RESUMEN
The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified â¼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
Asunto(s)
Factores de Ribosilacion-ADP , Fosfolipasa D , Transducción de Señal , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Humanos , Fosfolipasa D/metabolismo , Fosfolipasa D/genética , Células HEK293 , Animales , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Mapeo de Interacción de ProteínasRESUMEN
Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.
Asunto(s)
Vía de Señalización Hippo , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas , Ribonucleasa P , Humanos , Células HeLa , Vía de Señalización Hippo/fisiología , Ribonucleasa P/metabolismo , Células HEK293 , Subunidades de Proteína/metabolismoRESUMEN
BACKGROUND: Necrotizing enterocolitis (NEC) is a major challenge for premature infants in neonatal intensive care units and efforts toward the search for indicators that could be used to predict the development of the disease have given limited results until now. METHODS: In this study, stools from 132 very low birth weight infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction. Eight infants (~6%) received a stage 3 NEC diagnosis. Their stools collected up to 10 days before diagnosis were included and matched with 14 non-NEC controls and tested by ELISA for the quantitation of eight biomarkers. RESULTS: Biomarkers were evaluated in all available stool samples leading to the identification of lipocalin-2 and calprotectin as the two most reliable predicting markers over the 10-day period prior to NEC development. Pooling the data for each infant confirmed the significance of lipocalin-2 and calprotectin, individually and in combination 1 week in advance of the NEC clinical diagnosis. CONCLUSIONS: The lipocalin-2 and calprotectin tandem represents a significant biomarker signature for predicting NEC development. Although not yet fulfilling the "perfect biomarker" criteria, it represents a first step toward it. IMPACT: Stool biomarkers can be used to predict NEC development in very low birth weight infants more than a week before the diagnosis. LCN2 was identified as a new robust biomarker for predicting NEC development, which used in conjunction with CALPRO, allows the identification of more than half of the cases that will develop NEC in very low birth weight infants. Combining more stool markers with the LCN2/CALPRO tandem such as PGE2 can further improve the algorithm for the prediction of NEC development.
Asunto(s)
Enterocolitis Necrotizante/diagnóstico , Heces/química , Recien Nacido Prematuro , Complejo de Antígeno L1 de Leucocito/metabolismo , Lipocalina 2/metabolismo , Biomarcadores/metabolismo , Enterocolitis Necrotizante/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Recién Nacido , Unidades de Cuidado Intensivo Neonatal , MasculinoRESUMEN
Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-ß-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Ciclinas/metabolismo , Homeostasis , Magnesio/metabolismo , Factores de Ribosilacion-ADP/genética , Transporte Biológico , Ciclinas/genética , Glicosilación , Células HEK293 , Humanos , Modelos Moleculares , Unión ProteicaRESUMEN
Necrotizing enterocolitis (NEC) is a life-threatening condition for premature infants in neonatal intensive care units. Finding indicators that can predict NEC development before symptoms appear would provide more time to apply targeted interventions. In this study, stools from 132 very-low-birth-weight (VLBW) infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction using proteomics technology. Eight of the VLBW infants received a stage-3 NEC diagnosis. Stools collected from the NEC infants up to 10 days before their diagnosis were available for seven of them. Their samples were matched with those from seven pairs of non-NEC controls. The samples were processed for liquid chromatography-tandem mass spectrometry analysis using SWATH/DIA acquisition and cross-compatible proteomic software to perform label-free quantification. ROC curve and principal component analyses were used to explore discriminating information and to evaluate candidate protein markers. A series of 36 proteins showed the most efficient capacity with a signature that predicted all seven NEC infants at least a week in advance. Overall, our study demonstrates that multiplexed proteomic signature detection constitutes a promising approach for the early detection of NEC development in premature infants.
Asunto(s)
Enterocolitis Necrotizante , Enfermedades del Recién Nacido , Enfermedades del Prematuro , Biomarcadores/análisis , Enterocolitis Necrotizante/diagnóstico , Humanos , Lactante , Recién Nacido , Recién Nacido de muy Bajo Peso , Espectrometría de Masas , Estudios Prospectivos , ProteómicaRESUMEN
BACKGROUND AND OBJECTIVE: The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a broad spectrum of life-threatening adverse effects on the immature gastrointestinal tract. NSAID derivatives exploiting the beneficial effects of biologically active gases, such as hydrogen sulfide (H2S), have been developed. Herein, we determined the effects of ketoprofen and ATB-352, a H2S-releasing ketoprofen derivative, on selected metabolic pathways previously identified to be significantly altered by indomethacin in the human immature intestine. METHODS: Ketoprofen and ATB-352 were tested on human mid-gestation small intestinal explants maintained in a serum-free organ culture system for 48âhours. The expression levels of the representative genes involved in selected metabolic pathways were measured by real-time PCR after a treatment of 48âhours. RESULTS: Tested at a concentration that allows more than 80% inhibition of PGE2 production, ketoprofen was found to be less damaging than indomethacin at an equivalent dosage. However, based on the inducibility of cyclooxygenase-2 transcript expression, we were able to discriminate between responder individuals in which the deleterious effects observed with indomethacin were attenuated, and non-responder specimens in which the effects were similar to those observed with indomethacin. ATB-352 did not induce significant changes compared to ketoprofen on these metabolic pathways. CONCLUSIONS: These results show less damaging effects of ketoprofen compared to indomethacin on the immature intestine and indicate that the intestinal response to this NSAID significantly varies between individuals. However, the results did not allow us to demonstrate a specific beneficial effect of H2S release in organ culture.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Indometacina/farmacología , Intestino Delgado/efectos de los fármacos , Cetoprofeno/farmacología , Transducción de Señal/efectos de los fármacos , Ciclooxigenasa 2/efectos de los fármacos , Humanos , Intestino Delgado/embriología , Cetoprofeno/análogos & derivadosRESUMEN
Interactions between cells and the extracellular matrix regulate a wide range of cell processes such as proliferation and differentiation. Laminins are major components of the basement membrane that actively participate in most biological functions via their interactions with a variety of specific cell receptors. The α5-containing laminins (LAMA5) are one of the three main types of laminins identified at the epithelial basal lamina in the adult intestine. The aim of the present study was to investigate the role of α5-containing laminins on intestinal cell proliferation and differentiation. Using an shRNA targeting approach, the effects of knocking down the expression of LAMA5 were investigated in the enterocytic-like Caco-2/15 cell line, a well-characterized model for intestinal cell differentiation. Surprisingly, the abolition of the laminin α5 chain resulted in a drastic increase in the differentiation marker sucrase-isomaltase which was correctly expressed at the apical pole of the cells as observed by indirect immunofluorescence. Transient increases of dipeptidylpeptidase IV, villin, CDX2, HNF-1α, HNF-4α and transepithelial resistance as well as an apparent redistribution of the junctional components ZO-1 and E-cadherin were also observed at early stages of differentiation but no specific effect was observed on cell proliferation as evaluated by BrdU incorporation. Taken together, these data suggest that α5-containing laminins repress intestinal differentiation in its early stages.
Asunto(s)
Diferenciación Celular/fisiología , Enterocitos/citología , Enterocitos/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Intestinos/citología , Laminina/metabolismo , Animales , Células CACO-2 , Proliferación Celular/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Intestinos/fisiologíaRESUMEN
BACKGROUND AND AIM: NO synthase 2 (NOS2) was recently identified as one the most overexpressed genes in intestinal samples of premature infants with necrotizing enterocolitis (NEC). NOS2 is widely implicated in the processes of epithelial cell injury/apoptosis and host immune defense but its specific role in inflammation of the immature human intestinal mucosa remains unclear. Interestingly, factors that prevent NEC such as epidermal growth factor (EGF) attenuate the inflammatory response in the mid-gestation human small intestine using serum-free organ culture while drugs that are associated with NEC occurrence such as the non-steroidal anti-inflammatory drug, indomethacin (INDO), exert multiple detrimental effects on the immature human intestine. In this study we investigate the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 and its downstream pathways in the immature intestine. METHODS: Gene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1 µM INDO for 48 h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes. RESULTS: In the immature intestine, NOS2 expression was found to be increased by EGF and repressed by INDO. Bioinformatic analysis identified differentially regulated pathways where NOS2 is known to play an important role including citrulline/arginine metabolism, epithelial cell junctions and oxidative stress. At the individual gene level, we identified many differentially expressed genes of the citrulline/arginine metabolism pathway such as ARG1, ARG2, GLS, OAT and OTC in response to EGF and INDO. Gene expression of tight junction components such as CLDN1, CLDN2, CLDN7 and OCN and of antioxidant markers such as DUOX2, GPX2, SOD2 were also found to be differentially modulated by EGF and INDO. CONCLUSION: These results suggest that the protective effect of EGF and the deleterious influence of INDO on the immature intestine could be mediated via regulation of NOS2. Pathways downstream of NOS2 involved with these effects include metabolism linked to NO production, epithelial barrier permeability and antioxidant expression. These results suggest that NOS2 is a likely regulator of the inflammatory response in the immature human gut and may provide a mechanistic basis for the protective effect of EGF and the deleterious effects of INDO.
Asunto(s)
Antiinflamatorios/farmacología , Íleon/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Transducción de Señal/efectos de los fármacos , Arginina/metabolismo , Citrulina/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Investigación Fetal , Fármacos Gastrointestinales/farmacología , Humanos , Íleon/efectos de los fármacos , Íleon/enzimología , Indometacina/farmacología , Óxido Nítrico Sintasa de Tipo II/análisis , Técnicas de Cultivo de ÓrganosRESUMEN
The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ~3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
RESUMEN
Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1-/- mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.
Asunto(s)
Mioblastos , Transducción de Señal , Ratones , Animales , Mioblastos/metabolismo , Ratones Noqueados , Músculos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismoRESUMEN
Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/metabolismo , Secuencia de Aminoácidos/fisiología , Animales , Drosophila , Humanos , Unión Proteica/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Aberrant expression of receptor tyrosine kinase AXL is linked to metastasis. AXL can be activated by its ligand GAS6 or by other kinases, but the signaling pathways conferring its metastatic activity are unknown. Here, we define the AXL-regulated phosphoproteome in breast cancer cells. We reveal that AXL stimulates the phosphorylation of a network of focal adhesion (FA) proteins, culminating in faster FA disassembly. Mechanistically, AXL phosphorylates NEDD9, leading to its binding to CRKII which in turn associates with and orchestrates the phosphorylation of the pseudo-kinase PEAK1. We find that PEAK1 is in complex with the tyrosine kinase CSK to mediate the phosphorylation of PAXILLIN. Uncoupling of PEAK1 from AXL signaling decreases metastasis in vivo, but not tumor growth. Our results uncover a contribution of AXL signaling to FA dynamics, reveal a long sought-after mechanism underlying AXL metastatic activity, and identify PEAK1 as a therapeutic target in AXL positive tumors.
Asunto(s)
Movimiento Celular , Adhesiones Focales/metabolismo , Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Adhesiones Focales/genética , Humanos , Invasividad Neoplásica , Neoplasias/genética , Neoplasias/fisiopatología , Paxillin/genética , Paxillin/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal , Tirosina Quinasa del Receptor AxlRESUMEN
Cancer cells exploit the epithelial-to-mesenchymal transition (EMT) program to become metastatic. Cytoskeletal regulators are required in mesenchymal cells where they promote EMT and EMT-induced migration. In a search for regulators of metastasis, we conducted shRNA screens targeting the microtubule plus-end tracking proteins (+TIPs). We show that the +TIP ACF7 is essential both for the maintenance of the EMT program and to promote migration. We find that the E3 ubiquitin ligase HectD1 promotes ACF7-proteasome-mediated degradation. Depletion of HectD1 stabilized ACF7, and this enhanced EMT and migration. Decreased HectD1 expression increased metastases in mouse models and conferred increased resistance to the cytotoxic drug cisplatin. A retrospective analysis of biopsies from breast cancer patients also reveals a correlation between higher ACF7 or lower HectD1 expression with poor clinical outcomes. Together, these results suggest that the control of ACF7 levels by HectD1 modulates EMT and the efficiency of metastasis.
Asunto(s)
Proteínas de Microfilamentos/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Transición Epitelial-Mesenquimal , Humanos , Ratones , Ratones Desnudos , Proteínas de Microfilamentos/metabolismo , Metástasis de la Neoplasia , Transducción de SeñalRESUMEN
Myoblast fusion is tightly regulated during development and regeneration of muscle fibers. BAI3 is a receptor that orchestrates myoblast fusion via Elmo/Dock1 signaling, but the mechanisms regulating its activity remain elusive. Here we report that mice lacking BAI3 display small muscle fibers and inefficient muscle regeneration after cardiotoxin-induced injury. We describe two proteins that repress or activate BAI3 in muscle progenitors. We find that the secreted C1q-like1-4 proteins repress fusion by specifically interacting with BAI3. Using a proteomic approach, we identify Stabilin-2 as a protein that interacts with BAI3 and stimulates its fusion promoting activity. We demonstrate that Stabilin-2 activates the GPCR activity of BAI3. The resulting activated heterotrimeric G-proteins contribute to the initial recruitment of Elmo proteins to the membrane, which are then stabilized on BAI3 through a direct interaction. Collectively, our results demonstrate that the activity of BAI3 is spatiotemporally regulated by C1qL4 and Stabilin-2 during myoblast fusion.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Complemento C1q/metabolismo , Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/fisiología , Mioblastos Esqueléticos/fisiología , Proteínas del Tejido Nervioso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regeneración/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Fusión Celular , Membrana Celular/metabolismo , Células Cultivadas , Complemento C1q/genética , Proteínas del Citoesqueleto/metabolismo , Expresión Génica , Silenciador del Gen , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Modelos Biológicos , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/citología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Transducción de SeñalRESUMEN
AXL is activated by its ligand GAS6 and is expressed in triple-negative breast cancer cells. In the current study, we report AXL expression in HER2-positive (HER2+) breast cancers where it correlates with poor patient survival. Using murine models of HER2+ breast cancer, Axl, but not its ligand Gas6, was found to be essential for metastasis. We determined that AXL is required for intravasation, extravasation, and growth at the metastatic site. We found that AXL is expressed in HER2+ cancers displaying epithelial-to-mesenchymal transition (EMT) signatures where it contributes to sustain EMT. Interfering with AXL in a patient-derived xenograft (PDX) impaired transforming growth factor ß (TGF-ß)-induced cell invasion. Last, pharmacological inhibition of AXL specifically decreased the metastatic burden of mice developing HER2+ breast cancer. Our data identify AXL as a potential anti-metastatic co-therapeutic target for the treatment of HER2+ breast cancers.
Asunto(s)
Neoplasias de la Mama/mortalidad , Transición Epitelial-Mesenquimal , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor ErbB-2/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Xenoinjertos , Humanos , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Receptor ErbB-2/genética , Tirosina Quinasa del Receptor AxlRESUMEN
BACKGROUND: Necrotizing enterocolitis (NEC) is the most frequent life-threatening gastrointestinal disease experienced by premature infants in neonatal intensive care units. The challenge for neonatologists is to detect early clinical manifestations of NEC. One strategy would be to identify specific markers that could be used as early diagnostic tools to identify preterm infants most at risk of developing NEC or in the event of a diagnostic dilemma of suspected disease. As a first step in this direction, we sought to determine the specific gene expression profile of NEC. METHODS: Deep sequencing (RNA-Seq) was used to establish the gene expression profiles in ileal samples obtained from preterm infants diagnosed with NEC and non-NEC conditions. Data were analyzed with Ingenuity Pathway Analysis and ToppCluster softwares. RESULTS: Data analysis indicated that the most significant functional pathways over-represented in NEC neonates were associated with immune functions, such as altered T and B cell signaling, B cell development, and the role of pattern recognition receptors for bacteria and viruses. Among the genes that were strongly modulated in neonates with NEC, we observed a significant degree of similarity when compared with those reported in Crohn's disease, a chronic inflammatory bowel disease. CONCLUSIONS: Gene expression profile analysis revealed a predominantly altered immune response in the intestine of NEC neonates. Moreover, comparative analysis between NEC and Crohn's disease gene expression repertoires revealed a surprisingly high degree of similarity between these two conditions suggesting a new avenue for identifying NEC biomarkers.