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Exercise provides a robust physiological stimulus that evokes cross-talk among multiple tissues that when repeated regularly (i.e., training) improves physiological capacity, benefits numerous organ systems, and decreases the risk for premature mortality. However, a gap remains in identifying the detailed molecular signals induced by exercise that benefits health and prevents disease. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to address this gap and generate a molecular map of exercise. Preclinical and clinical studies will examine the systemic effects of endurance and resistance exercise across a range of ages and fitness levels by molecular probing of multiple tissues before and after acute and chronic exercise. From this multi-omic and bioinformatic analysis, a molecular map of exercise will be established. Altogether, MoTrPAC will provide a public database that is expected to enhance our understanding of the health benefits of exercise and to provide insight into how physical activity mitigates disease.
Assuntos
Exercício Físico/fisiologia , Resistência Física/fisiologia , Adolescente , Adulto , Animais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio , Projetos de Pesquisa , Adulto JovemRESUMO
Type 1 interferons (IFNs) promote inflammation in the skin but the mechanisms responsible for inducing these cytokines are not well understood. We found that IFN-ß was abundantly produced by epidermal keratinocytes (KCs) in psoriasis and during wound repair. KC IFN-ß production depended on stimulation of mitochondrial antiviral-signaling protein (MAVS) by the antimicrobial peptide LL37 and double stranded-RNA released from necrotic cells. MAVS activated downstream TBK1 (TANK-Binding Kinase 1)-AKT (AKT serine/threonine kinase 1)-IRF3 (interferon regulatory factor 3) signaling cascade leading to IFN-ß production and then promoted maturation of dendritic cells. In mice, the production of epidermal IFN-ß by LL37 required MAVS, and human wounded and/or psoriatic skin showed activation of MAVS-associated IRF3 and induction of MAVS and IFN-ß gene signatures. These findings show that KCs are an important source of IFN-ß and MAVS is critical to this function, and demonstrates how the epidermis triggers unwanted skin inflammation under disease conditions.
Assuntos
Catelicidinas/metabolismo , Células Dendríticas/fisiologia , Epiderme/patologia , Queratinócitos/imunologia , Mitocôndrias/metabolismo , Psoríase/imunologia , Ferimentos e Lesões/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/genética , Diferenciação Celular , Células Cultivadas , Humanos , Interferon beta/metabolismo , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Transdução de Sinais , CicatrizaçãoRESUMO
Sensory neurons are important in controlling cutaneous inflammation, but the role of neurons in host antimicrobial defense was relatively unknown. Kaplan and colleagues now demonstrate that nociceptive fibers within the dermis play a crucial role in antifungal defenses through their influence on dermal dendritic cells and induction of IL-17A.
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Células Dendríticas/imunologia , Imunidade/imunologia , Interleucina-23/imunologia , Células Receptoras Sensoriais/imunologia , Pele/imunologia , AnimaisRESUMO
The regulation of cutaneous inflammatory processes is essential for the human skin to maintain homeostasis in the presence of the dense communities of resident microbes that normally populate this organ. Forming the hair follicle-associated sebaceous gland, sebocytes are specialized lipid-producing cells that can release inflammatory mediators. Cytokine and chemokine expression by pilosebaceous epithelial cells (i.e., sebocytes and follicular keratinocytes) has been proposed to contribute to the common human skin disease acne vulgaris. The underlying mechanisms that drive inflammatory gene expression in acne-involved pilosebaceous epithelial cells are still unknown because almost all sebaceous follicles contain dense concentrations of bacteria yet only some show an inflammatory reaction. In this study, we hypothesized that metabolites from the abundant skin-resident microbe Propionibacterium acnes can influence cytokine expression from human sebocytes. We show that short-chain fatty acids produced by P. acnes under environmental conditions that favor fermentation will drive inflammatory gene expression from sebocytes. These molecules are shown to influence sebocyte behavior through two distinct mechanisms: the inhibition of histone deacetylase (HDAC) activity and the activation of fatty acid receptors. Depletion of HDAC8 and HDAC9 in human sebocytes resulted in an enhanced cytokine response to TLR-2 activation that resembled the transcriptional profile of an acne lesion. These data provide a new insight into the regulation of inflammatory gene expression in the skin, further characterize the contribution of sebocytes to epidermal immunity, and demonstrate how changes in the metabolic state of the skin microbiome can promote inflammatory acne.
Assuntos
Células Epiteliais/metabolismo , Ácidos Graxos Voláteis/metabolismo , Regulação da Expressão Gênica/fisiologia , Propionibacteriaceae/metabolismo , Glândulas Sebáceas/metabolismo , Acne Vulgar/imunologia , Acne Vulgar/metabolismo , Linhagem Celular , Epigênese Genética , Células Epiteliais/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Glândulas Sebáceas/imunologia , Pele/imunologia , Pele/metabolismo , Pele/microbiologiaRESUMO
A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor ß superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment.
Assuntos
Catelicidinas/farmacologia , Endotélio Vascular/metabolismo , Fibroblastos/metabolismo , Queratinócitos/metabolismo , RNA de Cadeia Dupla/genética , RNA não Traduzido/genética , Pele/metabolismo , Peptídeos Catiônicos Antimicrobianos , Western Blotting , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Pele/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Darier disease (DD) is a genetic skin disease that is associated with mutations in the ATP2A2 gene encoding the type 2 sarco/endoplasmic reticulum (ER) Ca2+ - ATPase (SERCA2). Mutations of this gene result in alterations of calcium homoeostasis, abnormal epidermal adhesion and dyskeratosis. Silencing of ATP2A2 in monolayer cell culture of keratinocytes reduces desmoplakin expression at the borders of cells and impacts cell adhesion. Here, we report establishment of a three-dimensional (3D) epidermal model of DD and use this model to evaluate peptide therapy with tuberoinfundibular peptide of 39 residues (TIP39) to normalize calcium transport. Gene silencing of ATP2A2 in keratinocytes grown in a 3D model resulted in dyskeratosis, partial parakeratosis and suprabasal clefts that resembled the histological changes seen in skin biopsies from patients with DD. TIP39, a peptide recently identified as a regulator of keratinocyte calcium transport, was then applied to this ATP2A2-silenced 3D epidermal model. In normal keratinocytes, TIP39 increased [Ca2+ ]i through the inositol trisphosphate (IP3) receptor pathway and stimulated differentiation. In monolayer ATP2A2-silenced keratinocytes, although TIP39 increased cytosolic calcium from the ER, the response was incomplete compared with its control. TIP39 was observed to reduce intercellular clefts of the gene-silenced epidermal model but did not significantly upregulate keratinocyte differentiation genes such as keratin 10 and filaggrin. These findings indicate that TIP39 is a modulator of ER calcium signalling and may be used as a potential strategy for improving aspects of DD.
Assuntos
Cálcio/metabolismo , Doença de Darier/metabolismo , Neuropeptídeos/metabolismo , Receptor Tipo 2 de Hormônio Paratireóideo/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Epiderme/metabolismo , Proteínas Filagrinas , Humanos , Queratinócitos/metabolismoRESUMO
The skin, the human body's largest organ, is home to a diverse and complex variety of innate and adaptive immune functions. Despite this potent immune system present at the cutaneous barrier, the skin encourages colonization by microorganisms. Characterization these microbial communities has enhanced our knowledge of the ecology of organisms present in normal skin; furthermore, studies have begun to bring to light the intimate relationships shared between host and resident microbes. In particular, it is apparent that just as host immunological factors and behaviors shape the composition of these communities, microbes present on the skin greatly impact the functions of human immunity. Thus, today the skin immune system should be considered a collective mixture of elements from the host and microbes acting in a mutualistic relationship. In this article we will review recent findings of the interactions of skin microbial communities with host immunity, and discuss the role that dysbiosis of these communities plays in diseases of the skin.
Assuntos
Microbiota/imunologia , Pele/imunologia , Pele/microbiologia , Animais , Disbiose , HumanosRESUMO
Mice with skeletal muscle-specific inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and thus could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was â¼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a â¼65% reduction in in vivo ankle dorsiflexion torque, as compared to wildtype (WT; i.e. Cre negative) littermates. Despite the profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca 2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca 2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice. New & Noteworthy: The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both p300 and CBP in skeletal muscle remains unknown. Here we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca 2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca 2+ and bypasses membrane depolarization.
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Mice with skeletal muscle-specific and inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and, thus, whether they could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was â¼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a â¼65% reduction in in vivo ankle dorsiflexion torque, as compared with wild type (WT; i.e., Cre negative) littermates. Despite this profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial-specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume, or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice.NEW & NOTEWORTHY The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both E1A binding protein p300 (p300) and cAMP-response element-binding protein binding protein (CBP) in skeletal muscle remains unknown. Here, we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca2+ and bypasses membrane depolarization.
Assuntos
Camundongos Knockout , Contração Muscular , Músculo Esquelético , Animais , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Músculo Esquelético/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional , Proteína p300 Associada a E1A/metabolismo , Proteína de Ligação a CREB/metabolismo , Masculino , Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , AcetilaçãoRESUMO
Exercise is firmly established as a key contributor to overall well-being and is frequently employed as a therapeutic approach to mitigate various health conditions. One pivotal aspect of the impact of exercise lies in the systemic transcriptional response, which underpins its beneficial adaptations. While extensive research has been devoted to understanding the transcriptional response to exercise, our knowledge of the protein constituents of nuclear processes that accompany gene expression in skeletal muscle remains largely elusive. We hypothesize that alterations in the nuclear proteome following exercise hold vital clues for comprehending the transcriptional regulation and other related nuclear functions. We isolated skeletal muscle nuclei from C57BL/6 mice both sedentary control and one-hour post 30-minute treadmill running, to gain insights into the nuclear proteome after exercise. A substantial number of the 2,323 proteins identified, were related to nuclear functions. For instance, we found 59 proteins linked to nucleocytoplasmic transport were higher in sedentary mice compared to exercise, hinting at an exercise-induced modulation to nuclear trafficking. Furthermore, 135 proteins exhibited increased abundance after exercise (FDR < 0.1) while 89 proteins decreased, with the most prominent changes in proteins linked to mRNA processing and splicing. Super resolution microscopy further highlights potential localization change in mRNA processing proteins post-exercise, further suggesting changes in nuclear transport dynamics. Nonetheless, our data provide important considerations for the study of the nuclear proteome and supports a paradigm through which exercise downregulated mRNA processing and splicing, offering valuable insights into the broader landscape of the impact from acute exercise. New & Noteworthy: Exercise plays a crucial role in promoting muscle health, but our understanding of nuclear proteins orchestrating exercise responses is limited. Isolation of skeletal muscle nuclei coupled with mass spectrometry enhanced the identification of nuclear proteins. This approach was used to investigate the effects of acute exercise, revealing changes in the muscle nuclear proteome 1-hour post-exercise, including proteins linked to post-transcriptional processing and splicing. Our findings offer insights into the exercise-induced changes within muscle nuclear proteins.
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Subcutaneous white adipose tissue (scWAT) is a dynamic storage and secretory organ that regulates systemic homeostasis, yet the impact of endurance exercise training (ExT) and sex on its molecular landscape is not fully established. Utilizing an integrative multi-omics approach, and leveraging data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we show profound sexual dimorphism in the scWAT of sedentary rats and in the dynamic response of this tissue to ExT. Specifically, the scWAT of sedentary females displays -omic signatures related to insulin signaling and adipogenesis, whereas the scWAT of sedentary males is enriched in terms related to aerobic metabolism. These sex-specific -omic signatures are preserved or amplified with ExT. Integration of multi-omic analyses with phenotypic measures identifies molecular hubs predicted to drive sexually distinct responses to training. Overall, this study underscores the powerful impact of sex on adipose tissue biology and provides a rich resource to investigate the scWAT response to ExT.
Assuntos
Tecido Adiposo Branco , Condicionamento Físico Animal , Caracteres Sexuais , Gordura Subcutânea , Animais , Masculino , Feminino , Ratos , Tecido Adiposo Branco/metabolismo , Gordura Subcutânea/metabolismo , Adipogenia , Ratos Sprague-Dawley , MultiômicaRESUMO
Building mechanistic models of kinase-driven signaling pathways requires quantitative measurements of protein phosphorylation across physiologically relevant conditions, but this is rarely done because of the insensitivity of traditional technologies. By using a multiplexed deep phosphoproteome profiling workflow, we were able to generate a deep phosphoproteomics dataset of the EGFR-MAPK pathway in non-transformed MCF10A cells across physiological ligand concentrations with a time resolution of <12 min and in the presence and absence of multiple kinase inhibitors. An improved phosphosite mapping technique allowed us to reliably identify >46,000 phosphorylation sites on >6600 proteins, of which >4500 sites from 2110 proteins displayed a >2-fold increase in phosphorylation in response to EGF. This data was then placed into a cellular context by linking it to 15 previously published protein databases. We found that our results were consistent with much, but not all previously reported data regarding the activation and negative feedback phosphorylation of core EGFR-ERK pathway proteins. We also found that EGFR signaling is biphasic with substrates downstream of RAS/MAPK activation showing a maximum response at <3ng/ml EGF while direct substrates, such as HGS and STAT5B, showing no saturation. We found that RAS activation is mediated by at least 3 parallel pathways, two of which depend on PTPN11. There appears to be an approximately 4-minute delay in pathway activation at the step between RAS and RAF, but subsequent pathway phosphorylation was extremely rapid. Approximately 80 proteins showed a >2-fold increase in phosphorylation across all experiments and these proteins had a significantly higher median number of phosphorylation sites (~18) relative to total cellular phosphoproteins (~4). Over 60% of EGF-stimulated phosphoproteins were downstream of MAPK and included mediators of cellular processes such as gene transcription, transport, signal transduction and cytoskeletal arrangement. Their phosphorylation was either linear with respect to MAPK activation or biphasic, corresponding to the biphasic signaling seen at the level of the EGFR. This deep, integrated phosphoproteomics data resource should be useful in building mechanistic models of EGFR and MAPK signaling and for understanding how downstream responses are regulated.
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Transcription factors (TFs) play a key role in regulating gene expression and responses to stimuli. We conducted an integrated analysis of chromatin accessibility, DNA methylation, and RNA expression across eight rat tissues following endurance exercise training (EET) to map epigenomic changes to transcriptional changes and determine key TFs involved. We uncovered tissue-specific changes and TF motif enrichment across all omic layers, differentially accessible regions (DARs), differentially methylated regions (DMRs), and differentially expressed genes (DEGs). We discovered distinct routes of EET-induced regulation through either epigenomic alterations providing better access for TFs to affect target genes, or via changes in TF expression or activity enabling target gene response. We identified TF motifs enriched among correlated epigenomic and transcriptomic alterations, DEGs correlated with exercise-related phenotypic changes, and EET-induced activity changes of TFs enriched for DEGs among their gene targets. This analysis elucidates the unique transcriptional regulatory mechanisms mediating diverse organ effects of EET.
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Subcutaneous white adipose tissue (scWAT) is a dynamic storage and secretory organ that regulates systemic homeostasis, yet the impact of endurance exercise training and sex on its molecular landscape has not been fully established. Utilizing an integrative multi-omics approach with data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we identified profound sexual dimorphism in the dynamic response of rat scWAT to endurance exercise training. Despite similar cardiorespiratory improvements, only male rats reduced whole-body adiposity, scWAT adipocyte size, and total scWAT triglyceride abundance with training. Multi-omic analyses of adipose tissue integrated with phenotypic measures identified sex-specific training responses including enrichment of mTOR signaling in females, while males displayed enhanced mitochondrial ribosome biogenesis and oxidative metabolism. Overall, this study reinforces our understanding that sex impacts scWAT biology and provides a rich resource to interrogate responses of scWAT to endurance training.
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In 2020, the Department of Energy established the National Virtual Biotechnology Laboratory (NVBL) to address key challenges associated with COVID-19. As part of that effort, Pacific Northwest National Laboratory (PNNL) established a capability to collect and analyze specimens from employees who self-reported symptoms consistent with the disease. During the spring and fall of 2021, 688 specimens were screened for SARS-CoV-2, with 64 (9.3%) testing positive using reverse-transcriptase quantitative PCR (RT-qPCR). Of these, 36 samples were released for research. All 36 positive samples released for research were sequenced and genotyped. Here, the relationship between patient age and viral load as measured by Ct values was measured and determined to be only weakly significant. Consensus sequences for each sample were placed into a global phylogeny and transmission dynamics were investigated, revealing that the closest relative for many samples was from outside of Washington state, indicating mixing of viral pools within geographic regions.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiologia , Teste para COVID-19 , Técnicas de Laboratório Clínico , Filogenia , RNA Viral/análise , Manejo de Espécimes , Local de Trabalho , WashingtonRESUMO
Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomicsâcurrently on the order of 25 mg wet tissue weight. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is ≥10 million peripheral blood mononuclear cells (PBMCs). Across large study cohorts, this requirement will exceed what is obtainable for many individual patients/time points. For this reason, we were interested in the impact of differential peptide loading across multiplex channels on proteomic data quality. To achieve this, we tested a range of channel loading amounts (approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification precision, and peptide/phosphopeptide detection in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, fewer missing values were observed in TMT channels with higher peptide loading amounts compared to lower loadings. Moreover, channels with a lower loading have greater quantitative variability than channels with higher loadings. A statistical analysis showed that decreased loading amounts result in an increase in the type I error rate. We then examined the impact of differential loading on the detection of known differences between distinct AML cell lines. Similar patterns of increased data missingness and higher quantitative variability were observed as loading was decreased resulting in fewer statistical differences; however, we found good agreement in features identified as differential, demonstrating the value of this approach.
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Fosfopeptídeos , Proteômica/métodos , Proteômica/normas , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , Células Cultivadas , Cromatografia de Afinidade , Confiabilidade dos Dados , Humanos , Marcação por Isótopo , Leucócitos Mononucleares/química , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificaçãoRESUMO
Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC-MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as "Mixing QC." This Mixing QC enables the detection of TMT labeling issues by LC-MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of logâ¯2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.
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Rosacea is a chronic skin disease characterized by photosensitivity, abnormal dermal vascular behavior, inflammation, and enhanced expression of the antimicrobial peptide LL-37. We observed that dermal endothelial cells in rosacea had an increased expression of VCAM1 and hypothesized that LL-37 could be responsible for this response. The digestion of double-stranded RNA from keratinocytes exposed to UVB blocked the capacity of these cells to induce adhesion molecules on dermal microvascular endothelial cells. However, a synthetic noncoding snoU1RNA was only capable of increasing adhesion molecules on endothelial cells in the presence of LL-37, suggesting that the capacity of UVB exposure to promote both double-stranded RNA and LL-37 was responsible for the endothelial response to keratinocytes. Sequencing of RNA from the endothelial cells uncovered the activation of Gene Ontology (GO) pathways relevant to the human disease, such as type I and II interferon signaling, cell-cell adhesion, leukocyte chemotaxis, and angiogenesis. Functional relevance was demonstrated as double-stranded RNA and LL-37 promoted adhesion and transmigration of monocytes across the endothelial cell monolayers. Gene knockdown of TLR3, RIGI, or IRF1 decreased monocyte adhesion in endothelial cells, confirming the role of the double-stranded RNA recognition pathways. These observations show how the expression of LL-37 can lead to enhanced sensitivity to UVB radiation in rosacea.
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Peptídeos Catiônicos Antimicrobianos/metabolismo , Imunidade Inata/imunologia , Transtornos de Fotossensibilidade/imunologia , Rosácea/complicações , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Biópsia , Adesão Celular/imunologia , Linhagem Celular , Movimento Celular/imunologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata/genética , Queratinócitos , Camundongos , Camundongos Transgênicos , Microvasos/citologia , Microvasos/metabolismo , Transtornos de Fotossensibilidade/patologia , RNA de Cadeia Dupla/metabolismo , RNA Nuclear Pequeno/metabolismo , Rosácea/imunologia , Rosácea/patologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/irrigação sanguínea , Pele/imunologia , Pele/patologia , Pele/efeitos da radiação , Células THP-1 , Raios Ultravioleta/efeitos adversos , CatelicidinasRESUMO
Netherton syndrome (NS) is a monogenic skin disease resulting from loss of function of lymphoepithelial Kazal-type-related protease inhibitor (LEKTI-1). In this study we examine if bacteria residing on the skin are influenced by the loss of LEKTI-1 and if interaction between this human gene and resident bacteria contributes to skin disease. Shotgun sequencing of the skin microbiome demonstrates that lesional skin of NS subjects is dominated by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Isolates of either species from NS subjects are able to induce skin inflammation and barrier damage on mice. These microbes promote skin inflammation in the setting of LEKTI-1 deficiency due to excess proteolytic activity promoted by S. aureus phenol-soluble modulin α as well as increased bacterial proteases staphopain A and B from S. aureus or EcpA from S. epidermidis. These findings demonstrate the critical need for maintaining homeostasis of host and microbial proteases to prevent a human skin disease.
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Síndrome de Netherton/microbiologia , Síndrome de Netherton/patologia , Peptídeo Hidrolases/metabolismo , Pele/microbiologia , Pele/patologia , Staphylococcus aureus/enzimologia , Staphylococcus epidermidis/enzimologia , Adolescente , Adulto , Animais , Toxinas Bacterianas/metabolismo , Criança , Contagem de Colônia Microbiana , Epiderme , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microbiota , Pessoa de Meia-Idade , Síndrome de Netherton/enzimologia , Fenóis , SolubilidadeRESUMO
Colonization of the skin by Staphylococcus aureus is associated with exacerbation of atopic dermatitis (AD), but any direct mechanism through which dysbiosis of the skin microbiome may influence the development of AD is unknown. Here, we show that proteases and phenol-soluble modulin α (PSMα) secreted by S. aureus lead to endogenous epidermal proteolysis and skin barrier damage that promoted inflammation in mice. We further show that clinical isolates of different coagulase-negative staphylococci (CoNS) species residing on normal skin produced autoinducing peptides that inhibited the S. aureus agr system, in turn decreasing PSMα expression. These autoinducing peptides from skin microbiome CoNS species potently suppressed PSMα expression in S. aureus isolates from subjects with AD without inhibiting S. aureus growth. Metagenomic analysis of the AD skin microbiome revealed that the increase in the relative abundance of S. aureus in patients with active AD correlated with a lower CoNS autoinducing peptides to S. aureus ratio, thus overcoming the peptides' capacity to inhibit the S. aureus agr system. Characterization of a S. hominis clinical isolate identified an autoinducing peptide (SYNVCGGYF) as a highly potent inhibitor of S. aureus agr activity, capable of preventing S. aureus-mediated epithelial damage and inflammation on murine skin. Together, these findings show how members of the normal human skin microbiome can contribute to epithelial barrier homeostasis by using quorum sensing to inhibit S. aureus toxin production.