions that affect cell homeostasis and when dysregulated trigger innate inflammatory pathways. This review focuses on updates on molecular pathways and diseases that cause predominantly IL-1ß and Type-I IFN-mediated autoinflammatory diseases.
Subject(s)
Autoimmune Diseases/immunology , Inflammation/immunology , Animals , Autoimmune Diseases/pathology , Humans , Immunity, Innate/immunology , Inflammation/pathology , Interferon Type I/immunologyABSTRACT
Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells.
ABSTRACT
BACKGROUND: HLA-DMB proteins are important for intracellular microbial metabolism in order other major histocompatibility complex (MHC) molecules present peptides to lymphocytes. In addition, HLA-DMB alleles have been found linked to diseases in some ethnic groups and HLA-DMB molecules may be important to explain HLA disease association. OBJECTIVE: To detect HLA-DMB alleles profile in Amerindians for the first time and compare them to other populations. This will establish the bases to study HLA-DMB linkage to disease in Amerindians. METHOD: A group of 168 voluntary Amerindians have been typed for HLA-DMB alleles. They have been characterized both, by genetic and genealogical bases. Cloning and automated HLA-DMB DNA (exons 2, 3 and 4) sequencing have been performed for allele assignation. RESULTS: HLA-DMB*01:01:01 and HLA-DMB*01:03:01 show the highest frequencies. These have been compared to other World wide populations. HLA-DMB*01:03:01 is tightly associated to certain specific HLA-DRB1 alleles in Amerindians. CONCLUSION: The specific Amerindian HLA-DMB allele frequencies and their linkage disequilibrium with other MHC alleles may be crucial to determine HLA-DMB World wide variation, evolution and specific linkage to disease in Amerindians and other populations.
Subject(s)
Alleles , HLA-D Antigens/genetics , HLA-DRB1 Chains/genetics , Indians, Central American , Indians, North American , Indians, South American , Linkage Disequilibrium , Evolution, Molecular , Gene Frequency , Genealogy and Heraldry , Genetic Predisposition to Disease , Genetics, Population , Histocompatibility Testing , Humans , Polymorphism, GeneticABSTRACT
HLA-G polymorphism has been found to be relatively low in all world populations. In the present paper two new HLA-G molecules are described in ancient American natives. A new HLA-G molecule from a Ecuador Amerindian individual (male) showed four codon changes with respect to HLA-G*01:01:01. Silent changes at α1 domain (residue 57, Pro, CCGâCCA) and α2 domain (residue 93, His, CACâCAT and residue 100, Gly, GGCâGGT) and one productive change in α3 domain (residue 219 changed from Arg to Trp). This α3 change may dramatically alter HLA-G interactions with beta-2 microglobulin, CD8, ILT-2 and ILT-4 ligands present in subsets of T, B, NK, monocytes, macrophages and dentritic cells. Another HLA-G new molecule was found in a woman from Hispaniola Island, Dominican Republic (Sto Domingo): it presented a silent change at α2 domain residue 107, Gly, GGAâGGT and non-silent change at residue 178, MetâThr (with respect to HLA-G*01:01:01) which is close to class I molecule/clonotypic T cell receptor interaction sites. Functional implications of these findings are discussed.
Subject(s)
HLA-G Antigens/genetics , Indians, Central American , Indians, South American , Alleles , Caribbean Region , Dominican Republic , Ecuador , Female , Genetics, Population , Histocompatibility Testing , Humans , Immunity/genetics , Male , Mutation/genetics , Polymorphism, GeneticABSTRACT
Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.
Subject(s)
Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Proteins/metabolism , Oncogene Proteins/metabolism , Receptors, Cell Surface/metabolism , Wiskott-Aldrich Syndrome Protein/metabolism , Adhesins, Bacterial/metabolism , Animals , Bacterial Adhesion , Fibroblasts/metabolism , Fibroblasts/microbiology , Gene Knockout Techniques , HeLa Cells , Humans , Hydroxamic Acids/pharmacology , Mice , Phosphorylation , Protein TransportABSTRACT
The Crk adaptor family of proteins comprises the alternatively spliced CrkI and CrkII isoforms, as well as the paralog Crk-like (CrkL) protein, which is encoded by a different gene. Initially thought to be involved in signaling during apoptosis and cell adhesion, this ubiquitously expressed family of proteins is now known to play essential roles in integrating signals from a wide range of stimuli. In this review, we describe the structure and function of the different Crk proteins. We then focus on the emerging roles of Crk adaptors during Enterobacteriaceae pathogenesis, with special emphasis on the important human pathogens Salmonella, Shigella, Yersinia, and enteropathogenic Escherichia coli. Throughout, we remark on opportunities for future research into this intriguing family of proteins.
Subject(s)
Cell Adhesion/genetics , Host-Pathogen Interactions/genetics , Proto-Oncogene Proteins c-crk/genetics , Alternative Splicing/genetics , Enteropathogenic Escherichia coli/genetics , Gene Expression Regulation , Humans , Phosphorylation , Protein Isoforms/genetics , Proto-Oncogene Proteins c-crk/biosynthesis , Proto-Oncogene Proteins c-crk/chemistry , Proto-Oncogene Proteins c-crk/metabolism , Signal Transduction/genetics , Structure-Activity RelationshipABSTRACT
The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.
Subject(s)
Actins/metabolism , Chromaffin Cells/metabolism , src-Family Kinases/metabolism , Actin Cytoskeleton/drug effects , Animals , Calcium/pharmacology , Cattle , Cells, Cultured , Chromaffin Cells/cytology , Chromaffin Cells/drug effects , Cytochalasin D/pharmacology , Exocytosis/drug effects , Kinetics , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , src-Family Kinases/chemistry , src-Family Kinases/geneticsABSTRACT
Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.
Subject(s)
Actins/metabolism , Escherichia coli Infections/metabolism , Escherichia coli Infections/pathology , Escherichia coli Proteins/metabolism , Oncogene Protein v-crk/metabolism , Receptors, Cell Surface/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Electroporation , Enteropathogenic Escherichia coli/metabolism , HeLa Cells , Humans , Microscopy, Fluorescence , Oncogene Proteins/metabolism , Protein Isoforms/metabolism , RNA, Small Interfering , TransfectionABSTRACT
BACKGROUND: Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. Coxiella burnetii, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood. METHODOLOGY/PRINCIPAL FINDING: Here we analyzed the role of different cortactin motifs in the internalization of C. burnetii by non-phagocytic cells. C. burnetii internalization into HeLa cells was significantly reduced when the cells expressed GFP-cortactin W525K, which carries a mutation in the SH3 domain that renders the protein unable to bind targets such as N-WASP. However, internalization was unaffected when the cells expressed the W22A mutant, which has a mutation in the N-terminal acidic region that destroys the protein's ability to bind and activate Arp2/3. We also determined whether the phosphorylation status of cortactin is important for internalization. Expression of GFP-cortactin 3F, which lacks phosphorylatable tyrosines, significantly increased internalization of C. burnetii, while expression of GFP-cortactin 3D, a phosphotyrosine mimic, did not affect it. In contrast, expression of GFP-cortactin 2A, which lacks phosphorylatable serines, inhibited C. burnetii internalization, while expression of GFP-cortactin SD, a phosphoserine mimic, did not affect it. Interestingly, inhibitors of Src kinase and the MEK-ERK kinase pathway blocked internalization. In fact, both kinases reached maximal activity at 15 min of C. burnetii infection, after which activity decreased to basal levels. Despite the decrease in kinase activity, cortactin phosphorylation at Tyr421 reached a peak at 1 h of infection. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the SH3 domain of cortactin is implicated in C. burnetii entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation at tyrosine favor C. burnetii internalization. We present evidence that ERK and Src kinases play a role early in infection by this pathogen.
Subject(s)
Actin Cytoskeleton/metabolism , Cortactin/metabolism , Coxiella burnetii/metabolism , Q Fever/microbiology , Actin-Related Protein 2-3 Complex/metabolism , Endocytosis , Extracellular Signal-Regulated MAP Kinases/metabolism , HeLa Cells , Humans , Phagocytes/metabolism , Phosphorylation , Q Fever/metabolism , src-Family Kinases/metabolismABSTRACT
BACKGROUND: Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. CONCLUSIONS/SIGNIFICANCE: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.
Subject(s)
Cortactin/metabolism , Tyrosine/metabolism , Acetylation , Animals , Cell Line , Cortactin/genetics , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Histone Deacetylases/deficiency , Humans , Mice , Phosphorylation , Protein Binding , Protein Transport/genetics , Substrate Specificity , Transfection , src-Family Kinases/metabolismABSTRACT
Cell migration and invasion require the coordinated regulation of cytoskeletal architectural changes by signaling factors, including the actin-binding protein cortactin. Bacterial and viral pathogens subvert these signaling factors to promote their uptake, spread and dissemination. We show that the gastric pathogen Helicobacter pylori (Hp) targets cortactin by two independent processes leading to its tyrosine dephosphorylation and serine phosphorylation to regulate cell scattering and elongation. The phosphorylation status of cortactin dictates its subcellular localization and signaling partners. Upon infection, cortactin was found to interact with and stimulate the kinase activity of focal adhesion kinase (FAK). This interaction required the SH3 domain and phosphorylation of cortactin at serine 405 and a proline-rich sequence in FAK. Using Hp as a model, this study unravels a previously unrecognized FAK activation pathway. We propose that Hp targets cortactin to protect the gastric epithelium from excessive cell lifting and ensure sustained infection in the stomach.
Subject(s)
Cell Movement , Cortactin/metabolism , Focal Adhesion Kinase 1/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori/physiology , Serine/metabolism , Animals , Cell Line, Tumor , Cortactin/genetics , Focal Adhesion Kinase 1/chemistry , Focal Adhesion Kinase 1/genetics , Helicobacter Infections/enzymology , Helicobacter Infections/microbiology , Helicobacter Infections/physiopathology , Helicobacter pylori/genetics , Humans , Mice , Mice, Knockout , Phosphorylation , Protein Structure, Tertiary , Serine/genetics , Signal TransductionABSTRACT
Many Gram-negative pathogenic bacteria possess type-III or type-IV secretion systems to inject 'effector' proteins directly into host cells to modulate cellular processes in their favour. A common target is the actin-cytoskeleton linked to the delivery of a single (CagA) effector by Helicobacter pylori and multiple effectors by enteropathogenic Escherichia coli (EPEC) respectively. Here we report co-infection as a powerful strategy for defining effector protein function and mechanisms by which they modulate cellular responses. This is exemplified by our finding that EPEC inhibits H. pylori-induced AGS cell elongation, a disease-related event linked to Rac1 activation. While this inhibitory process is dependent on the translocated Intimin receptor, Tir, and the outer-membrane protein, Intimin, it unexpectedly revealed evidence for Tir signalling independent of Intimin interaction and tyrosine phosphorylation of Tir. Furthermore, the work defined a long awaited role for protein kinase A (PKA)-mediated phosphorylation of Tir at serine-434 and serine-463. Our data are consistent with a model whereby EPEC activates PKA for Tir phosphorylation. Activated PKA then phosphorylates Rac1 at serine-71 associated with reduced GTP-load and inhibited cell elongation. Thus, the co-infection approach is a powerful strategy for defining novel effector function with important implications for characterizing mechanisms and regulatory signalling pathways in bacterial pathogenesis.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Enteropathogenic Escherichia coli/metabolism , Escherichia coli Infections/complications , Escherichia coli Proteins/metabolism , Helicobacter Infections/complications , Receptors, Cell Surface/metabolism , Serine/metabolism , rac1 GTP-Binding Protein/metabolism , Adhesins, Bacterial/metabolism , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Cell Line, Tumor , Cytoskeleton/metabolism , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Proteins/chemistry , Helicobacter pylori/metabolism , Helicobacter pylori/pathogenicity , Host-Pathogen Interactions , Humans , Phosphorylation , Receptors, Cell Surface/chemistry , Serine/chemistry , VirulenceABSTRACT
BACKGROUND: Cortactin activates the actin-related 2/3 (Arp2/3) complex promoting actin polymerization to remodel cell architecture in multiple processes (e.g. cell migration, membrane trafficking, invadopodia formation etc.). Moreover, it was called the Achilles' heel of the actin cytoskeleton because many pathogens hijack signals that converge on this oncogenic scaffolding protein. Cortactin is able to modulate N-WASP activation in vitro in a phosphorylation-dependent fashion. Thus Erk-phosphorylated cortactin is efficient in activating N-WASP through its SH3 domain, while Src-phosphorylated cortactin is not. This could represent a switch on/off mechanism controlling the coordinated action of both nucleator promoting factors (NPFs). Pedestal formation by enteropathogenic Escherichia coli (EPEC) requires N-WASP activation. N-WASP is recruited by the cell adapter Nck which binds a major tyrosine-phosphorylated site of a bacterial injected effector, Tir (translocated intimin receptor). Tir-Nck-N-WASP axis defines the current major pathway to actin polymerization on pedestals. In addition, it was recently reported that EPEC induces tyrosine phosphorylation of cortactin. RESULTS: Here we demonstrate that cortactin phosphorylation is absent on N-WASP deficient cells, but is recovered by re-expression of N-WASP. We used purified recombinant cortactin and Tir proteins to demonstrate a direct interaction of both that promoted Arp2/3 complex-mediated actin polymerization in vitro, independently of cortactin phosphorylation. CONCLUSION: We propose that cortactin binds Tir through its N-terminal part in a tyrosine and serine phosphorylation independent manner while SH3 domain binding and activation of N-WASP is regulated by tyrosine and serine mediated phosphorylation of cortactin. Therefore cortactin could act on Tir-Nck-N-WASP pathway and control a possible cycling activity of N-WASP underlying pedestal formation.
ABSTRACT
Mother-to-child transmission during pregnancy provides a unique system for studying the correlation between HLA phenotype and susceptibility to HIV infection. We studied this relationship in a Spanish cohort. We determined frequencies of HLA class I and II alleles in 120 infants born to HIV-infected mothers and 67 HIV-infected mothers. Although there was no statistical difference in the frequency of HLA-B35 between transmitting and non-transmitting mothers, the allele was more frequent in infected children than in uninfected children. HLA-B35 has been consistently reported as a risk factor in the progression to AIDS. In addition, it has been proposed that whether a given allele can confer susceptibility to, or protection against, progression depends on maternal versus paternal inheritance patterns, since the child inherits a virus that reflects the history of CTL encounters of the mother. Our results on vertical HIV transmission combine for the first time the 'HLA-B35 disadvantage' and the 'pattern of inheritance' theories.
Subject(s)
HIV Infections/transmission , HLA-B35 Antigen/genetics , HLA-C Antigens/genetics , Infectious Disease Transmission, Vertical , Adult , Cohort Studies , Disease Susceptibility , Female , Gene Frequency , Humans , Infant, Newborn , Risk Factors , Spain , Young AdultABSTRACT
The Arp2/3 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP family members and by the acidic N-terminal and F-actin-binding repeat region of cortactin, which possesses a C-terminal SH3 domain. Cortactin is a target for phosphorylation by Src tyrosine kinases and by serine/threonine kinases that include Erk. Here we demonstrate that cortactin binds N-WASP and WASP via its SH3 domain, induces in vitro N-WASP-mediated actin polymerization, and colocalizes with N-WASP and WASP at sites of active actin polymerization. Erk phosphorylation and a mimicking S405,418D double mutation enhanced cortactin binding and activation of N-WASP. In contrast, Src phosphorylation inhibited the ability of cortactin previously phosphorylated by Erk, and that of S405,418D double mutant cortactin, to bind and activate N-WASP. Furthermore, Y-->D mutation of three tyrosine residues targeted by Src (Y421, Y466, and Y482) inhibited the ability of S405,418D cortactin to activate N-WASP. We propose that Erk phosphorylation liberates the SH3 domain of cortactin from intramolecular interactions with proline-rich regions, causing it to synergize with WASP and N-WASP in activating the Arp2/3 complex, and that Src phosphorylation terminates cortactin activation of N-WASP and WASP.
Subject(s)
Microfilament Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Nerve Tissue Proteins/metabolism , src-Family Kinases/metabolism , Actin-Related Protein 2 , Actin-Related Protein 3 , Actins/metabolism , Animals , Binding Sites , Cell Line , Cortactin , Cytoskeletal Proteins/metabolism , Humans , Jurkat Cells , Mice , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Mutagenesis, Site-Directed , Nerve Tissue Proteins/genetics , Phosphorylation , Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Swiss 3T3 Cells , Wiskott-Aldrich Syndrome Protein , Wiskott-Aldrich Syndrome Protein, Neuronal , src Homology DomainsABSTRACT
The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.