RESUMO
Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.
Assuntos
Integrase de HIV , HIV-1 , Integração Viral , Humanos , DNA Viral/química , Integrase de HIV/química , HIV-1/enzimologia , Modelos Moleculares , Nucleoproteínas/química , Multimerização ProteicaRESUMO
Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.
Assuntos
Cisteína , Mucinas , Mucinas/metabolismo , Cisteína/metabolismo , Transporte Biológico , Vesículas Secretórias/metabolismoRESUMO
Biomaterials with antimicrobial activity are gaining attention due to their biodegradability and efficacy in interacting with a wide range of microorganisms. A new cellulose nano-biomaterial, endospermic nanocellulose crystals (ENC) obtained from parenchymal tissue of ivory nut endosperm, has a natural capacity as a universal binder. This feature is enhanced when it is chemically functionalized, and can be exploited in the fight against microbes. We tested the ability of sulfated ENC in aqueous suspension to encapsulate viruses through a crosslinking reaction mediated by cations. 0.25% w/v ENC suspensions efficiently encapsulated spike (S) protein, preventing its interaction with ACE2 receptor. ENC was further able to encapsulate SARS-CoV-2 pseudoviruses and prevent infection of 293T-hsACE2 cells. ENC also suppressed infection of MT-4 cells with HIV-1LAI.04. This antiviral activity of sulfated ENC is due to the irreversible interaction of ENC with viral particles mediated by crosslinking, as antiviral activity was less effective in the absence of cations. Additionally, ENC was used as a matrix to immobilize recombinant ACE2 receptors and anti-S IgG, creating molecular lures that efficiently inhibited SARS-CoV-2 infections in vitro. These results show that sulfated ENC from ivory nuts can be used as an efficient antiviral material.
Assuntos
COVID-19 , HIV-1 , Humanos , SARS-CoV-2/metabolismo , COVID-19/prevenção & controle , HIV-1/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Sulfatos , Endosperma/metabolismo , Ligação Proteica , Antivirais/farmacologiaRESUMO
Biomaterials with antimicrobial activity are gaining attention due to their biodegradability and efficacy in interacting with a wide range of microorganisms. A new cellulose nano-biomaterial, endospermic nanocellulose crystals (ENC) obtained from parenchymal tissue of ivory nut endosperm, has a natural capacity as a universal binder. This feature is enhanced when it is chemically functionalized, and can be exploited in the fight against microbes. We tested the ability of sulfated ENC in aqueous suspension to encapsulate viruses through a crosslinking reaction mediated by cations. 0.25% w/v ENC suspensions efficiently encapsulated spike (S) protein, preventing its interaction with ACE2 receptor. ENC was further able to encapsulate SARS-CoV-2 pseudoviruses and prevent infection of 293T-ACE2 cells. ENC also suppressed infection of MT-4 cells with HIV-1 LAI.04 . This antiviral activity of sulfated ENC is due to the irreversible interaction of ENC with viral particles mediated by crosslinking, as antiviral activity was less effective in the absence of cations. Additionally, ENC was used as a matrix to immobilize recombinant ACE2 receptors and anti-S IgG, creating molecular lures that efficiently inhibited SARS-CoV-2 infections in vitro . These results show that sulfated ENC from ivory nuts can be used as an efficient antiviral material.
RESUMO
Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; â¼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up â¼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófago T4/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Viral/química , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Escherichia coli , Interações Hospedeiro-Patógeno , RegulonRESUMO
Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19-469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.
Assuntos
Monofenol Mono-Oxigenase/química , Nanopartículas/química , Vias Biossintéticas , Catálise , Fracionamento Químico , Expressão Gênica , Melaninas/biossíntese , Microscopia de Força Atômica , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/isolamento & purificaçãoRESUMO
The interplay between transcription factors, chromatin remodelers, 3-D organization, and mechanical properties of the chromatin fiber controls genome function in eukaryotes. Besides the canonical histones which fold the bulk of the chromatin into nucleosomes, histone variants create distinctive chromatin domains that are thought to regulate transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether histone variants translate distinctive biochemical or biophysical properties to their associated chromatin structures, and whether these properties impact chromatin dynamics as the genome undergoes a multitude of transactions, is an important question in biology. Here, we describe single-molecule nanoindentation tools that we developed specifically to determine the mechanical properties of histone variant nucleosomes and their complexes. These methods join an array of cutting-edge new methods that further our quantitative understanding of the response of chromatin to intrinsic and extrinsic forces which act upon it during biological transactions in the nucleus.
Assuntos
Montagem e Desmontagem da Cromatina , Histonas/química , Nucleossomos/química , Células HeLa , Histonas/metabolismo , Histonas/ultraestrutura , Humanos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Análise EspectralRESUMO
Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (<25 °C). At higher temperatures, >31 °C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates.
Assuntos
Melanossomas/metabolismo , Oxirredutases/metabolismo , Humanos , Melaninas/metabolismo , Modelos Moleculares , Oxirredutases/química , Agregados Proteicos , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins, which discriminate between histone variant nucleosomes, suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Taken together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.
Assuntos
Histonas/química , Nucleossomos/química , Proteína Centromérica A/química , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Simulação por Computador , Reparo do DNA , Replicação do DNA , Histonas/fisiologia , Simulação de Dinâmica Molecular , Nucleossomos/fisiologia , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
Durotaxis is a type of directed cell migration in which cells respond to a gradient of extracellular stiffness. Using automated tracking of positional data for large sample sizes of single migrating cells, we investigated 1) whether cancer cells can undergo durotaxis; 2) whether cell durotactic efficiency varies depending on the regional compliance of stiffness gradients; 3) whether a specific cell migration parameter such as speed or time of migration correlates with durotaxis; and 4) whether Arp2/3, previously implicated in leading edge dynamics and migration, contributes to cancer cell durotaxis. Although durotaxis has been characterized primarily in nonmalignant mesenchymal cells, little is known about its role in cancer cell migration. Diffusible factors are known to affect cancer cell migration and metastasis. However, because many tumor microenvironments gradually stiffen, we hypothesized that durotaxis might also govern migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing substrate stiffness gradients mirroring the physiological stiffness encountered by cells in a variety of tissues. Automated cell tracking permitted rapid acquisition of positional data and robust statistical analyses for migrating cells. These durotaxis assays demonstrated that all cancer cell lines tested (two glioblastoma, metastatic breast cancer, and fibrosarcoma) migrated directionally in response to changes in extracellular stiffness. Unexpectedly, all cancer cell lines tested, as well as noninvasive human fibroblasts, displayed the strongest durotactic migratory response when migrating on the softest regions of stiffness gradients (2-7 kPa), with decreased responsiveness on stiff regions of gradients. Focusing on glioblastoma cells, durotactic forward migration index and displacement rates were relatively stable over time. Correlation analyses showed the expected correlation with displacement along the gradient but much less with persistence and none with cell speed. Finally, we found that inhibition of Arp2/3, an actin-nucleating protein necessary for lamellipodial protrusion, impaired durotactic migration.
Assuntos
Movimento Celular , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Humanos , CinéticaRESUMO
Human tyrosinase (hTyr) is a Type 1 membrane bound glycoenzyme that catalyzes the initial and rate-limiting steps of melanin production in the melanosome. Mutations in the Tyr gene are linked to oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder. Currently, the application of enzyme replacement therapy for a treatment of OCA1 is hampered by the absence of pure hTyr. Here, full-length hTyr (residues 1-529) was overexpressed in Trichoplusia ni larvae infected with a baculovirus, solubilized with detergent and purified using chromatography. Michaelis-Menten kinetics, enzymatic specific activity, and analytical ultracentrifugation were used to compare the hTyr in detergent with the soluble recombinant intra-melanosomal domain, hTyrCtr (residues 19-469). Active hTyr is monomeric in detergent micelles suggesting no stable interactions between protein molecules. Both, hTyr and hTyrCtr, exhibited similar enzymatic activity and ligand affinity in L-DOPA and L-Tyrosine reactions. In addition, expression in larvae is a scalable process that will allow high yield protein production. Thus, larval production of enzymatically active human tyrosinase potentially could be a useful tool in developing a cure for OCA1.
Assuntos
Monofenol Mono-Oxigenase/química , Albinismo Oculocutâneo/enzimologia , Albinismo Oculocutâneo/genética , Albinismo Oculocutâneo/terapia , Terapia de Reposição de Enzimas , Humanos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/uso terapêutico , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêuticoRESUMO
OBJECTIVE: Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. APPROACH AND RESULTS: Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. CONCLUSIONS: To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.
Assuntos
Colesterol/metabolismo , Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Células Cultivadas , Matriz Extracelular/ultraestrutura , Humanos , Macrófagos/ultraestrutura , Masculino , Microdomínios da Membrana/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Microscopia de Força Atômica , Microscopia Eletroquímica de Varredura , Microscopia de FluorescênciaRESUMO
Cartilage is composed of cells and an extracellular matrix, the latter being a composite of a collagen mesh interpenetrated by proteoglycans responsible for tissue osmotic swelling. The matrix composition and structure vary through the tissue depth. Mapping such variability requires tissue sectioning to gain access. The resulting surface roughness, and concomitant proteoglycan loss contribute to large uncertainties in elastic modulus estimates. To extract elasticity values for the bulk matrix which are not obfuscated by the indeterminate surface layer, we developed a novel experimental and data analysis methodology. We analyzed the surface roughness to optimize the probe size, and performed high-resolution (1 µm) elasticity mapping on thin (â¼12 µm), epiphyseal newborn mouse cartilage sections cut parallel to the bone longitudinal axis or normal to the articular surface. Mild fixation prevented the major proteoglycan loss observed in unfixed specimens but not the stress release that resulted in thickness changes in the sectioned matrix. Our novel data analysis method introduces a virtual contact point as a fitting parameter for the Hertz model, to minimize the effects of surface roughness and corrects for the finite section thickness. Our estimates of cartilage elasticity converge with increasing indentation depth and, unlike previous data interpretations, are consistent with linearly elastic material. A high cell density that leaves narrow matrix septa between cells may cause the underestimation of elastic moduli, whereas fixation probably causes an overestimation. The proposed methodology has broader relevance to nano- and micro-indentation of soft materials with multiple length scales of organization and whenever surface effects (including roughness, electrostatics, van der Waals forces, etc.) become significant.
RESUMO
Deposition of amyloid-ß plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-ß peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aß fibrils increases ß-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aß aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-ß-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Amiloide/toxicidade , Neurotoxinas/toxicidade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Amiloide/química , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Dicroísmo Circular , Imunofluorescência , Humanos , Camundongos Transgênicos , Microscopia de Força Atômica , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/química , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress.
Assuntos
Proteína BRCA1/química , Proteínas de Ligação a DNA/química , Pontos de Checagem da Fase S do Ciclo Celular , Animais , Sítios de Ligação , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Aberrações Cromossômicas , Reparo do DNA , Replicação do DNA/genética , Replicação do DNA/fisiologia , Humanos , Camundongos , Microscopia de Força Atômica , Mutagênese Sítio-Dirigida , Domínios Proteicos , Análise de Sequência de ProteínaRESUMO
Hydrogels are of intense recent interest in connection with biomedical applications ranging from 3-D cell cultures and stem cell differentiation to regenerative medicine, controlled drug delivery and tissue engineering. This prototypical form of soft matter has many emerging material science applications outside the medical field. The physical processes underlying this type of solidification are incompletely understood and this limits design efforts aimed at optimizing these materials for applications. We address this general problem by applying multiple techniques (e.g., NMR, dynamic light scattering, small angle neutron scattering, rheological measurements) to the case of a peptide derivative hydrogelator (molecule 1, NapFFKYp) over a broad range of concentration and temperature to characterize both the formation of individual nanofibers and the fiber network. We believe that a better understanding of the hierarchical self-assembly process and control over the final morphology of this kind of material should have broad significance for biological and medicinal applications utilizing hydrogels.
RESUMO
UNLABELLED: Repeated extragenic palindromes (REPs) in the enterobacterial genomes are usually composed of individual palindromic units separated by linker sequences. A total of 355 annotated REPs are distributed along the Escherichia coli genome. RNA sequence (RNAseq) analysis showed that almost 80% of the REPs in E. coli are transcribed. The DNA sequence of REP325 showed that it is a cluster of six repeats, each with two palindromic units capable of forming cruciform structures in supercoiled DNA. Here, we report that components of the REP325 element and at least one of its RNA products play a role in bacterial nucleoid DNA condensation. These RNA not only are present in the purified nucleoid but bind to the bacterial nucleoid-associated HU protein as revealed by RNA IP followed by microarray analysis (RIP-Chip) assays. Deletion of REP325 resulted in a dramatic increase of the nucleoid size as observed using transmission electron microscopy (TEM), and expression of one of the REP325 RNAs, nucleoid-associated noncoding RNA 4 (naRNA4), from a plasmid restored the wild-type condensed structure. Independently, chromosome conformation capture (3C) analysis demonstrated physical connections among various REP elements around the chromosome. These connections are dependent in some way upon the presence of HU and the REP325 element; deletion of HU genes and/or the REP325 element removed the connections. Finally, naRNA4 together with HU condensed DNA in vitro by connecting REP325 or other DNA sequences that contain cruciform structures in a pairwise manner as observed by atomic force microscopy (AFM). On the basis of our results, we propose molecular models to explain connections of remote cruciform structures mediated by HU and naRNA4. IMPORTANCE: Nucleoid organization in bacteria is being studied extensively, and several models have been proposed. However, the molecular nature of the structural organization is not well understood. Here we characterized the role of a novel nucleoid-associated noncoding RNA, naRNA4, in nucleoid structures both in vivo and in vitro. We propose models to explain how naRNA4 together with nucleoid-associated protein HU connects remote DNA elements for nucleoid condensation. We present the first evidence of a noncoding RNA together with a nucleoid-associated protein directly condensing nucleoid DNA.
Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , RNA não Traduzido/genética , DNA Bacteriano/genética , DNA Super-Helicoidal , Sequências Repetidas Invertidas , Análise em Microsséries , Microscopia de Força Atômica , Modelos Moleculares , Análise de Sequência de RNA/métodosRESUMO
Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.
Assuntos
Extensões da Superfície Celular/fisiologia , Colágenos Fibrilares/fisiologia , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Galinhas , Humanos , Integrina alfa2beta1/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin decompaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina and die of cardiac malfunction. Chromatin decompaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions.
Assuntos
Cardiomiopatia Dilatada/genética , Núcleo Celular/metabolismo , Proteínas HMGN/genética , Heterocromatina/química , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Núcleo Celular/ultraestrutura , Tamanho Celular , Elasticidade , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas HMGN/metabolismo , Dureza , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , Histonas/genética , Histonas/metabolismo , Integrases/genética , Integrases/metabolismo , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Miócitos Cardíacos/patologia , Células NIH 3T3 , Lâmina Nuclear/metabolismo , Lâmina Nuclear/ultraestrutura , Cultura Primária de CélulasRESUMO
Mannobiose-modified polyethylenimines (PEI) are used in gene therapy to generate nanoparticles of DNA that can be targeted to the antigen-presenting cells of the immune system. We report that the sugar modification alters the DNA organization within the nanoparticles from homogenous to shell-like packing. The depth-dependent packing of DNA within the nanoparticles was probed using AFM nano-indentation. Unmodified PEI-DNA nanoparticles display linear elastic properties and depth-independent mechanics, characteristic of homogenous materials. Mannobiose-modified nanoparticles, however, showed distinct force regimes that were dependent on indentation depth, with 'buckling'-like response that is reproducible and not due to particle failure. By comparison with theoretical studies of spherical shell mechanics, the structure of mannobiosylated particles was deduced to be a thin shell with wall thickness in the order of few nanometers, and a fluid-filled core. The shell-core structure is also consistent with observations of nanoparticle denting in altered solution conditions, with measurements of nanoparticle water content from AFM images, and with images of DNA distribution in Transmission Electron Microscopy.