Georgios Noutsios - Academia.edu (original) (raw)

Papers by Georgios Noutsios

Journal of molecular biochemistry, Jan 20, 2013

The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that c... more The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that contains 11 motifs. To study the role of this region on SP-B mRNA splicing, minigenes were generated by systematic removal of motifs from either the 5' or 3' end. These were transfected in CHO cells to study their splicing efficiency. The latter was determined as the ratio of completely to incompletely spliced SP-B RNA. Our results indicate that SP-B intron 4 motifs differentially affect splicing. Motifs 8 and 9 significantly enhanced and reduced splicing of intron 4, respectively. RNA mobility shift assays performed with a Motif 8 sequence that contains a CAUC cis-element and cell extracts resulted in a RNA:protein shift that was lost upon mutation of the element. Furthermore, in silico analysis of mRNA secondary structure stability for minigenes with and without motif 8 indicated a correlation between mRNA stability and splicing ratio. We conclude that differential loss of speci...

AJP: Lung Cellular and Molecular Physiology, 2013

Noutsios GT, Silveyra P, Bhatti F, Floros J. Exon B of human surfactant protein A2 mRNA, alone or... more Noutsios GT, Silveyra P, Bhatti F, Floros J. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure. Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5= untranslated (5=UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5=UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 ␤/␣ isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence-and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA. 5= untranslated regions; translation; trans-acting factors; 14-3-3 family of proteins; electromobility shift assays

Pneumon, Mar 20, 2014

Nowadays we know a great deal about the lung. We understand its major functions, how it achieves ... more Nowadays we know a great deal about the lung. We understand its major functions, how it achieves most of these, how it looks microscopically, and other physiological attributes such as that adequate amounts of pulmonary surfactant in the prematurely born infant are essential for lung function and consequently for life. In this review, we summarize highlights of the history, i.e. the journey of pulmonary surfactant discovery and how it moved from the lab bench to the patient's bedside. Pneumon 2013, 26(4):1-6.

American Journal of Physiology - Lung Cellular and Molecular Physiology, 2015

Human surfactant protein A, an innate immunity molecule, is encoded by two genes SFTPA1 and SFTPA... more Human surfactant protein A, an innate immunity molecule, is encoded by two genes SFTPA1 and SFTPA2. The 5' untranslated splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation, and contains cis regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains 7 isoforms that were found by mass spectrometry to be present in eB electromobility shift assays (REMSA) (AJPLC MP 304:L722, 2013). We investigated whether 14-3-3 isoforms bind directly to eB using four different approaches. i) eB RNA pull down assays showed that 14-3-3 isoforms bind specifically eB. ii) REMSA complexes were formed using purified 14-3-3 isoforms (β, γ, ε, η, σ, τ) with wild type eB RNA, but not with isoform ζ. Both iii) RNA affinity chromatography assays and vi) Surface Plasmon Resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not ζ, bind specifically and directly eB. Inibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels, leaving the SP-A1 levels unaffected. However, inhibition of 14-3-3 σ isoform was correlated with lower levels of both SP-A1 and SP-A2. Inhibition of 14-3-3 ζ/δ isoform that does not bind eB had no effect on the expression levels of SP-A1 and SP-A2. In conclusion the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'UTR mRNA.

Journal of molecular biochemistry, Jan 20, 2013

The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that c... more The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that contains 11 motifs. To study the role of this region on SP-B mRNA splicing, minigenes were generated by systematic removal of motifs from either the 5' or 3' end. These were transfected in CHO cells to study their splicing efficiency. The latter was determined as the ratio of completely to incompletely spliced SP-B RNA. Our results indicate that SP-B intron 4 motifs differentially affect splicing. Motifs 8 and 9 significantly enhanced and reduced splicing of intron 4, respectively. RNA mobility shift assays performed with a Motif 8 sequence that contains a CAUC cis-element and cell extracts resulted in a RNA:protein shift that was lost upon mutation of the element. Furthermore, in silico analysis of mRNA secondary structure stability for minigenes with and without motif 8 indicated a correlation between mRNA stability and splicing ratio. We conclude that differential loss of speci...

Swiss Medical Weekly, 2014

Childhood asthma is an umbrella of multifactorial diseases with similar clinical features such as... more Childhood asthma is an umbrella of multifactorial diseases with similar clinical features such as mast cell and eosinophil infiltration causing airway hyper responsiveness, inflammation, and airway obstruction. There are various factors that are implicated in childhood asthma pathogenesis. A combined contribution of genetic predisposition, environmental insults, and epigenetic changes account for polarisation of the immune system towards T helper (Th) type 2 cell responses that include production of pro-inflammatory cytokines, IgE, and eosinophil infiltrates, shown to associate with asthma. Environmental cues in prenatal, perinatal, and early childhood seem to determine development of asthma incidence or protection against it. Mode of birth delivery, use of antibiotics, oxidative stress, exposure to tobacco smoke and an industrialised lifestyle are significant contributors to childhood asthma exacerbation. Environmental stimuli such as exposure to maternal antibodies through breast milk, and certain early infections favour Th1 cell responses, leading to the production of anti-inflammatory cytokines that protect from asthma. Aside from the Th cell responses the role of innate immunity in the context of alveolar macrophages, dendritic cells, and surfactant protein A (SP-A) and SP-D is discussed. SP-A and SP-D enhance pathogen phagocytosis and cytokine production by alveolar macrophages, bind and clear pathogens, and interact with dendritic cells to mediate adaptive immunity responses. Further study of the interactions between genetic variants of genes of interest (SP-A and SP-D) and the environment may provide valuable knowledge about the underlying mechanisms of various interactions that differentially affect asthma susceptibility, disease severity, and reveal potential points for therapeutic interventions.

Experimental Lung Research, 2014

Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innat... more Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II cells (ATII), one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3′UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII vs. ATI, 12 of which predicted to bind SP-A 3′UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SPA expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.

Veterinary Research Communications, 2012

AJP: Lung Cellular and Molecular Physiology, 2013

Journal of molecular biochemistry, Jan 20, 2013

The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that c... more The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that contains 11 motifs. To study the role of this region on SP-B mRNA splicing, minigenes were generated by systematic removal of motifs from either the 5' or 3' end. These were transfected in CHO cells to study their splicing efficiency. The latter was determined as the ratio of completely to incompletely spliced SP-B RNA. Our results indicate that SP-B intron 4 motifs differentially affect splicing. Motifs 8 and 9 significantly enhanced and reduced splicing of intron 4, respectively. RNA mobility shift assays performed with a Motif 8 sequence that contains a CAUC cis-element and cell extracts resulted in a RNA:protein shift that was lost upon mutation of the element. Furthermore, in silico analysis of mRNA secondary structure stability for minigenes with and without motif 8 indicated a correlation between mRNA stability and splicing ratio. We conclude that differential loss of speci...

AJP: Lung Cellular and Molecular Physiology, 2013

Noutsios GT, Silveyra P, Bhatti F, Floros J. Exon B of human surfactant protein A2 mRNA, alone or... more Noutsios GT, Silveyra P, Bhatti F, Floros J. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure. Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5= untranslated (5=UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5=UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 ␤/␣ isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence-and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA. 5= untranslated regions; translation; trans-acting factors; 14-3-3 family of proteins; electromobility shift assays

Pneumon, Mar 20, 2014

Nowadays we know a great deal about the lung. We understand its major functions, how it achieves ... more Nowadays we know a great deal about the lung. We understand its major functions, how it achieves most of these, how it looks microscopically, and other physiological attributes such as that adequate amounts of pulmonary surfactant in the prematurely born infant are essential for lung function and consequently for life. In this review, we summarize highlights of the history, i.e. the journey of pulmonary surfactant discovery and how it moved from the lab bench to the patient's bedside. Pneumon 2013, 26(4):1-6.

American Journal of Physiology - Lung Cellular and Molecular Physiology, 2015

Human surfactant protein A, an innate immunity molecule, is encoded by two genes SFTPA1 and SFTPA... more Human surfactant protein A, an innate immunity molecule, is encoded by two genes SFTPA1 and SFTPA2. The 5' untranslated splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation, and contains cis regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains 7 isoforms that were found by mass spectrometry to be present in eB electromobility shift assays (REMSA) (AJPLC MP 304:L722, 2013). We investigated whether 14-3-3 isoforms bind directly to eB using four different approaches. i) eB RNA pull down assays showed that 14-3-3 isoforms bind specifically eB. ii) REMSA complexes were formed using purified 14-3-3 isoforms (β, γ, ε, η, σ, τ) with wild type eB RNA, but not with isoform ζ. Both iii) RNA affinity chromatography assays and vi) Surface Plasmon Resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not ζ, bind specifically and directly eB. Inibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels, leaving the SP-A1 levels unaffected. However, inhibition of 14-3-3 σ isoform was correlated with lower levels of both SP-A1 and SP-A2. Inhibition of 14-3-3 ζ/δ isoform that does not bind eB had no effect on the expression levels of SP-A1 and SP-A2. In conclusion the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'UTR mRNA.

Journal of molecular biochemistry, Jan 20, 2013

The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that c... more The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that contains 11 motifs. To study the role of this region on SP-B mRNA splicing, minigenes were generated by systematic removal of motifs from either the 5' or 3' end. These were transfected in CHO cells to study their splicing efficiency. The latter was determined as the ratio of completely to incompletely spliced SP-B RNA. Our results indicate that SP-B intron 4 motifs differentially affect splicing. Motifs 8 and 9 significantly enhanced and reduced splicing of intron 4, respectively. RNA mobility shift assays performed with a Motif 8 sequence that contains a CAUC cis-element and cell extracts resulted in a RNA:protein shift that was lost upon mutation of the element. Furthermore, in silico analysis of mRNA secondary structure stability for minigenes with and without motif 8 indicated a correlation between mRNA stability and splicing ratio. We conclude that differential loss of speci...

Swiss Medical Weekly, 2014

Childhood asthma is an umbrella of multifactorial diseases with similar clinical features such as... more Childhood asthma is an umbrella of multifactorial diseases with similar clinical features such as mast cell and eosinophil infiltration causing airway hyper responsiveness, inflammation, and airway obstruction. There are various factors that are implicated in childhood asthma pathogenesis. A combined contribution of genetic predisposition, environmental insults, and epigenetic changes account for polarisation of the immune system towards T helper (Th) type 2 cell responses that include production of pro-inflammatory cytokines, IgE, and eosinophil infiltrates, shown to associate with asthma. Environmental cues in prenatal, perinatal, and early childhood seem to determine development of asthma incidence or protection against it. Mode of birth delivery, use of antibiotics, oxidative stress, exposure to tobacco smoke and an industrialised lifestyle are significant contributors to childhood asthma exacerbation. Environmental stimuli such as exposure to maternal antibodies through breast milk, and certain early infections favour Th1 cell responses, leading to the production of anti-inflammatory cytokines that protect from asthma. Aside from the Th cell responses the role of innate immunity in the context of alveolar macrophages, dendritic cells, and surfactant protein A (SP-A) and SP-D is discussed. SP-A and SP-D enhance pathogen phagocytosis and cytokine production by alveolar macrophages, bind and clear pathogens, and interact with dendritic cells to mediate adaptive immunity responses. Further study of the interactions between genetic variants of genes of interest (SP-A and SP-D) and the environment may provide valuable knowledge about the underlying mechanisms of various interactions that differentially affect asthma susceptibility, disease severity, and reveal potential points for therapeutic interventions.

Experimental Lung Research, 2014

Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innat... more Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II cells (ATII), one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3′UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII vs. ATI, 12 of which predicted to bind SP-A 3′UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SPA expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.

Veterinary Research Communications, 2012

AJP: Lung Cellular and Molecular Physiology, 2013