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2.
BMC Infect Dis ; 23(1): 747, 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37907849

RESUMO

BACKGROUND: While existing evidence suggests less severe clinical manifestations and lower mortality are associated with the Omicron variant as compared to the Delta variant. However, these studies fail to control for differences in health systems facilities and providers. By comparing patients hospitalized on a single medical service during the Delta and Omicron surges we were able to conduct a more accurate comparison of the two varaints' clinical manifestations and outcomes. METHODS: We conducted a prospective study of 364 Omicron (BA.1) infected patients on a single hospitalist service and compared these findings to a retrospective analysis of 241 Delta variant infected patients managed on the same service. We examined differences in symptoms, laboratory measures, and clinical severity between the two variants and assessed potential risk drivers for case mortality. FINDINGS: Patients infected with Omicron were older and had more underlying medical conditions increasing their risk of death. Although they were less severely ill and required less supplemental oxygen and dexamethasone, in-hospital mortality was similar to Delta cases, 7.14% vs. 4.98% for Delta (q-value = 0.38). Patients older than 60 years or with immunocompromised conditions had much higher risk of death during hospitalization, with estimated odds ratios of 17.46 (95% CI: 5.05, 110.51) and 2.80 (1.03, 7.08) respectively. Neither vaccine history nor variant type played a significant role in case fatality. The Rothman score, NEWS-2 score, level of neutrophils, level of care, age, and creatinine level at admission were highly predictive of in-hospital death. INTERPRETATION: In hospitalized patients, the Omicron variant is less virulent than the Delta variant but is associated with a comparable mortality. Clinical and laboratory features at admission are informative about the risk of death.


Assuntos
COVID-19 , Médicos Hospitalares , Humanos , Mortalidade Hospitalar , Estudos Prospectivos , Estudos Retrospectivos , SARS-CoV-2
3.
Nat Cell Biol ; 3(10): 897-904, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11584271

RESUMO

The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.


Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Desenvolvimento Embrionário e Fetal , Proteínas do Tecido Nervoso/fisiologia , Animais , Linhagem Celular , Linhagem Celular Transformada , Fibroblastos , Marcação de Genes , Listeria/fisiologia , Camundongos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Recombinação Genética , Shigella flexneri/fisiologia , Vaccinia virus/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich
4.
J Cell Biol ; 154(4): 775-84, 2001 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-11514591

RESUMO

Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a null Capg allele engineered into mice. Both CapG-null mice and CapG/gelsolin double-null mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG-null macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin-null macrophages and no additive effects are observed in CapG/gelsolin double-null macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.


Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Gelsolina/genética , Macrófagos/fisiologia , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Membranas Intracelulares/fisiologia , Macrófagos/citologia , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Neutrófilos/citologia , Neutrófilos/fisiologia , Fagocitose/fisiologia
5.
Obstet Gynecol ; 98(6): 1140-2, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11755567

RESUMO

An academic physician gives a personal account of his wife's progressive deterioration during her hospitalization at a university medical center. He encounters physicians who are too distracted by other academic pursuits to care adequately for his wife. Her hospital course is complicated by bilateral pulmonary emboli which occur during inadequate heparin therapy and are followed by myocardial infarction, shock, and the adult respiratory distress syndrome. His experience exemplifies the importance of closely supervised care. He calls on the leaders of academic medical centers to make excellence in patient care a top priority and recommends that clinical as well as research skills be rewarded.


Assuntos
Cuidados Críticos , Hospitais Universitários/normas , Corpo Clínico Hospitalar , Qualidade da Assistência à Saúde , Carga de Trabalho , Anedotas como Assunto , Feminino , Florida , História do Século XX , Humanos , Recursos Humanos
7.
Cell Motil Cytoskeleton ; 45(4): 272-8, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10744860

RESUMO

Mounting evidence supports the role of truncated vinculin in the intracellular actin-based motility of Shigella flexneri. Vinculin's role was recently questioned by Goldberg [1997: Cell Motil Cytoskeleton 37:44-53] who observed Shigella motility in mouse embryonal carcinoma 5.51 cells, a genetically modified cell line that reputedly lacked vinculin. That challenge implicitly relied on the assumption that 5.51 cells had no detectable vinculin polypeptide and lacked full-length vinculin mRNA. Despite the appearance of being an unambiguous test of vinculin's role in Shigella motility, 5.51 cells were shown to contain adequate amounts of truncated vinculin (as well as the corresponding mRNA transcript) to support bacterial locomotion. We also examined Shigella locomotion in gamma229 cells, a related embryonal carcinoma cell line containing approximately one-half the vinculin content found in 5.51 cells. We observed that there was a commensurate twofold decrease in the Shigella motility rate, as compared to 5.51 cells; this finding raises the possibility that vinculin can become a rate-limiting factor under some circumstances. Immunofluorescence microscopy using vin 11-5 monoclonal antibody directed against the vinculin head domain showed intense staining of Shigella rocket tails in both gamma229 and 5.51 cells. Our findings clearly demonstrate that motility in 5.51 cells cannot be regarded as a valid criterion for evaluating the role of truncated vinculin in Shigella motility.


Assuntos
Actinas/metabolismo , Shigella flexneri/fisiologia , Vinculina/fisiologia , Animais , Western Blotting , Movimento Celular , Sistema Livre de Células , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Shigella flexneri/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Vinculina/farmacologia
8.
J Biol Chem ; 274(52): 36963-72, 1999 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-10601251

RESUMO

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1. Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin. Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin. Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4. Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4. Actin and ushers actin as a Profilin. Actin complex onto growing barbed filament ends.


Assuntos
Actinas/química , Proteínas Contráteis , Proteínas dos Microfilamentos/farmacologia , Animais , Humanos , Luz , Polímeros/química , Profilinas , Coelhos , Espalhamento de Radiação , Termodinâmica , Timosina/farmacologia
9.
Mol Cell Biol Res Commun ; 1(3): 176-81, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10425223

RESUMO

Actin-Based Motility motifs [ABM-1 sequence = (D/E)FPPPPX(D/E), where X = P or T, and ABM-2 sequence = XPPPPP, where X denotes G, A, L, P, and S] facilitate assembly of an activated motility complex. Potent inhibition of intracellular motility of pathogens by ABM-1 and ABM-2 peptide analogues has served as a criterion for investigating actin-based motility. To assess the specificity of ABM-1 peptide inhibitors, we microinjected proline-rich peptides into Listeria-infected PtK2 host cells. Use of a combinatorial ABM-1 peptide library (empirical formula = D1E2F2P4T1) demonstrated that high-potency inhibition requires a precise sequence, and not merely a particular amino acid composition. Calculated concentrations of specific sequences in this library indicate that the entire (D/E)FPPPPX(D/E) motif is needed to achieve high-affinity inhibition in living cells. The failure of the well known proline-rich SH3 binding antagonists VSL-12 or APP-12 to inhibit Listeria motility also indicates that SH3 interactions are unlikely to control actin-based motility directly.


Assuntos
Actinas/fisiologia , Proteínas de Bactérias/fisiologia , Listeria monocytogenes/citologia , Listeria monocytogenes/fisiologia , Aderência Bacteriana , Peptídeos/fisiologia
11.
Proc Natl Acad Sci U S A ; 95(23): 13917-22, 1998 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-9811901

RESUMO

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 microM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.


Assuntos
Actinas/fisiologia , Vaccinia virus/fisiologia , Animais , Transporte Biológico Ativo , Citoesqueleto/virologia , Células HeLa , Humanos , Fragmentos de Peptídeos , Coelhos , Replicação Viral
12.
Infect Immun ; 66(8): 3775-82, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9673261

RESUMO

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Gelsolina/metabolismo , Listeria monocytogenes/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Células 3T3 , Animais , Anticorpos/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Gelsolina/genética , Gelsolina/imunologia , Humanos , Camundongos , Proteínas dos Microfilamentos/imunologia , Microinjeções , Proteínas Nucleares/imunologia , Coelhos , Transfecção
14.
J Cell Biol ; 138(6): 1255-64, 1997 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-9298981

RESUMO

To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotein (VASP) (Brindle, N.P.J., M. R. Hold, J.E. Davies, C.J. Price, and D.R. Critchley. 1996. Biochem. J. 318:753-757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.


Assuntos
Proteínas de Bactérias/metabolismo , Movimento Celular/fisiologia , Disenteria Bacilar/microbiologia , Proteínas de Membrana/metabolismo , Shigella flexneri/citologia , Vinculina/metabolismo , Actinas/fisiologia , Animais , Especificidade de Anticorpos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Plaquetas/química , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Reações Cruzadas , Disenteria Bacilar/metabolismo , Imunofluorescência , Humanos , Rim/citologia , Macropodidae , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Proteínas dos Microfilamentos/metabolismo , Microinjeções , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Fosfoproteínas/metabolismo , Prolina/metabolismo , Shigella flexneri/química , Vinculina/química , Vinculina/farmacologia
15.
Biochemistry ; 36(27): 8384-92, 1997 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-9204886

RESUMO

Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.


Assuntos
Actinas/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Listeria monocytogenes/fisiologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Actinas/farmacologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas Contráteis , Humanos , Proteínas dos Microfilamentos/química , Movimento/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Cloreto de Potássio/farmacologia , Profilinas , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência
16.
Biochem Biophys Res Commun ; 231(3): 686-91, 1997 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-9070872

RESUMO

Actin-based motility involves a cascade of binding interactions designed to assemble actin regulatory proteins into functional locomotory units. Listeria ActA surface protein contains a series of nearly identical EFPPPPTDE-type oligoproline sequences for binding vasodilator-stimulated phosphoprotein (VASP). The latter is a tetrameric protein with numerous GPP-PPP docking sites for profilin, a 15 kDa regulatory protein that promotes actin filament assembly. Analysis of known actin regulatory proteins led to the identification of distinct Actin-Based Motility homology sequences ABM-1; (D/E)FPPPPX(D/E); and ABM-2, XPPPPP (where X denotes G, A, L, and S).


Assuntos
Actinas/química , Proteínas de Bactérias/química , Moléculas de Adesão Celular/química , Proteínas Contráteis , Proteínas de Membrana/química , Proteínas dos Microfilamentos/química , Fosfoproteínas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Consenso , Humanos , Dados de Sequência Molecular , Profilinas , Prolina/química , Ligação Proteica
17.
J Cell Biol ; 133(1): 49-59, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8601612

RESUMO

The gram negative rod Shigella flexneri uses it surface protein IcsA to induce host cell actin assembly and to achieve intracellular motility. Yet, the IcsA protein lacks the oligoproline sequences found in ActA, the surface protein required for locomotion of the gram positive rod Listeria monocytogenes. Microinjection of a peptide matching the second ActA oligoproline repeat (FEFPPPPTDE) stops Listeria locomotion (Southwick, F.S., and D.L. Purich. 1994a. Proc. Natl. Acad. Sci. USA. 91:5168-5172), and submicromolar concentrations (intracellular concentration 80-800 nM) similarly arrest Shigella rocket-tail assembly and intracellular motility. Coinjection of a binary solution containing profilin and the ActA analogue increased the observed rates of intracellular motility by a factor of three (mean velocity 0.90 +/- 0.07 mu m/s, SD n=16 before injection vs 0.3 +/- 0.1 mu m/s, n=33 postinjection, intracellular concentration = 80 nM profilin plus 80 nM ActA analogue). Recent evidence suggests the ActA analogue may act by displacing the profilin-binding protein VASP (Pistor, S.C., T. Chakaborty, V. Walter, and J. Wehland. 1995. Curr. Biol. 5:517-525). At considerably higher intracellular concentrations (10 muM), the VASP oligoproline sequence (GPPPPP)3 thought to represent the profilin-binding site (Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B.M. Jockusch, and U. Walter. 1995. EMBO (Eur. Mol. Biol. Organ.) J. 14:1583-1589) also inhibited Shigella movement. A binary mixture of the VASP analogue and profilin (each 10 muM intracellular concentration) led to a doubling of Shigella intracellular migration velocity (0.09 +/- 0.06 mu m/s, n = 25 preinjection vs 0.18 +/- 0.10 mu m/s, n = 61 postinjection). Thus, the two structurally divergent bacteria, Listeria and Shigella, have adopted convergent mechanisms involving profilin recognition of VASP oligoproline sequences and VASP recognition of oligoproline sequences in ActA or an ActA-like host protein to induce host cell actin assembly and to provide the force for intracellular locomotion and cell-cell spread.


Assuntos
Actinas/metabolismo , Proteínas Contráteis , Proteínas dos Microfilamentos/fisiologia , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Shigella flexneri/fisiologia , Actinina/análise , Actinas/análise , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Moléculas de Adesão Celular/química , Linhagem Celular , Epitélio/microbiologia , Listeria monocytogenes/química , Listeria monocytogenes/citologia , Macropodidae , Proteínas de Membrana/química , Proteínas dos Microfilamentos/farmacologia , Microinjeções , Dados de Sequência Molecular , Movimento , Oligopeptídeos/síntese química , Peptídeos/síntese química , Fosfoproteínas/química , Profilinas , Shigella flexneri/química , Shigella flexneri/citologia
19.
Biochemistry ; 35(11): 3518-24, 1996 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-8639502

RESUMO

In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL(-1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Quimiotaxia de Leucócito , Proteínas do Citoesqueleto , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/isolamento & purificação , Neuropeptídeos , Neutrófilos/química , Fatores de Despolimerização de Actina , Proteína de Capeamento de Actina CapZ , Movimento Celular , Destrina , Gelsolina/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Peso Molecular , Proteínas Musculares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Espectrina/metabolismo
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