RESUMO
Viral genomes are packaged into "procapsids" by powerful molecular motors. We report the crystal structure of the DNA packaging motor protein, gene product 17 (gp17), in bacteriophage T4. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. We show that another function of the C-terminal domain is to translocate the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a "tensed state." A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 shows that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a "relaxed state." These structures suggest a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors.
Assuntos
Bacteriófago T4/metabolismo , Empacotamento do DNA , Proteínas Virais/química , Proteínas Virais/metabolismo , Montagem de Vírus , Cristalografia por Raios X , Modelos Moleculares , Eletricidade EstáticaRESUMO
DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.
Assuntos
Adenosina Trifosfatases/metabolismo , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Proteínas do Capsídeo/metabolismo , Empacotamento do DNA , DNA Viral/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacteriófago T4/química , Sítios de Ligação , Transporte Biológico , Endopeptidases/metabolismo , Ordem dos Genes , Sequências Hélice-Alça-Hélice , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Proteínas do Core Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Persistent fetal vasculature (PFV) occurs as a result of a failure of fetal vasculature to undergo normal programmed involution. During development, before the formation of retinal vessels, the lens and the inner retina are nourished by the hyaloid vasculature. Hyaloid vessels extend from the optic nerve and run through the vitreous to encapsulate the lens. As fetal retinal vessels develop, hyaloid vasculature naturally regresses. Failure of regression of the hyaloid artery has been shown to lead to severe congenital pathologies. Studies on childhood blindness and visual impairment in the United States have shown that PFV accounts for 4.8% of total blindness. Although PFV is a serious developmental disease affecting the normal visual development pathway, the exact regulatory mechanism responsible for the regression of the hyaloid artery is still unknown. In this review, we have summarized the cellular defects associated with different knockout models that manifest features of persistent fetal vasculature. Based on similar cellular defects observed in different knockouts (KO)s such as altered migration, increased proliferation and decreased apoptosis and, the known role of integrins in the regulation of these cellular behaviors, we propose here that integrins may play a significant role in the pathophysiology of persistent fetal vasculature disease.
Assuntos
Modelos Animais de Doenças , Feto/irrigação sanguínea , Integrinas/fisiologia , Vítreo Primário Hiperplásico Persistente/fisiopatologia , Animais , Proteínas da Matriz Extracelular/fisiologia , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
ßA3/A1-crystallin is an abundant structural protein of the lens that is very critical for lens function. Many different genetic mutations have been shown to associate with different types of cataracts in humans and in animal models. ßA3/A1-crystallin has four Greek key-motifs that organize into two crystallin domains. It shown to bind calcium with moderate affinity and has putative calcium-binding site. Other than in the lens, ßA3/A1 is also expressed in retinal astrocytes, retinal pigment epithelial (RPE) cells, and retinal ganglion cells. The function of ßA3/A1-crystallin in the retinal cell types is well studied; however, a clear understanding of the function of this protein in the lens has not yet been established. In the current study, we generated the ßA3/A1-crystallin knockout (KO) mouse and explored the function of ßA3/A1-crystallin in lens development. Our results showed that ßA3-KO mice develop congenital nuclear cataract and exhibit persistent fetal vasculature condition. At the cellular level KO lenses show defective lysosomal clearance and accumulation of nuclei, mitochondria, and autophagic cargo in the outer cortical region of the lens. In addition, the calcium level and the expression and activity of calpain-3 were increased in KO lenses. Taken together, these results suggest the lack of ßA3-crystallin function in lenses, alters calcium homeostasis which in turn causes lysosomal defects and calpain activation. These defects are responsible for the development of nuclear cataract in KO lenses.
Assuntos
Calpaína/metabolismo , Catarata/genética , Cristalinas/genética , Cristalino/patologia , Lisossomos/metabolismo , Alelos , Motivos de Aminoácidos , Animais , Autofagia , Sítios de Ligação , Cálcio/metabolismo , Catarata/metabolismo , Núcleo Celular/metabolismo , Cristalinas/metabolismo , Citoplasma/metabolismo , Feminino , Regulação da Expressão Gênica , Heterozigoto , Homeostase , Imuno-Histoquímica , Cristalino/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia , Mitocôndrias/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Cadeia A de beta-CristalinaRESUMO
Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 µL and (iv) 200 µL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.
Assuntos
Colágeno/metabolismo , Meios de Cultura Livres de Soro/química , Técnicas de Cultura de Órgãos/instrumentação , Esclera/citologia , Animais , Reatores Biológicos , Sobrevivência Celular , Miopia/metabolismo , Refração Ocular , Esclera/metabolismo , TupaiidaeRESUMO
Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and ß-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with ßA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with ßA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for ßA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for ßA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with ßA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ßA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with ßA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor ßA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.
Assuntos
Amidas/metabolismo , Cristalinas/metabolismo , Processamento de Proteína Pós-Traducional , Cadeia B de alfa-Cristalina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalinas/química , Cristalinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cristalino/química , Cristalino/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/genética , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/genéticaRESUMO
PURPOSE: The CRYAAN101D transgenic mouse model expressing deamidated αA-crystallin (deamidation at N101 position to D) develops cortical cataract at the age of 7 to 9 months. The present study was carried out to explore the molecular mechanism that leads to the development of cortical opacity in CRYAAN101D lenses. METHODS: RNA sequence analysis was carried out on 2- and 4-month-old αA-N101D and wild type (WT) lenses. To understand the biologic relevance and function of significantly altered genes, Ingenuity Pathway Analysis (IPA) was done. To elucidate terminal differentiation defects, immunohistochemical, and Western blot analyses were carried out. RESULTS: RNA sequence and IPA data suggested that the genes belonging to gene expression, cellular assembly and organization, and cell cycle and apoptosis networks were altered in N101D lenses. In addition, the tight junction signaling and Rho A signaling were among the top three canonical pathways that were affected in N101D mutant. Immunohistochemical analysis identified a series of terminal differentiation defects in N101D lenses, specifically, increased proliferation and decreased differentiation of lens epithelial cells (LEC) and decreased denucleation of lens fiber cells (LFC). The expression of Rho A was reduced in different-aged N101D lenses, and, conversely, Cdc42 and Rac1 expressions were increased in the N101D mutants. Moreover, earlier in development, the expression of major membrane-bound molecular transporter Na,K-ATPase was drastically reduced in N101D lenses. CONCLUSIONS: The results suggest that the terminal differentiation defects, specifically, increased proliferation and decreased denucleation are responsible for the development of lens opacity in N101D lenses.
Assuntos
Catarata/genética , Regulação da Expressão Gênica , Córtex do Cristalino/metabolismo , RNA Mensageiro/genética , Cadeia A de alfa-Cristalina/genética , Animais , Western Blotting , Catarata/metabolismo , Catarata/patologia , Proliferação de Células , Modelos Animais de Doenças , Imuno-Histoquímica , Córtex do Cristalino/patologia , Camundongos , Camundongos Transgênicos , Cadeia A de alfa-Cristalina/biossínteseRESUMO
La7, an immunogenic outer membrane lipoprotein of Borrelia burgdorferi, produced during infection, has been shown to play a redundant role in mammalian infectivity. Here we show that La7 facilitates pathogen survival in all tested phases of the vector-specific spirochete life cycle, including tick-to-host transmission. Unlike wild type or la7-complemented isolates, isogenic La7-deficient spirochetes are severely impaired in their ability to persist within feeding ticks during acquisition from mice, in quiescent ticks during larval-nymphal inter-molt, and in subsequent pathogen transmission from ticks to naïve hosts. Analysis of gene expression during the major stages of the tick-rodent infection cycle showed increased expression of la7 in the vector and a swift downregulation in the mammalian hosts. Co-immunoprecipitation studies coupled with liquid chromatography-mass spectrometry analysis further suggested that La7, a highly conserved and abundant inner membrane protein, is involved in protein-protein interaction with a discrete set of borrelial ligands although biological significance of such interactions remains unclear. Further characterization of vector-induced membrane antigens like La7 and its interacting partners will likely aid in our understanding of the molecular details of B. burgdorferi persistence and transmission through a complex enzootic cycle.