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1.
Cell ; 184(10): 2696-2714.e25, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33891876

RESUMO

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Autofagia Mediada por Chaperonas/fisiologia , Neurônios/metabolismo , Proteostase , Envelhecimento/patologia , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Caseína Quinase I/genética , Autofagia Mediada por Chaperonas/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Neurônios/patologia , Proteoma
2.
Immunity ; 54(4): 721-736.e10, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33725478

RESUMO

Hyperglycemia and hyperlipidemia are often observed in individuals with type II diabetes (T2D) and related mouse models. One dysmetabolic biochemical consequence is the non-enzymatic reaction between sugars, lipids, and proteins, favoring protein glycation, glycoxidation, and lipoxidation. Here, we identified oxidative alterations in key components of the major histocompatibility complex (MHC) class II molecule antigen processing and presentation machinery in vivo under conditions of hyperglycemia-induced metabolic stress. These modifications were linked to epitope-specific changes in endosomal processing efficiency, MHC class II-peptide binding, and DM editing activity. Moreover, we observed some quantitative and qualitative changes in the MHC class II immunopeptidome of Ob/Ob mice on a high-fat diet compared with controls, including changes in the presentation of an apolipoprotein B100 peptide associated previously with T2D and metabolic syndrome-related clinical complications. These findings highlight a link between glycation reactions and altered MHC class II antigen presentation that may contribute to T2D complications.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Estresse Fisiológico/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 2/imunologia , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Ligação Proteica/imunologia
3.
Bioorg Chem ; 123: 105744, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35349830

RESUMO

While interstrand crosslinks (ICLs) have been considered as one type of DNA damage in the past, there is mounting evidence suggesting that these highly cytotoxic lesions are processed differently by the cellular machinery depending upon the ICL structure. In this study, we examined the crosslinking ability of three mitomycins, the structure of the ICLs they produce and the cytotoxicity of the drugs toward three different cell lines. The drugs are: mitomycin C (1), decarbamoylmitomycin C (2), and a mitomycin-conjugate (3) whose mitosane moiety is linked to a N-methylpyrrole carboxamide. We found that, overall, both MC and compound 3 show strong similarities regarding their alkylation of DNA, while DMC alkylating behavior is markedly different. To gain further insight into the mode of action of these drugs, we performed high throughput gene expression and gene ontology analysis to identify gene expression and cellular pathways most impacted by each drug treatment in MCF-7 cell lines. We observed that the novel mitomycin derivative (3) specifically causes changes in the expression of genes encoding proteins involved in cell integrity and tissue structure. Further analysis using bioinformatics (IPA) indicated that the new derivative (3) displays a stronger downregulation of major signaling networks that regulate the cell cycle, DNA damage response and cell proliferation when compared to MC and DMC. Collectively, these findings demonstrate that cytotoxic mechanisms of all three drugs are complex and are not solely related to their crosslinking abilities or the structure of the ICLs they produce.


Assuntos
Adutos de DNA , Mitomicina , Alquilação , DNA/química , Dano ao DNA , Humanos , Mitomicina/química , Mitomicina/farmacologia , Mitomicinas/química , Mitomicinas/farmacologia
4.
J Immunol ; 203(8): 2339-2350, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31519866

RESUMO

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.


Assuntos
Cateterismo , Linfonodos/imunologia , Linfa/imunologia , Vasos Linfáticos/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley
5.
Microcirculation ; 26(1): e12512, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30383330

RESUMO

OBJECTIVE: Using primary LMCs in vitro, we sought to characterize the impact of LMC remodeling on their functional and molecular response to mechanical loading and culture conditions. METHODS: Primary "wounded leg" LMCs were derived from the hindlimb of three sheep who underwent lymphatic injury 6 weeks prior, while "control leg" LMCs were derived from the contralateral, unwounded, limb. Function of the LMCs was characterized in response to media of variable levels of serum (10% vs 0.2%) and glucose (4.5 vs 1 g/L). Functional and proteomic data were evaluated in LMCs exposed to cyclic stretch (0.1 Hz, 7.5% elongation) for 1 week. RESULTS: LMCs were sensitive to changes in serum levels, significantly reducing overall activity and collagen synthesis under low serum conditions. LMCs from the remodeled vessel had higher baseline levels of metabolic activity but not collagen synthesis. Cyclic loading induced cellular alignment perpendicular to the axis of stretch and alterations in signaling pathways associated with metabolism. Remodeled LMCs had consistently higher levels of metabolic activity and were more resistant to strain-induced apoptosis. CONCLUSIONS: LMCs exist on a functional spectrum, becoming more active in response to stretching and maintaining phenotypic remodeling in response to local lymphatic/tissue damage.


Assuntos
Sistema Linfático/citologia , Células Musculares/fisiologia , Remodelação Vascular , Animais , Fenômenos Biomecânicos , Células Cultivadas , Glucose/farmacologia , Extremidade Inferior , Células Musculares/metabolismo , Proteômica , Soro , Ovinos , Cicatrização
6.
Chemistry ; 24(23): 6030-6035, 2018 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-29504661

RESUMO

Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug.


Assuntos
DNA/química , Mitomicina/farmacologia , Mitomicinas/química , Alquilação , Reagentes de Ligações Cruzadas/química , Adutos de DNA , Dano ao DNA , Humanos , Estereoisomerismo
7.
J Biol Chem ; 291(11): 5576-5595, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26740625

RESUMO

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin ß4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.


Assuntos
Células Dendríticas/metabolismo , Antígeno HLA-DR1/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Colágeno Tipo II/química , Colágeno Tipo II/metabolismo , Complemento C3/química , Complemento C3/metabolismo , Células Dendríticas/química , Gelsolina/química , Gelsolina/metabolismo , Antígeno HLA-DR1/química , Humanos , Linfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Proteoma/química , Proteômica , Transdução de Sinais , Timosina/química , Timosina/metabolismo
8.
J Biol Chem ; 291(35): 18096-106, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27405763

RESUMO

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 µm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Membranas Intracelulares/metabolismo , Fosfatidilserinas/metabolismo , Animais , Linhagem Celular , Endossomos/química , Endossomos/genética , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/genética , Membranas Intracelulares/química , Camundongos , Fosfatidilserinas/química , Fosfatidilserinas/genética
9.
Int Immunol ; 27(5): 219-27, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25788586

RESUMO

During the last 20 years a deeper understanding of the lymphatic circulatory system, lymph formation and composition has emerged. This review will examine the current knowledge on the organization of the lymphatic vascular tree, the formation of lymph from the extracellular fluid, lymph circulation and the lymph proteomic composition during physiological and pathological conditions. Formation of the lymph fluid is dependent on pressure gradients in the capillary beds and the composition of the endothelial cell glycocalyx, which acts as a molecular sieve. Fluid propulsion toward the draining node is dependent on the intrinsic pumping mechanism of the lymphangions and their unidirectional valves. The lymph 'omics' composition is dependent on the ultrafiltration of plasma proteins as well as proteins and molecules derived from the metabolic and catabolic activities of each parenchymal organ from which the lymph drains. Altogether, these new insights have brought about a new awareness of the importance of the lymphatic system in human physiology and pathology.


Assuntos
Células Endoteliais/imunologia , Glicocálix/imunologia , Linfonodos/imunologia , Linfa/metabolismo , Vasos Linfáticos/imunologia , Animais , Apresentação de Antígeno , Circulação Sanguínea , Humanos , Proteômica
10.
Trends Immunol ; 32(1): 6-11, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21123113

RESUMO

Prenodal lymph is generated from the interstitial fluid that surrounds organs, and thus contains products of organ metabolism and catabolism. New proteomic analyses of lymph have identified proteins and peptides that are derived from capillary extravasation and tissue-specific proteins. Many of these peptides are detected at nanomolar concentrations in the lymph before passage through a regional lymph node. Before entering the node and once inside, proteins and processed peptides are filtered from the lymph by circulating immature dendritic cells (DCs) or non-activated nodal antigen-presenting cells (APCs) (macrophages, B cells and immature DCs). Here, we suggest that this process ensures organ-specific self-antigens are displayed to circulating and nodal APCs, thus contributing to the maintenance of peripheral tolerance.


Assuntos
Autoantígenos/imunologia , Tolerância Imunológica , Linfa/química , Linfa/imunologia , Proteoma/análise , Animais , Humanos , Sistema Linfático/química , Complexo Principal de Histocompatibilidade , Peptídeos/análise
11.
Chem Biol Interact ; 395: 111007, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38642817

RESUMO

Mitomycin C (MC) is an anti-cancer drug which functions by forming interstrand crosslinks (ICLs) between opposing DNA strands. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger cytotoxic effects on cancer cells with TP53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (TP53-proficient) and K562 (TP53 deficient) cells in a TP53-independent mode. We also found that MC/DMC regulate AKT activation in a TP53-dependent manner and that AKT deactivation is not associated with the activation of p21WAF1/CIP1 in response to MC/DMC treatment. RAS proteins are known players in the upstream mediated signaling of p21WAF1/CIP1 activation that leads to control of cell proliferation and cell death. Thus, this prompted us to investigate the effect of both drugs on the expression of RAS proteins and regulation of the MAPK/ERK signaling pathways in MCF-7 and K562 cancer cells. To accomplish this goal, we performed comparative label free proteomics profiling coupled to bioinformatics/complementary phosphoprotein arrays and Western blot validations of key signaling molecules. The MAPK/ERK pathway exhibited an overall downregulation upon MC/DMC treatment in MCF-7 cells but only DMC exhibited a mild downregulation of that same pathway in TP53 mutant K562 cells. Furthermore, treatment of MCF-7 and K562 cell lines with oligonucleotides containing the interstrand crosslinks (ICLs) formed by MC or DMC shows that both ICLs had a stronger effect on the downregulation of RAS protein expression in mutant TP53 K562 cells. We discuss the implication of this regulation of the MAPK/ERK pathway in relation to cellular TP53 status.


Assuntos
Sistema de Sinalização das MAP Quinases , Mitomicina , Proteínas ras , Humanos , Mitomicina/farmacologia , Células K562 , Proteínas ras/metabolismo , Células MCF-7 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética
12.
Cell Rep ; 43(6): 114311, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38848214

RESUMO

The lymphatic fluid is the conduit by which part of the tissue "omics" is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics, herein we investigate the lymph proteomic composition, uncovering that its composition varies according to the tissue of origin. Tissue specificity is also reflected in the dendritic cell-major histocompatibility complex class II-eluted immunopeptidome harvested from the cervical and mesenteric nodes. Following inflammatory disruption of the gut barrier, the lymph antigenic and inflammatory loads are analyzed in both mice and subjects with inflammatory bowel diseases. Gastrointestinal tissue damage reflects the lymph inflammatory and damage-associated molecular pattern signatures, microbiome-derived by-products, and immunomodulatory molecules, including metabolites of the gut-brain axis, mapped in the afferent mesenteric lymph. Our data point to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance.


Assuntos
Antígenos , Inflamação , Linfonodos , Linfa , Camundongos Endogâmicos C57BL , Animais , Camundongos , Linfa/metabolismo , Linfa/imunologia , Inflamação/imunologia , Inflamação/patologia , Inflamação/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Humanos , Antígenos/metabolismo , Antígenos/imunologia , Masculino , Feminino , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo
13.
Aging Cell ; 22(11): e13979, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37749958

RESUMO

Senolytic drugs are designed to selectively clear senescent cells (SnCs) that accumulate with injury or aging. In a mouse model of osteoarthritis (OA), senolysis yields a pro-regenerative response, but the therapeutic benefit is reduced in aged mice. Increased oxidative stress is a hallmark of advanced age. Therefore, here we investigate whether senolytic treatment differentially affects joint oxidative load in young and aged animals. We find that senolysis by a p53/MDM2 interaction inhibitor, UBX0101, reduces protein oxidative modification in the aged arthritic knee joint. Mass spectrometry coupled with protein interaction network analysis and biophysical stability prediction of extracted joint proteins revealed divergent responses to senolysis between young and aged animals, broadly suggesting that knee regeneration and cellular stress programs are contrarily poised to respond as a function of age. These opposing responses include differing signatures of protein-by-protein oxidative modification and abundance change, disparate quantitative trends in modified protein network centrality, and contrasting patterns of oxidation-induced folding free energy perturbation between young and old. We develop a composite sensitivity score to identify specific key proteins in the proteomes of aged osteoarthritic joints, thereby nominating prospective therapeutic targets to complement senolytics.


Assuntos
Osteoartrite , Senoterapia , Masculino , Camundongos , Animais , Modelos Animais de Doenças , Estresse Oxidativo , Envelhecimento/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Senescência Celular
14.
Cell Rep ; 42(12): 113529, 2023 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-38060380

RESUMO

Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.


Assuntos
Autofagia Mediada por Chaperonas , Microautofagia , Autofagia , Endossomos/metabolismo , Lisossomos/metabolismo , Chaperonas Moleculares/metabolismo
15.
Proc Natl Acad Sci U S A ; 106(20): 8368-73, 2009 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-19416831

RESUMO

Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G(2)-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-kappaB, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.


Assuntos
Benzodioxóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Purinas/farmacologia , Antineoplásicos/farmacologia , Benzodioxóis/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Complexos Multiproteicos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Purinas/uso terapêutico , Receptor ErbB-2/deficiência , Receptores de Estrogênio/deficiência , Receptores de Progesterona/deficiência , Indução de Remissão
16.
Sci Rep ; 12(1): 5012, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35322079

RESUMO

The lymphatic vasculature is critical for lung function, but defects in lymphatic function in the pathogenesis of lung disease is understudied. In mice, lymphatic dysfunction alone is sufficient to cause lung injury that resembles human emphysema. Whether lymphatic function is disrupted in cigarette smoke (CS)-induced emphysema is unknown. In this study, we investigated the effect of CS on lung lymphatic function. Analysis of human lung tissue revealed significant lung lymphatic thrombosis in patients with emphysema compared to control smokers that increased with disease severity. In a mouse model, CS exposure led to lung lymphatic thrombosis, decreased lymphatic drainage, and impaired leukocyte trafficking that all preceded the development of emphysema. Proteomic analysis demonstrated an increased abundance of coagulation factors in the lymph draining from the lungs of CS-exposed mice compared to control mice. In addition, in vitro assays demonstrated a direct effect of CS on lymphatic endothelial cell integrity. These data show that CS exposure results in lung lymphatic dysfunction and a shift in thoracic lymph towards a prothrombic state. Furthermore, our data suggest that lymphatic dysfunction is due to effects of CS on the lymphatic vasculature that precede emphysema. These studies demonstrate a novel component of CS-induced lung injury that occurs early in the pathogenesis of emphysema.


Assuntos
Enfisema , Lesão Pulmonar , Enfisema Pulmonar , Fumaça , Trombose , Poluição por Fumaça de Tabaco , Animais , Enfisema/patologia , Humanos , Pulmão/patologia , Lesão Pulmonar/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteômica , Enfisema Pulmonar/patologia , Fumaça/efeitos adversos , Lesão por Inalação de Fumaça , Trombose/patologia , Nicotiana/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversos
17.
Sci Immunol ; 7(74): eabl3795, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35984892

RESUMO

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4+ immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.


Assuntos
Autoimunidade , Diabetes Mellitus Tipo 2 , Fígado , Isomerases de Dissulfetos de Proteínas , Animais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epitopos , Antígenos de Histocompatibilidade Classe II , Fígado/patologia , Camundongos , Peptídeos , Isomerases de Dissulfetos de Proteínas/imunologia , Isomerases de Dissulfetos de Proteínas/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-21206024

RESUMO

The serine protease thrombin plays a major role in thrombosis and haemostasis. This has driven interest in thrombin inhibitors as potential antithrombotic drugs. Here, the crystallization and preliminary crystallographic analysis of human α-thrombin in complex with three noncovalent peptide inhibitors of the general sequence D-Phe-Pro-D-Arg-P1'-CONH2 are reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and diffracted to beyond 1.3 Šresolution.


Assuntos
Peptídeos/química , Inibidores de Serina Proteinase/química , Trombina/antagonistas & inibidores , Trombina/química , Cristalização , Cristalografia por Raios X , Humanos , Peptídeos/genética , Trombina/genética
19.
STAR Protoc ; 2(3): 100648, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34278334

RESUMO

A detailed quantification of antigen processing by endosomal compartments provides important information on the pattern of protein fragmentation. Here, we describe a protocol that combines gradient purified endosomes, incubated with antigens, followed by hot spot analysis of MS/MS-sequenced peptides. The analysis identifies differences in endosomal antigen processing by dendritic cells under diverse experimental conditions. For complete details on the use and execution of this protocol, please refer to Clement et al. (2021).


Assuntos
Antígenos/metabolismo , Endossomos/metabolismo , Biologia Molecular/métodos , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endossomos/imunologia , Proteínas de Membrana/administração & dosagem , Camundongos , Peptídeos/imunologia , Peptídeos/metabolismo , Espectrometria de Massas em Tandem
20.
Front Immunol ; 12: 658601, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995376

RESUMO

Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-Ab presented immunopeptidome of the thymus of C57BL/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of <1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-Ab binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.


Assuntos
Apresentação de Antígeno/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Peptídeos/imunologia , Timo/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mapeamento de Epitopos/métodos , Epitopos/química , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Ligação Proteica , Baço/imunologia , Baço/metabolismo , Timo/metabolismo
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