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1.
EMBO J ; 40(6): e105543, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33586810

RESUMO

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Vírus da Influenza A/patogenicidade , Proteínas Associadas aos Microtúbulos/metabolismo , Infecções por Orthomyxoviridae/genética , Deleção de Sequência , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Animais , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Embrião de Galinha , Citocinas/metabolismo , Cães , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Domínios Proteicos , Replicação Viral
2.
Pharmacol Res ; 107: 229-233, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27025785

RESUMO

This review synthesizes examples of pharmacological agents who have off-target effects of an epigenetic nature. We expand upon the paradigm of epigenetics to include "quasi-epigenetic" mechanisms. Quasi-epigenetics includes mechanisms of drugs acting upstream of epigenetic machinery or may themselves impact transcription factor regulation on a more global scale. We explore these avenues with four examples of conventional pharmaceuticals and their unintended, but not necessarily adverse, biological effects. The quasi-epigenetic drugs identified in this review include the use of beta-lactam antibiotics to alter glutamate receptor activity and the action of cyclosporine on multiple transcription factors. In addition, we report on more canonical epigenome changes associated with pharmacological agents such as lithium impacting autophagy of aberrant proteins, and opioid drugs whose chronic use increases the expression of genes associated with addictive phenotypes. By expanding our appreciation of transcriptomic regulation and the effects these drugs have on the epigenome, it is possible to enhance therapeutic applications by exploiting off-target effects and even repurposing established pharmaceuticals. That is, exploration of "pharmacoepigenetic" mechanisms can expand the breadth of the useful activity of a drug beyond the traditional drug targets such as receptors and enzymes.


Assuntos
Analgésicos Opioides/farmacologia , Ciclosporina/farmacologia , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos de Lítio/farmacologia , beta-Lactamas/farmacologia , Sistema X-AG de Transporte de Aminoácidos/genética , Antibacterianos/farmacologia , Imunossupressores/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores Opioides/metabolismo
3.
J Virol ; 88(18): 10340-53, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24965446

RESUMO

UNLABELLED: The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for N(pro) through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a proviral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. IMPORTANCE: Although the pestivirus N-terminal protease, N(pro), has been shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We used proteomic mass spectrometry to identify novel interacting proteins and have shown that N(pro) is present in ribosomal and ribonucleoprotein particles (RNPs), indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to N(pro). A proviral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon.


Assuntos
Vírus da Diarreia Viral Bovina Tipo 1/enzimologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Infecções por Pestivirus/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Vírus da Diarreia Viral Bovina Tipo 1/química , Vírus da Diarreia Viral Bovina Tipo 1/genética , Interações Hospedeiro-Patógeno , Humanos , Peptídeo Hidrolases/genética , Infecções por Pestivirus/virologia , Ligação Proteica , Estrutura Terciária de Proteína , Ribonucleoproteínas/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas Virais/genética
4.
JID Innov ; 4(4): 100283, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38827330

RESUMO

The skin is a multifunctional organ, forming a barrier between the external and internal environment, thereby functioning as a safeguard against extrinsic factors. Autophagy has been implicated in epidermal differentiation and in preserving skin homeostasis. LC3-associated phagocytosis (LAP) uses some but not all components of autophagy. The Atg16l1 (Δ WD) mouse model lacks the WD40 domain required for LAP and has been widely used to study the effects of LAP deficiency and autophagy on tissue homeostasis and response to infection. In this study, the Δ WD model was used to study the relationship between LAP and skin homeostasis by determining whether LAP-deficient mice display a cutaneous phenotype. Skin histology of wild-type and Δ WD mice aged 1 year revealed minor morphological differences in the tail skin dermal layer. RT-qPCR and western blot analysis showed no differences in key keratin expression between genotypes. Skin barrier formation, assessed by dye permeation assays, demonstrated full and proper formation of the skin barrier at embryonic day 18.5 in both genotypes. Biomechanical analysis of the skin showed decreased skin elasticity in aged Δ WD but not wild-type mice. In summary, the LAP-deficient Δ WD mice displayed subtle alterations in dermal histology and age-related biomechanical changes.

5.
Autophagy ; : 1-13, 2024 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-39216469

RESUMO

The WD40 domain (WDD) of ATG16L1 plays a pivotal role in non-canonical autophagy. This study examined the role of recently identified LAP-like non-canonical autophagy (LNCA) in acute pancreatitis. LNCA involves rapid single-membrane LC3 conjugation to endocytic vacuoles in pancreatic acinar cells. The rationale for this study was the previously observed presence of trypsin in the organelles undergoing LNCA; aberrant trypsin formation is an important factor in pancreatitis development. Here we report that the deletion of WDD (attained in ATG16L1[E230] mice) eliminated LNCA, aggravated caerulein-induced acute pancreatitis and suppressed the fast trypsin degradation observed in both a rapid caerulein-induced disease model and in caerulein-treated isolated pancreatic acinar cells. These experiments indicate that LNCA is a WDD-dependent mechanism and suggest that it plays not an activating but a protective role in acute pancreatitis. Furthermore, palmitoleic acid, another inducer of experimental acute pancreatitis, strongly inhibited LNCA, suggesting a novel mechanism of pancreatic lipotoxicity.Abbreviation: AMY: amylase; AP: acute pancreatitis; CASM: conjugation of Atg8 to single membranes; CCK: cholecystokinin; FAEE model: fatty acid and ethanol model; IL6: interleukin 6; LA: linoleic acid; LAP: LC3-associated phagocytosis; LMPO: lung myeloperoxidase; LNCA: LAP-like non-canonical autophagy; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MPO: myeloperoxidase; PMPO: pancreatic myeloperoxidase; POA: palmitoleic acid; WDD: WD40 domain; WT: wild type.

6.
PLoS One ; 18(8): e0289407, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37531384

RESUMO

BACKGROUND: Chronic Rhinosinusitis (CRS) affects approximately 1 in 10 UK adults and impacts quality of life quality of life significantly. Response to treatment may be driven by individual CRS endotypes and therefore work to delineate biomarker clusters that may separate responders from non-responders is needed. The ongoing MACRO three-arm parallel-group trial randomises adult CRS patients to endoscopic sinus surgery, macrolide therapy or placebo. AIM: This study aims to correlate CRS endotypes with clinical parameters from the ongoing MACRO trial, including olfactory function and outcomes in terms of response to treatment using core biomarkers sets. METHODS: Adult CRS patients enrolled into the MACRO trial will be recruited from participating UK otorhinolaryngology departments. Nasal tissue samples and swabs will be obtained in theatre or clinic from patients randomised to all three trial arms. Nasal tissue will be analysed with multiplex electrochemiluminescence for 32 cytokines including IL-5, IL-13, IgE and periostin. Bacterial swabs will be analysed using illumina miSeq 16S amplicon sequencing. Mean expression for each biomarker will be reported for treatment responder and non-responder groups. Correlation of biomarkers with MACRO trial outcome data such as endoscopic evaluation scores and quality-of-life improvement scores will be reported. DISCUSSION: Defining clear endotypes in CRS will contribute to refining patient pathways for the efficient use of clinical resources. This work may lay the groundwork for future studies to predict which patients might respond to medical or surgical therapy.


Assuntos
Pólipos Nasais , Rinite , Sinusite , Adulto , Humanos , Estudos de Coortes , Qualidade de Vida , Biomarcadores/análise , Pólipos Nasais/metabolismo , Doença Crônica , Ensaios Clínicos Controlados Aleatórios como Assunto
7.
J Neurol Sci ; 437: 120268, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35486970

RESUMO

BACKGROUND/OBJECTIVES: While the clinical manifestations of myasthenia gravis (MG) are well understood, its humanistic impact is not. The objective of this systematic literature review (SLR) was to provide a comprehensive understanding of the humanistic burden of MG with regards to psychological symptoms and health-related quality of life (HRQoL) according to patients and caregivers. METHODS: A systematic search was conducted on December 27, 2019, in MEDLINE and Embase to identify English-language studies that were published from January 1, 2009-December 27, 2019 and presented relevant information on the humanistic burden among adults with MG and their caregivers. Title/abstract and full-text screening was performed by two investigators, with any discrepancies resolved by a third investigator. RESULTS: Sixty-seven publications were included in the SLR. Compared with the general population, patients with MG experienced worse HRQoL. Studies reporting on psychological symptoms of MG, including depression, anxiety, fatigue, and sleep, were heterogeneous in terms of the scales and instruments used to assess patients, as well as the patient populations themselves. However, in general those with more severe symptoms and hospitalization days had worse depression and anxiety, and fatigue and sleep improved with disease remission and/or improvement. Scores were worse for females compared with males and where evaluated, HRQoL scores generally improved following treatment. CONCLUSION: While the literature demonstrates that symptoms associated with MG get better with disease improvement and remission, additional options in efficacious therapy that adequately address the disease-related symptoms and also improve HRQoL may contribute to beneficial outcomes in a greater number of patients with MG.


Assuntos
Miastenia Gravis , Qualidade de Vida , Adulto , Ansiedade , Cuidadores/psicologia , Fadiga/etiologia , Feminino , Humanos , Masculino , Miastenia Gravis/terapia
8.
Sci Adv ; 8(43): eabn3298, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36288298

RESUMO

The delivery of pathogens to lysosomes for degradation provides an important defense against infection. Degradation is enhanced when LC3 is conjugated to endosomes and phagosomes containing pathogens to facilitate fusion with lysosomes. In phagocytic cells, TLR signaling and Rubicon activate LC3-associated phagocytosis (LAP) where stabilization of the NADPH oxidase leads to sustained ROS production and raised vacuolar pH. Raised pH triggers the assembly of the vacuolar ATPase on the vacuole membrane where it binds ATG16L1 to recruit the core LC3 conjugation complex (ATG16L1:ATG5-12). This V-ATPase-ATG16L1 axis is also activated in nonphagocytic cells to conjugate LC3 to endosomes containing extracellular microbes. Pathogens provide additional signals for recruitment of LC3 when they raise vacuolar pH with pore-forming toxins and proteins, phospholipases, or specialized secretion systems. Many microbes secrete virulence factors to inhibit ROS production and/or the V-ATPase-ATG16L1 axis to slow LC3 recruitment and avoid degradation in lysosomes.

9.
J Clin Invest ; 132(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34990402

RESUMO

The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation, and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for remaining apoptotic debris in regulating the immunologic response to and growth of solid tumors. Here, we investigated the role of macrophage LC3-associated phagocytosis (LAP) within the BM microenvironment of AML. Depletion of BM macrophages (BMMs) increased AML growth in vivo. We show that LAP is the predominate method of BMM phagocytosis of dead and dying cells in the AML microenvironment. Targeted inhibition of LAP led to the accumulation of apoptotic cells (ACs) and apoptotic bodies (ABs), resulting in accelerated leukemia growth. Mechanistically, LAP of AML-derived ABs by BMMs resulted in stimulator of IFN genes (STING) pathway activation. We found that AML-derived mitochondrial damage-associated molecular patterns were processed by BMMs via LAP. Moreover, depletion of mitochondrial DNA (mtDNA) in AML-derived ABs showed that it was this mtDNA that was responsible for the induction of STING signaling in BMMs. Phenotypically, we found that STING activation suppressed AML growth through a mechanism related to increased phagocytosis. In summary, we report that macrophage LAP of apoptotic debris in the AML BM microenvironment suppressed tumor growth.


Assuntos
Medula Óssea , Leucemia Mieloide Aguda , Medula Óssea/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Macrófagos/metabolismo , Fagocitose , Microambiente Tumoral
10.
J Biol Chem ; 285(29): 22414-25, 2010 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-20463008

RESUMO

The protein kinase C (PKC) signaling pathway is a major regulator of cellular functions and is implicated in pathologies involving extracellular matrix remodeling. Inflammatory joint disease is characterized by excessive extracellular matrix catabolism, and here we assess the role of PKC in the induction of the collagenases, matrix metalloproteinase (MMP)-1 and MMP-13, in human chondrocytes by the potent cytokine stimulus interleukin-1 (IL-1) in combination with oncostatin M (OSM). IL-1 + OSM-stimulated collagenolysis and gelatinase activity were ameliorated by pharmacological PKC inhibition in bovine cartilage, as was collagenase gene induction in human chondrocytes. Small interfering RNA-mediated silencing of PKC gene expression showed that both novel (nPKC delta, nPKC eta) and atypical (aPKC zeta, aPKC iota) isoforms were involved in collagenase induction by IL-1. However, MMP1 and MMP13 induction by IL-1 + OSM was inhibited only by aPKC silencing, suggesting that only atypical isoforms play a significant role in complex inflammatory milieus. Silencing of either aPKC led to diminished IL-1 + OSM-dependent extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) 3 phosphorylation, and c-fos expression. STAT3 gene silencing or ERK pathway inhibition also resulted in loss of IL-1 + OSM-stimulated c-fos and collagenase expression. Silencing of c-fos and c-jun expression was sufficient to abrogate IL-1 + OSM-stimulated collagenase gene induction, and overexpression of both c-fos and c-jun was sufficient to drive transcription from the MMP1 promoter in the absence of a stimulus. Our data identify atypical PKC isozymes as STAT and ERK activators that mediate c-fos and collagenase expression during IL-1 + OSM synergy in human chondrocytes. aPKCs may constitute potential therapeutic targets for inflammatory joint diseases involving increased collagenase expression.


Assuntos
Cartilagem Articular/patologia , Colagenases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/enzimologia , Bovinos , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Condrócitos/patologia , Colagenases/biossíntese , Colagenases/genética , Indução Enzimática/efeitos dos fármacos , Gelatinases/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Interleucina-1/farmacologia , Isoenzimas/antagonistas & inibidores , Oncostatina M/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Fator de Transcrição STAT3/deficiência , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo
11.
J Exp Pharmacol ; 13: 797-806, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34429664

RESUMO

PURPOSE: One of the most serious and devastating complications of diabetes mellitus is diabetic ulcers. They are difficult to treat and often result in limb loss. Topical sucralfate and platelet-rich plasma have the potential to improve the healing outcomes of chronic ulcers, including diabetic ulcers. This research aims to determine the effectiveness of sucralfate and platelet-rich plasma therapy for the improvement of diabetic ulcer wound healing. PATIENTS AND METHODS: Ninety Wistar rats were used in this study and were classified into five groups. Four of the five groups were diabetic induced and were treated with topical sucralfate only, platelet-rich plasma only, combination of topical sucralfate and platelet-rich plasma, and diabetic control group which received standard therapy only. The non-diabetic control group did not receive any therapy. We observed macrophage amount, platelet-derived growth factor, vascular endothelial growth factor, and hypoxia-inducible factor as a biomarker. Rats were terminated after 7th and 14th days and were subjected to immunohistochemistry staining and examination. RESULTS: We found that topical sucralfate and platelet-rich plasma increase macrophage levels, vascular endothelial growth factor expression and platelet-derived growth factor expression in diabetic wound cells. We also found a reduction in hypoxia inducible factor-1α expression. Combination of topical sucralfate and platelet-rich plasma for 14 days gave the most significant improvement in terms of wound healing compared to topical sucralfate or platelet-rich plasma alone. CONCLUSION: The combination of topical sucralfate and platelet-rich plasma therapy results in the best improvement in diabetic ulcer wound healing compared to sucralfate or platelet-rich plasma monotherapy or conventional wound healing therapy.

12.
Rheumatology (Oxford) ; 49(11): 2043-53, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20634235

RESUMO

OBJECTIVES: To determine the effects and mechanism of action of lithium chloride (LiCl) on cartilage destruction induced by the pro-inflammatory cytokines IL-1, IL-1 + oncostatin M and TNF-α. METHODS: The release of collagen was assessed in bovine cartilage explant cultures, whereas collagenolytic activities (active and total) in conditioned culture supernatants were determined by bioassay. The expression and production of MMP from chondrocytes were analysed by real-time RT-PCR and ELISA. Signalling pathway analysis was performed using a phospho-antibody array and standard immunoblotting. RESULTS: LiCl, but not selective glycogen synthase kinase 3 (GSK-3) inhibitor compounds SB-415286 and TDZD-8, significantly decreased pro-inflammatory cytokine-induced collagen release from bovine cartilage via the down-regulation of collagenolytic activity. Furthermore, MMP-1 and MMP-13 expression was reduced in both bovine and human chondrocytes. Pathway analysis revealed that LiCl selectively inhibited activation of the p38 mitogen-activated protein kinase pathway; effects that were recapitulated by specific p38 pathway inhibition. CONCLUSIONS: This study demonstrates for the first time that LiCl can protect against cartilage damage induced by pro-inflammatory cytokines, and indicates that LiCl-mediated cartilage protection is not via a GSK-3-dependent mechanism, but potentially via inhibition of the p38 pathway. These data indicate that lithium administration may represent a potential therapy for arthritis.


Assuntos
Cartilagem/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Cloreto de Lítio/farmacologia , Metaloproteinases da Matriz/metabolismo , Osteoartrite/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Idoso , Animais , Cartilagem/metabolismo , Bovinos , Condrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Cloreto de Lítio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Viruses ; 12(1)2019 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-31905741

RESUMO

Sindbis virus (SINV) infection induces eIF2α phosphorylation, which leads to stress granule (SG) assembly. SINV infection also stimulates autophagy, which has an important role in controlling the innate immune response. The importance of autophagy to virus-induced translation arrest is not well understood. In this study, we show that the autophagy protein ATG16L1 not only regulates eIF2α phosphorylation and the translation of viral and antiviral proteins, but also controls SG assembly. Early in infection (2hpi), capsids were recruited by host factors Cytotoxic Granule-Associated RNA Binding Protein (TIA1), Y-box binding protein 1 (YBX1), and vasolin-containing protein 1 (VCP), to a single perinuclear body, which co-localized with the viral pattern recognition sensors, double stranded RNA-activated protein-kinase R (PKR) and RIG-I. By 6hpi, there was increased eIF2α phosphorylation and viral protein synthesis. However, in cells lacking the autophagy protein ATG16L1, SG assembly was inhibited and capsid remained in numerous small foci in the cytoplasm containing YBX1, TIA1 with RIG-I, and these persisted for over 8hpi. In the absence of ATG16L1, there was little phosphorylation of eIF2α and low levels of viral protein synthesis. Compared to wild type cells, there was potentiated interferon protein and interferon-stimulated gene (ISG) mRNA expression. These results show that ATG16L1 is required for maximum eIF2α phosphorylation, proper SG assembly into a single perinuclear focus, and for attenuating the innate immune response. Therefore, this study shows that, in the case of SINV, ATG16L1 is pro-viral, required for SG assembly and virus replication.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Grânulos Citoplasmáticos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fibroblastos/virologia , Interações Hospedeiro-Patógeno , Animais , Autofagia , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos , Fosforilação , Biossíntese de Proteínas , Sindbis virus , Proteínas Virais/genética
14.
Biomater Sci ; 7(3): 809-821, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30663733

RESUMO

Immunosenescence poses a formidable challenge in designing effective influenza vaccines for aging populations. While approved vaccines against influenza viruses exist, their efficacy in older adults is significantly decreased due to the diminished capabilities of innate and adaptive immune responses. In this work, the ability of a combination nanovaccine containing both recombinant hemagglutinin and nucleoprotein to provide protection against seasonal influenza virus infection was examined in young and aged mice. Vaccine formulations combining two nanoadjuvants, polyanhydride nanoparticles and pentablock copolymer micelles, were shown to enhance protection against challenge compared to each adjuvant alone in young mice. Nanoparticles were shown to enhance in vitro activation of dendritic cells isolated from aged mice, while both nanoadjuvants did not induce proinflammatory cytokine secretion which may be detrimental in aged individuals. In addition, the combination nanovaccine platform was shown to induce demonstrable antibody titers in both young and aged mice that correlated with the maintenance of body weight post-challenge. Collectively, these data demonstrate that the combination nanovaccine platform is a promising technology for influenza vaccines for older adults.


Assuntos
Envelhecimento , Vacinas contra Influenza/imunologia , Nanopartículas/química , Infecções por Orthomyxoviridae/prevenção & controle , Adjuvantes Imunológicos/química , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/química , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Polianidridos/química , Polímeros/química , Taxa de Sobrevida
15.
Autophagy ; 15(4): 599-612, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30403914

RESUMO

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP-/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.


Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose , Animais , Autofagia/genética , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Proteínas de Transporte/metabolismo , Citocinas/sangue , Feminino , Fibroblastos/metabolismo , Homeostase/genética , Homeostase/fisiologia , Rim/citologia , Rim/crescimento & desenvolvimento , Rim/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Longevidade/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Músculos/citologia , Músculos/metabolismo , Músculos/patologia , Fagocitose/genética , Fagocitose/fisiologia , Fagossomos/genética , Fagossomos/metabolismo , Repetições WD40/genética
16.
Dis Model Mech ; 12(3)2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30814064

RESUMO

Paneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.


Assuntos
Autofagia , Intestinos/fisiologia , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/metabolismo , Proteômica , Transcriptoma/genética , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Exocitose , Feminino , Masculino , Camundongos Endogâmicos C57BL , Proteólise , Reprodutibilidade dos Testes
17.
Physiol Rep ; 6(23): e13941, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30548229

RESUMO

The double-stranded RNA-dependent protein kinase (PKR) contributes to inflammatory cytokine expression and disease pathogenesis in many conditions. Limited data are available on the efficacy of the PKR inhibitor imoxin to prevent lipopolysaccharide (LPS)-induced inflammation in skeletal muscle in vivo. The aim of this study was to evaluate the effect of imoxin, a PKR inhibitor, on inflammatory and atrophy signaling in skeletal muscle in response to an acute inflammatory insult with LPS. Six-week old C57BL/6J mice received vehicle (saline) or 0.5 mg/kg imoxin 24 and 2 h prior to induction of inflammation via 1 mg/kg LPS. Gastrocnemius muscles were collected 24 h post-LPS and mRNA and protein expression were assessed. LPS lead to a loss of body weight, which was similar in Imoxin+LPS. There were no differences in muscle weight among groups. LPS increased gastrocnemius mRNA expression of TNF-α and IL-1ß, and protein levels of NLRP3, all of which were attenuated by imoxin. Similarly, IL-6 mRNA and IL-1ß protein were suppressed in Imoxin+LPS compared to LPS alone. LPS increased mRNA of the atrogenes, MuRF1 and MAFbx, and imoxin attenuated the LPS-induced increase in MuRF1 mRNA, and lowered MuRF1 protein. Imoxin+LPS increased p-Akt compared to saline or LPS, whereas p-mTOR was unaltered. FoxO1 was upregulated and p-FoxO1/FoxO1 reduced by LPS, both of which were prevented by imoxin. Both LPS and Imoxin+LPS had diminished p-FoxO3/FoxO3 compared to control. These results demonstrate the potential anti-inflammatory and anti-atrophy effects of imoxin on skeletal muscle in vivo.


Assuntos
Anti-Inflamatórios/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Proteínas com Motivo Tripartido/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/genética
18.
Genes Dis ; 2(3): 247-254, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30258868

RESUMO

This review considers available evidence for mechanisms of conferred adaptive advantages in the face of specific infectious diseases. In short, we explore a number of genetic conditions, which carry some benefits in adverse circumstances including exposure to infectious agents. The examples discussed are conditions known to result in resistance to a specific infectious disease, or have been proposed as being associated with resistance to various infectious diseases. These infectious disease-genetic disorder pairings include malaria and hemoglobinopathies, cholera and cystic fibrosis, tuberculosis and Tay-Sachs disease, mycotic abortions and phenylketonuria, infection by enveloped viruses and disorders of glycosylation, infection by filoviruses and Niemann-Pick C1 disease, as well as rabies and myasthenia gravis. We also discuss two genetic conditions that lead to infectious disease hypersusceptibility, although we did not cover the large number of immunologic defects leading to infectious disease hypersusceptibilities. Four of the resistance-associated pairings (malaria/hemogloginopathies, cholera/cystic fibrosis, tuberculosis/Tay-Sachs, and mycotic abortions/phenylketonuria) appear to be a result of selection pressures in geographic regions in which the specific infectious agent is endemic. The other pairings do not appear to be based on selection pressure and instead may be serendipitous. Nonetheless, research investigating these relationships may lead to treatment options for the aforementioned diseases by exploiting established mechanisms between genetically affected cells and infectious organisms. This may prove invaluable as a starting point for research in the case of diseases that currently have no reliably curative treatments, e.g., HIV, rabies, and Ebola.

19.
PLoS One ; 9(2): e88838, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24551175

RESUMO

The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway.


Assuntos
Fator Regulador 3 de Interferon/metabolismo , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Pestivirus/enzimologia , Serina Endopeptidases/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose , Biomarcadores/metabolismo , Caspases/metabolismo , Bovinos , Células HeLa , Humanos , Camundongos , Proteólise , Estresse Fisiológico , Ubiquitina/metabolismo
20.
Med Hypotheses ; 83(3): 343-5, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24986645

RESUMO

This manuscript considers available evidence that a specific Salmonella strain could be used as an effective orally-administered option for cancer therapy involving the brain. It has been established that Salmonella preferentially colonizes neoplastic tissue and thrives as a facultative anaerobe in the intra-tumor environment. Although Salmonella accumulates in tumors by passive processes, it is still possible for lipopolysaccharide to cause sepsis and endotoxic shock during the migration of bacteria to the tumor site. An LPS-free version of a recently identified Salmonella isolate may have the capability to circumvent the blood brain barrier and provide a safer method of reaching brain tumors. This isolate merits further research as a "Trojan horse" for future oral biotherapy of brain cancer.


Assuntos
Neoplasias Encefálicas/microbiologia , Salmonella/fisiologia , Animais , Antineoplásicos/administração & dosagem , Barreira Hematoencefálica , Encéfalo/patologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/terapia , Bovinos , Modelos Animais de Doenças , Humanos , Hipóxia , Lipopolissacarídeos/química , Mutação , Neoplasias/complicações , Neoplasias/microbiologia , Neoplasias/terapia , Sepse/fisiopatologia , Choque Séptico/fisiopatologia , Suínos
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