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1.
Nat Commun ; 11(1): 4659, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938936

RESUMO

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.


Assuntos
Butiratos/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Integrinas/antagonistas & inibidores , Naftiridinas/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Administração por Inalação , Animais , Antígenos de Neoplasias/metabolismo , Bleomicina/toxicidade , Butiratos/administração & dosagem , Butiratos/metabolismo , Butiratos/farmacocinética , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/patologia , Integrinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Naftiridinas/administração & dosagem , Naftiridinas/metabolismo , Naftiridinas/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirrolidinas/administração & dosagem , Pirrolidinas/metabolismo , Pirrolidinas/farmacocinética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tomografia Computadorizada de Emissão de Fóton Único , Fator de Crescimento Transformador beta/metabolismo , Pesquisa Médica Translacional
2.
Eur Rev Med Pharmacol Sci ; 24(14): 7816-7825, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32744709

RESUMO

Currently, the outbreak and spread of coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), are increasing worldwide. Furthermore, it has been considered as a major challenge, which threatens human beings and affects all aspects of their life. Understanding the cellular and molecular pathophysiology of the disease is currently under the focus of investigations. Accordingly, this turns the human scientific community attention to find a solution for addressing the challenge. The development of vaccines and efficient therapeutic modality is critical. So, both primary and clinical scientists are not only trying to decipher the structure of SARS-CoV-2, but also attempting to understand the underlying molecular mechanisms that cause tissues and cell injuries. SARS-CoV and SARS-CoV2 are highly homologous and share a highly similar function and behavior patterns. Therefore, this might guide us toward decoding the molecular mechanisms that are behind the SARS-CoV2 pathologic effects. It is noteworthy to mention that, the undesired host immune reactions play important roles in the pathophysiology of the disease, and it also seems that, renin-angiotensin signaling (RAS) is a key contributor in this regard. In this review, we provided a vision, highlight as well as discussing on potential therapeutic targets that might be considered to address the COVID-19 challenge.


Assuntos
Betacoronavirus/fisiologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/patologia , Basigina/metabolismo , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Integrinas/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Sistema Renina-Angiotensina , Vírus da SARS/isolamento & purificação , Síndrome Respiratória Aguda Grave/virologia , Troponina I/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(32): 19033-19044, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709748

RESUMO

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).


Assuntos
Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/citologia , Microgéis/química , Neovascularização Fisiológica , Animais , Proliferação de Células , Células Imobilizadas/química , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Membro Posterior/cirurgia , Humanos , Integrinas/genética , Integrinas/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus
4.
PLoS Biol ; 18(7): e3000755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644996

RESUMO

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Multimerização Proteica , Movimento Celular , Humanos , Integrinas/metabolismo , Células K562 , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
5.
PLoS Genet ; 16(6): e1008717, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479493

RESUMO

Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. We find that elimination of integrins from follicle cells impairs F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, we find that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. We thus propose that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces.


Assuntos
Actomiosina/metabolismo , Forma Celular , Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Integrinas/metabolismo , Fibras de Estresse/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila , Células Epiteliais/citologia , Fibras de Estresse/ultraestrutura
6.
Invest Ophthalmol Vis Sci ; 61(5): 10, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32396631

RESUMO

Purpose: A burst in phagocytosis of spent photoreceptor outer fragments by RPE is a rhythmic process occurring 1 to 2 hours after the onset of light. This phenomenon is considered crucial for the health of the photoreceptors and RPE. We have recently reported that dopamine, via dopamine 2 receptor (D2R), shifts the circadian rhythm in the RPE. Methods: Here, we first investigated the impact of the removal of D2R on the daily peak of phagocytosis by RPE and then we analyzed the function and morphology of retina and RPE in the absence of D2R. Results: D2R knockout (KO) mice do not show a daily burst of phagocytic activity after the onset of light. RNA sequencing revealed a total of 394 differentially expressed genes (DEGs) between ZT 23 and ZT 1 in the control mice, whereas in D2R KO mice, we detected 1054 DEGs. Pathway analysis of the gene expression data implicated integrin signaling to be one of the upregulated pathways in control but not in D2R KO mice. Consistent with the gene expression data, phosphorylation of focal adhesion kinase (FAK) did not increase significantly in KO mice at ZT 1. No difference in retinal thickness, visual function, or morphology of RPE cells was observed between wild-type (WT) and D2R KO mice at the age of 3 and 12 months. Conclusions: Our data suggest that removal of D2R prevents the burst of phagocytosis and a related increase in the phosphorylation of FAK after light onset. The pathway analysis points toward a putative role of D2R in controlling integrin signaling, which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Our data also indicate that the absence of the burst of phagocytic activity in the early morning does not produce any apparent deleterious effect on the retina or RPE up to 1 year of age.


Assuntos
Fagocitose , Receptores de Dopamina D2/fisiologia , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/fisiologia , Animais , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/patologia , Fosforilação/fisiologia , Tomografia de Coerência Óptica , Regulação para Cima/fisiologia
7.
Proc Natl Acad Sci U S A ; 117(24): 13329-13338, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461372

RESUMO

Two-dimensional (2D) molybdenum disulfide (MoS2) nanomaterials are an emerging class of biomaterials that are photoresponsive at near-infrared wavelengths (NIR). Here, we demonstrate the ability of 2D MoS2 to modulate cellular functions of human stem cells through photothermal mechanisms. The interaction of MoS2 and NIR stimulation of MoS2 with human stem cells is investigated using whole-transcriptome sequencing (RNA-seq). Global gene expression profile of stem cells reveals significant influence of MoS2 and NIR stimulation of MoS2 on integrins, cellular migration, and wound healing. The combination of MoS2 and NIR light may provide new approaches to regulate and direct these cellular functions for the purposes of regenerative medicine as well as cancer therapy.


Assuntos
Dissulfetos/efeitos da radiação , Células-Tronco Mesenquimais/efeitos da radiação , Molibdênio/efeitos da radiação , Nanoestruturas/efeitos da radiação , Adesão Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Sobrevivência Celular , Dissulfetos/química , Dissulfetos/metabolismo , Perfilação da Expressão Gênica , Humanos , Raios Infravermelhos , Integrinas/genética , Integrinas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Molibdênio/química , Molibdênio/metabolismo , Nanoestruturas/química , Fármacos Fotossensibilizantes , Transdução de Sinais/efeitos da radiação
8.
Int J Nanomedicine ; 15: 2501-2513, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368037

RESUMO

Purpose: The extracellular matrix (ECM) labyrinthine network secreted by mesenchymal stem cells (MSCs) provides a microenvironment that enhances cell adherence, proliferation, viability, and differentiation. The potential of graphene-based nanomaterials to mimic a tissue-specific ECM has been recognized in designing bone tissue engineering scaffolds. In this study, we investigated the expression of specific ECM proteins when human fat-derived adult MSCs adhered and underwent osteogenic differentiation in the presence of functionalized graphene nanoparticles. Methods: Graphene nanoparticles with 6-10% oxygen content were prepared and characterized by XPS, FTIR, AFM and Raman spectroscopy. Calcein-am and crystal violet staining were performed to evaluate viability and proliferation of human fat-derived MSCs on graphene nanoparticles. Alizarin red staining and quantitation were used to determine the effect of graphene nanoparticles on osteogenic differentiation. Finally, immunofluorescence assays were used to investigate the expression of ECM proteins during cell adhesion and osteogenic differentiation. Results: Our data show that in the presence of graphene, MSCs express specific integrin heterodimers and exhibit a distinct pattern of the corresponding bone-specific ECM proteins, primarily fibronectin, collagen I and vitronectin. Furthermore, MSCs undergo osteogenic differentiation spontaneously without any chemical induction, suggesting that the physicochemical properties of graphene nanoparticles might trigger the expression of bone-specific ECM. Conclusion: Understanding the cell-graphene interactions resulting in an osteogenic niche for MSCs will significantly improve the application of graphene nanoparticles in bone repair and regeneration.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Grafite/farmacologia , Células-Tronco Mesenquimais/metabolismo , Nanopartículas/química , Osteogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Integrinas/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxigênio/química , Espectroscopia Fotoeletrônica , Multimerização Proteica
9.
Nat Commun ; 11(1): 2416, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415208

RESUMO

Chemoresistance is a major obstacle in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype. Here we identify hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key inducer of chemoresistance by developing chemoresistant TNBC tumors in vivo and characterizing their transcriptomes by RNA-sequencing. Inhibiting LOX reduces collagen cross-linking and fibronectin assembly, increases drug penetration, and downregulates ITGA5/FN1 expression, resulting in inhibition of FAK/Src signaling, induction of apoptosis and re-sensitization to chemotherapy. Similarly, inhibiting FAK/Src results in chemosensitization. These effects are observed in 3D-cultured cell lines, tumor organoids, chemoresistant xenografts, syngeneic tumors and PDX models. Re-expressing the hypoxia-repressed miR-142-3p, which targets HIF1A, LOX and ITGA5, causes further suppression of the HIF-1α/LOX/ITGA5/FN1 axis. Notably, higher LOX, ITGA5, or FN1, or lower miR-142-3p levels are associated with shorter survival in chemotherapy-treated TNBC patients. These results provide strong pre-clinical rationale for developing and testing LOX inhibitors to overcome chemoresistance in TNBC patients.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/enzimologia , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Colágeno/química , Regulação para Baixo , Matriz Extracelular/metabolismo , Feminino , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia , Integrinas/metabolismo , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Transplante de Neoplasias , RNA-Seq , Transdução de Sinais
10.
PLoS One ; 15(5): e0233116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407363

RESUMO

Kaposi Sarcoma (KS) is among the most angiogenic cancers in humans and an AIDS-defining condition. KS-associated herpesvirus (KSHV) is necessary for KS development, as is vascular endothelial growth factor (VEGF-A). DLX1008 is a novel anti-VEGF-A antibody single-chain variable fragment (scFv) with low picomolar affinity for VEGF-A. In vivo imaging techniques were used to establish the efficacy of DLX1008 and to establish the mechanism of action; this included non-invasive imaging by ultrasound and optical fluorescence, verified by post-mortem histochemistry. The results showed that DLX1008 was efficacious in a KS mouse model. The NSG mouse xenografts suffered massive internal necrosis or involution, consistent with a lack of blood supply. We found that imaging by ultrasound was superior to external caliper measurements in the validation of the angiogenesis inhibitor DLX1008. Further development of DLX1008 against VEGF-dependent sarcomas is warranted.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/patologia , Anticorpos de Cadeia Única/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Meia-Vida , Integrinas/metabolismo , Masculino , Camundongos , Reprodutibilidade dos Testes , Sarcoma de Kaposi/diagnóstico por imagem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
11.
J Neurosci ; 40(17): 3360-3373, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32265259

RESUMO

The Drosophila nervous system is ensheathed by a layer of outer glial cells, the perineurial glia, and a specialized extracellular matrix, the neural lamella. The function of perineurial glial cells and how they interact with the extracellular matrix are just beginning to be elucidated. Integrin-based focal adhesion complexes link the glial membrane to the extracellular matrix, but little is known about integrin's regulators in the glia. The transmembrane Ig domain protein Basigin/CD147/EMMPRIN is highly expressed in the perineurial glia surrounding the Drosophila larval nervous system. Here we show that Basigin associates with integrin at the focal adhesions to uphold the structure of the glia-extracellular matrix sheath. Knockdown of Basigin in perineurial glia using RNAi results in significant shortening of the ventral nerve cord, compression of the glia and extracellular matrix in the peripheral nerves, and reduction in larval locomotion. We determined that Basigin is expressed in close proximity to integrin at the glial membrane, and that expression of the extracellular integrin-binding domain of Basigin is sufficient to rescue peripheral glial compression. We also found that a reduction in expression of integrin at the membrane rescues the ventral nerve cord shortening, peripheral glial compression, and locomotor phenotypes, and that reduction in the integrin-binding protein Talin can partially rescue glial compression. These results identify Basigin as a potential negative regulator of integrin in the glia, supporting proper glial and extracellular matrix ensheathment of the nervous system.SIGNIFICANCE STATEMENT The glial cells and extracellular matrix play important roles in supporting and protecting the nervous system, but the interactions between these components have not been well characterized. Our study identified expression of a conserved Ig superfamily protein, Basigin, at the glial membrane of Drosophila where it associates with the integrin-based focal adhesion complexes to ensure proper ensheathment of the CNS and PNS. Loss of Basigin in the glia results in an overall compression of the nervous system due to integrin dysregulation, which causes locomotor defects in the animals. This underlies the importance of glia-matrix communication for structural and functional support of the nervous system.


Assuntos
Proteínas de Drosophila/metabolismo , Integrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neuroglia/metabolismo , Nervos Periféricos/metabolismo , Animais , Adesão Celular/fisiologia , Drosophila melanogaster , Matriz Extracelular/metabolismo , Larva/metabolismo , Locomoção/fisiologia , Neuroglia/citologia , Nervos Periféricos/citologia , Interferência de RNA
12.
J Biosci Bioeng ; 130(1): 98-105, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32278672

RESUMO

Although various types of artificial skeletal muscle tissue have been reported, the contractile forces generated by tissue-engineered artificial skeletal muscles remain to be improved for biological model and clinical applications. In this study, we investigated the effects of extracellular matrix (ECM) and supplementation of a small molecule, which has been reported to enhance α7ß1 integrin expression (SU9516), on cell migration speed, cell fusion rate, myoblast (mouse C2C12 cells) differentiation and contractile force generation of tissue-engineered artificial skeletal muscles. When cells were cultured on varying ECM coated-surfaces, we observed significant enhancement in the migration speed, while the myotube formation (differentiation ratio) decreased in all except for cells cultured on Matrigel coated-surfaces. In contrast, SU9516 supplementation resulted in an increase in both the myotube width and differentiation ratio. Following combined culture with a Matrigel-coated surface and SU9516 supplementation, myotube width was further increased. Additionally, contractile forces produced by the tissue-engineered artificial skeletal muscles was augmented following combined culture. These findings indicate that regulation of the cell-ECM interaction is a promising approach to improve the function of tissue-engineered artificial skeletal muscles.


Assuntos
Matriz Extracelular/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Colágeno/metabolismo , Combinação de Medicamentos , Integrinas/genética , Integrinas/metabolismo , Laminina/metabolismo , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteoglicanas/metabolismo
13.
Food Chem ; 324: 126892, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32339789

RESUMO

To investigate calpain's effect on protein degradation, myowater properties, and the water-holding capacity (WHC), porcine longissimus muscles were incubated with control buffer, PD150,606 (calpain-specific inhibitor) and MG-262 (multiple-protease inhibitor) and assigned to an ageing period of 1, 4 or 7 d. Over 7 d of storage, no significant differences (P > 0.05) were observed in desmin or integrin expression between the MG-262 and PD150,606 groups, which indicated that calpain played a major role in protein proteolysis. Compared to those in the control group, muscle samples subjected to PD150,606 and MG-262 exhibited higher water mobility and a poorer WHC. Additionally, there were no significant differences in myowater properties or the WHC between the two groups at 1 d postmortem (P > 0.05). Calpain regulated the distribution and mobility of myowater, which contributed to a higher WHC in the early postmortem period (before 4 d), but other proteases tended to take over at a later stage.


Assuntos
Calpaína/metabolismo , Músculo Esquelético/química , Água/metabolismo , Acrilatos/química , Acrilatos/metabolismo , Animais , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Calpaína/antagonistas & inibidores , Desmina/metabolismo , Armazenamento de Alimentos , Integrinas/metabolismo , Carne/análise , Músculo Esquelético/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Proteólise , Suínos , Água/análise
16.
Nat Cell Biol ; 22(4): 498-511, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203420

RESUMO

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.


Assuntos
Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Integrinas/genética , Mecanotransdução Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células COS , Adesão Celular , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Biologia Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Pan troglodytes , Domínios Proteicos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/classificação , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
17.
Nat Commun ; 11(1): 1114, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111837

RESUMO

Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Movimento Celular/imunologia , Quimiocinas/metabolismo , Integrinas/metabolismo , Linfonodos/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Endotélio Linfático/fisiologia , Integrinas/genética , Linfa/citologia , Linfonodos/citologia , Ativação Linfocitária , Camundongos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de Retorno de Linfócitos/metabolismo
18.
Immunity ; 52(3): 513-527.e8, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32187519

RESUMO

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.


Assuntos
Complemento C3/imunologia , Integrinas/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Linfócitos/imunologia , Monócitos/imunologia , Migração Transendotelial e Transepitelial/imunologia , Adulto , Idoso , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Criança , Pré-Escolar , Complemento C3/genética , Complemento C3/metabolismo , Feminino , Humanos , Integrinas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Monócitos/metabolismo , Transdução de Sinais/imunologia
19.
PLoS One ; 15(3): e0230380, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163511

RESUMO

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes.


Assuntos
Membrana Basal/metabolismo , Epiderme/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Queratinócitos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Morfogênese/fisiologia , Actinas/metabolismo , Animais , Membrana Basal/citologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Adesões Focais/metabolismo , Integrinas/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Transgênicos
20.
Adv Exp Med Biol ; 1202: 129-149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32034712

RESUMO

Tumor cell invasiveness is a critical challenge in the clinical management of glioma patients. In addition, there is accumulating evidence that current therapeutic modalities, including anti-angiogenic therapy and radiotherapy, can enhance glioma invasiveness. Glioma cell invasion is stimulated by both autocrine and paracrine factors that act on a large array of cell surface-bound receptors. Key signaling elements that mediate receptor-initiated signaling in the regulation of glioblastoma invasion are Rho family GTPases, including Rac, RhoA and Cdc42. These GTPases regulate cell morphology and actin dynamics and stimulate cell squeezing through the narrow extracellular spaces that are typical of the brain parenchyma. Transient attachment of cells to the extracellular matrix is also necessary for glioblastoma cell invasion. Interactions with extracellular matrix components are mediated by integrins that initiate diverse intracellular signalling pathways. Key signaling elements stimulated by integrins include PI3K, Akt, mTOR and MAP kinases. In order to detach from the tumor mass, glioma cells secrete proteolytic enzymes that cleave cell surface adhesion molecules, including CD44 and L1. Key proteases produced by glioma cells include uPA, ADAMs and MMPs. Increased understanding of the molecular mechanisms that control glioma cell invasion has led to the identification of molecular targets for therapeutic intervention in this devastating disease.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Invasividade Neoplásica , Transdução de Sinais , Animais , Movimento Celular , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Integrinas/metabolismo
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