Jochen Deckert - Academia.edu (original) (raw)
Papers by Jochen Deckert
Die katalytische Aktivierung des spleißosomalen B Komplexes ist eine mechanistische Voraussetzung... more Die katalytische Aktivierung des spleißosomalen B Komplexes ist eine mechanistische Voraussetzung für den Vorgang des Prä-mRNA Spleißens. Bisherige Untersuchungen dieses Komplexes wurden in Anwesenheit von Heparin durchgeführt, wodurch weniger stabil assoziierte Faktoren bei der Komplexisolierung verlorengehen. Zur Ermittlung der Protein-Zusammensetzung des B Komplexes unter milden, nativen Bedingungen, wurde dieser mittels zweier verschiedener Methoden isoliert. In beiden Fällen wurden RNA Bindungssequenzen für das MS2-Hüllprotein oder das Aminoglykosid Tobramycin in das Prä-mRNA Substrat eingefügt, die eine Markierung der RNA für Affinitätsselektionen bereitstellten. Die erste Prozedur kombiniert Dichtegradientenzentrifugation und Affinitätsselektion mittels Maltose-bindendem Protein (fusioniert mit MS2 Hüllprotein), wogegen das zweite Isolierungsverfahren sich der Tobramycin Affinitätsselektion, gefolgt von Immunaffinitätsselektion, bedient. In beiden Fällen enthielten die isolie...
iii 2.2.4.11 MS2 affinity selection of spliceosomal B complexes 2.2.4.12 In vitro splicing assays... more iii 2.2.4.11 MS2 affinity selection of spliceosomal B complexes 2.2.4.12 In vitro splicing assays with MS2 affinity-selected B complexes isolated at 150 mM salt conditions 2.2.4.13 Electron microscopy 2.2.4.14 Mass spectrometry 3. Results 37 3.2.4 In vitro translated 61kDa (hPrp31) protein is specifically recognized by cognate affinity-purified antibodies 3.2.5 Affinity purified anti-61kDa (hPrp31) antibodies efficiently precipitate U4/U6.U5 tri-snRNP from HeLa nuclear extract 3.2.6 Tobramycin affinity selection of spliceosomal B complex 3.2.7 Mass spectrometry of spliceosomal B complexes purified by tobramycin affinity selection followed by immunoprecipitation 3.3 Purification of spliceosomes in the presence of NIPP1 3.3.1 Purification of spliceosomal intermediate complexes by dominant negative protein variants 3.3.2 MS2 affinity selection of NIPP1 stalled spliceosomal B complex 3.3.3 MS of MS2 affinity purified spliceosomal complexes stalled with NIPP1 (1-311) 3.4 Electron microscopy of spliceosomal complexes 3.4.1 Structure analysis of spliceosomal complexes by electron microscopy 3.4.2 Electron microscopy of purified native B complex 4. Discussion 4.1 Purification of precatalytic B complexes using two independent approaches 4.2 Determining the stoichiometry of certain proteins within different spliceosomal complexes is a major task in the future 4.3 A number of proteins were detected only once in our purifications 4.4 MS2 and tobramycin affinity-purified B complexes are highly pure 4.5 Members of the exon junction complex are recruited after B complex formation 4.6 A large number of B complex proteins are lost upon treatment with heparin 4.7 The vast majority of A complex proteins are also present in the B complex 4.8 A large number of proteins, including the hPrp19/CDC5 complex, are recruited during B complex formation 4.9 Analysis of the factor requirements for catalytic activation and step I of splicing using MS2 affinity-selected B complexes v 4.10 Spliceosome assembly can be stalled with dominant negative protein variants 98 4.11 Electron microscopy of MS2 affinity-purified B complexes 99 5. References 101 6. Appendix
Exon, intron and splice site locations in the spliceosomal B complex
This article cites 52 articles, 27 of which can be accessed free
The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. ... more The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins— and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total wewere able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. TheMS-based quantification approaches classify the m...
La presente invention concerne de molecules de petits ARN interferents (arnsi) dirigees contre le... more La presente invention concerne de molecules de petits ARN interferents (arnsi) dirigees contre le gene SOD1, des vecteurs du virus adeno-associe (VAA) codant pour des molecules d'ARNsi et des methodes de traitement de la sclerose laterale amyotrophique (SLA) a l'aide des molecules d'ARNsi et des vecteurs VAA.
La presente invention concerne un acide ribonucleique bicatenaire (ARNdb) servant a inhiber l'... more La presente invention concerne un acide ribonucleique bicatenaire (ARNdb) servant a inhiber l'expression d'un gene du virus de l'hepatite B. L'invention concerne egalement une composition pharmaceutique contenant l'ARNdb, ou des molecules d'acides nucleiques ou des vecteurs le codant, avec un support pharmaceutiquement acceptable ; des methodes de traitement de maladies provoquees par une infection par le virus de l'hepatite B au moyen de ladite composition pharmaceutique ; et des methodes d'inhibition de l'expression d'un gene du virus de l'hepatite B dans une cellule.
Scientific reports, Apr 28, 2016
The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initi... more The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize ...
Zeitschrift für Gastroenterologie, 2015
Journal of Hepatology, 2015
POSTERS from Gpnmb − mice are able to ingest the cargo but not to process it. As in vivo model of... more POSTERS from Gpnmb − mice are able to ingest the cargo but not to process it. As in vivo model of liver fibrosis we have used the chronic administration of CCl 4. After CCl 4 withdraw mice experience a fibrotic phase dominated by inflammatory macrophages, followed by a phase of fibrosis remodelling characterised by restorative macrophages. Results: Gpnmb − mice show a higher level of plasma transaminases in the phase of fibrosis remodelling, associated with higher proliferation of hepatocytes and higher levels of inflammatory cytokines in the tissue. Concomitantly, Gpnmb − mice show a lower number of macrophages and T cells. Flow cytometry analysis unveils a skew to a pro-inflammatory phenotype in the recovering liver but not in circulating monocytes. The role of IL6 as pro-proliferative to hepatocytes is established but its regulatory role on phagocytosis is unknown. Both in vivo and in vitro IL6 shows a pro-phagocytic effect and this effect seems to be mediated by the activation of the STAT3 pathway. After a single CCl4 injection the fraction of phagocytic macrophages in the liver of Gpnmb − mice increases from 25% to 85% when IL6 is co-administered (n = 5, p < 0.01, two-way ANOVA followed by Bonferroni's post-hoc test). Conclusions: Our data suggest that phagocytosis could be the gatekeeper between the phase of initial inflammation and the phase of tissue remodelling and that IL6 could mediate this activity.
Zeitschrift für Gastroenterologie, 2014
International Journal of Infectious Diseases, 2012
Background: Hepatitis E virus (HEV)infectionis endemic in India. The disease manifestation ranges... more Background: Hepatitis E virus (HEV)infectionis endemic in India. The disease manifestation ranges fromasymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). The pathogenesis of Hepatitis E is poorly understood. High mobility group box 1 (HMGB-I) is a non-histone chromosomal protein with recently discovered proinflamatory and immunomodulatory effect in sepsis. Hypothesizing the damage in HEV is immune mediated; the present study was designed to elucidate the role of circulating HMGB1 in serum and its gene expression in peripheral blood mono-nuclear cells (PBMCs). Methods: Approximately 10 ml of venous blood was collected from 47 HEV patients (33 AVH and 14 ALF), confirmed by anti-HEV IgM and/or HEV RNA positivity. The control group (n= 20) comprised of age and sex matched apparently healthy volunteers. Serum was separated and PBMCs were isolated using Ficoll. Approximately 2x105 PBMCs/well were cultured in RPMI-1640 and pulsed with recombinant HEV ORF2 protein (452-617 a.a). Routine Biochemical investigations were performed and levels of circulating HMGB1 were estimated by quantitative micro ELISA. Gene expression levels in the patient PBMCs were checked using Real time RT-PCR. Lymphocyte proliferation was estimated using Colorimetric MTT assay. Results: Mean circulating HMGB1 levels in healthy controls (HC), AVH and ALF patient groups were found to be 18.29±16.68 ng/ml, 155.4±89.51 ng/ml and 306±70.38 ng/ml respectively. The ALF patients had significantly higher levels than AVH patients (p<0.0001). Interestingly 90% of the patients who succumbed to disease, had circulating HMGB1 levels > 250 ng/ml. However the gene expression in the PBMCs between ALF and AVH patients in comparison to HC were upregulated to 2.5 and 2.0 folds respectively. A significantly low proliferation index was observed in ALF (2.188±0.722) Vs AVH (3.040±0.667) patients (p=0.008). Strong negative correlation was found between the HMGB1 levels and prothrombin time index (PTI) in the ALF patients. Conclusion: This suggests, massive destruction of hepatocytes might lead to excessive accumulation of extracellular HMGB1 which downregulates T cell proliferation. Thus, excessive circulating HMGB1 protein might play a key role in ALF patients towards immunosupression and fulminant disease course following HEV infection.
Molecular and Cellular Biology, 2006
The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA ... more The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5 end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5 end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNAprotein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.
The Embo Journal, Jul 1, 2009
In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structu... more In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structure maps of several spliceosomal complexes. However, owing to their limited resolution, little is known at present about the location of the pre-mRNA, the spliceosomal small nuclear ribonucleoprotein or the spliceosome's active site within these structures. In this work, we used EM to localise the intron and the 5 0 and 3 0 exons of a model pre-mRNA, as well as the U2-associated protein SF3b155, in pre-catalytic spliceosomes (i.e. B complexes) by labelling them with an antibody that bears colloidal gold. Our data reveal that the intron and both exons, together with SF3b155, are located in specific regions of the head domain of the B complex. These results represent an important first step towards identifying functional sites in the spliceosome. The goldlabelling method adopted here can be applied to other spliceosomal complexes and may thus contribute significantly to our overall understanding of the pre-mRNA splicing process.
Clinical Proteomics, 2013
Background: Alzheimer's disease (AD) is the most common type of dementia affecting people over 65... more Background: Alzheimer's disease (AD) is the most common type of dementia affecting people over 65 years of age. The hallmarks of AD are the extracellular deposits known as amyloid β plaques and the intracellular neurofibrillary tangles, both of which are the principal players involved in synaptic loss and neuronal cell death. Tau protein and Aβ fragment 1-42 have been investigated so far in cerebrospinal fluid as a potential AD biomarkers. However, an urgent need to identify novel biomarkers which will capture disease in the early stages and with better specificity remains. High-throughput proteomic and pathway analysis of hippocampal tissue provides a valuable source of disease-related proteins and biomarker candidates, since it represents one of the earliest affected brain regions in AD. Results: In this study 2954 proteins were identified (with at least 2 peptides for 1203 proteins) from both control and AD brain tissues. Overall, 204 proteins were exclusively detected in AD and 600 proteins in control samples. Comparing AD and control exclusive proteins with cerebrospinal fluid (CSF) literature-based proteome, 40 out of 204 AD related proteins and 106 out of 600 control related proteins were also present in CSF. As most of these proteins were extracellular/secretory origin, we consider them as a potential source of candidate biomarkers that need to be further studied and verified in CSF samples. Conclusions: Our semiquantitative proteomic analysis provides one of the largest human hippocampal proteome databases. The lists of AD and control related proteins represent a panel of proteins potentially involved in AD pathogenesis and could also serve as prospective AD diagnostic biomarkers.
Molecular and Cellular Biology, 2006
The spliceosomal B complex is the substrate that undergoes catalytic activation leading to cataly... more The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including ϳ50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 Å and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.
The EMBO Journal, 2007
Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing... more Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.
Die katalytische Aktivierung des spleißosomalen B Komplexes ist eine mechanistische Voraussetzung... more Die katalytische Aktivierung des spleißosomalen B Komplexes ist eine mechanistische Voraussetzung für den Vorgang des Prä-mRNA Spleißens. Bisherige Untersuchungen dieses Komplexes wurden in Anwesenheit von Heparin durchgeführt, wodurch weniger stabil assoziierte Faktoren bei der Komplexisolierung verlorengehen. Zur Ermittlung der Protein-Zusammensetzung des B Komplexes unter milden, nativen Bedingungen, wurde dieser mittels zweier verschiedener Methoden isoliert. In beiden Fällen wurden RNA Bindungssequenzen für das MS2-Hüllprotein oder das Aminoglykosid Tobramycin in das Prä-mRNA Substrat eingefügt, die eine Markierung der RNA für Affinitätsselektionen bereitstellten. Die erste Prozedur kombiniert Dichtegradientenzentrifugation und Affinitätsselektion mittels Maltose-bindendem Protein (fusioniert mit MS2 Hüllprotein), wogegen das zweite Isolierungsverfahren sich der Tobramycin Affinitätsselektion, gefolgt von Immunaffinitätsselektion, bedient. In beiden Fällen enthielten die isolie...
iii 2.2.4.11 MS2 affinity selection of spliceosomal B complexes 2.2.4.12 In vitro splicing assays... more iii 2.2.4.11 MS2 affinity selection of spliceosomal B complexes 2.2.4.12 In vitro splicing assays with MS2 affinity-selected B complexes isolated at 150 mM salt conditions 2.2.4.13 Electron microscopy 2.2.4.14 Mass spectrometry 3. Results 37 3.2.4 In vitro translated 61kDa (hPrp31) protein is specifically recognized by cognate affinity-purified antibodies 3.2.5 Affinity purified anti-61kDa (hPrp31) antibodies efficiently precipitate U4/U6.U5 tri-snRNP from HeLa nuclear extract 3.2.6 Tobramycin affinity selection of spliceosomal B complex 3.2.7 Mass spectrometry of spliceosomal B complexes purified by tobramycin affinity selection followed by immunoprecipitation 3.3 Purification of spliceosomes in the presence of NIPP1 3.3.1 Purification of spliceosomal intermediate complexes by dominant negative protein variants 3.3.2 MS2 affinity selection of NIPP1 stalled spliceosomal B complex 3.3.3 MS of MS2 affinity purified spliceosomal complexes stalled with NIPP1 (1-311) 3.4 Electron microscopy of spliceosomal complexes 3.4.1 Structure analysis of spliceosomal complexes by electron microscopy 3.4.2 Electron microscopy of purified native B complex 4. Discussion 4.1 Purification of precatalytic B complexes using two independent approaches 4.2 Determining the stoichiometry of certain proteins within different spliceosomal complexes is a major task in the future 4.3 A number of proteins were detected only once in our purifications 4.4 MS2 and tobramycin affinity-purified B complexes are highly pure 4.5 Members of the exon junction complex are recruited after B complex formation 4.6 A large number of B complex proteins are lost upon treatment with heparin 4.7 The vast majority of A complex proteins are also present in the B complex 4.8 A large number of proteins, including the hPrp19/CDC5 complex, are recruited during B complex formation 4.9 Analysis of the factor requirements for catalytic activation and step I of splicing using MS2 affinity-selected B complexes v 4.10 Spliceosome assembly can be stalled with dominant negative protein variants 98 4.11 Electron microscopy of MS2 affinity-purified B complexes 99 5. References 101 6. Appendix
Exon, intron and splice site locations in the spliceosomal B complex
This article cites 52 articles, 27 of which can be accessed free
The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. ... more The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins— and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total wewere able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. TheMS-based quantification approaches classify the m...
La presente invention concerne de molecules de petits ARN interferents (arnsi) dirigees contre le... more La presente invention concerne de molecules de petits ARN interferents (arnsi) dirigees contre le gene SOD1, des vecteurs du virus adeno-associe (VAA) codant pour des molecules d'ARNsi et des methodes de traitement de la sclerose laterale amyotrophique (SLA) a l'aide des molecules d'ARNsi et des vecteurs VAA.
La presente invention concerne un acide ribonucleique bicatenaire (ARNdb) servant a inhiber l'... more La presente invention concerne un acide ribonucleique bicatenaire (ARNdb) servant a inhiber l'expression d'un gene du virus de l'hepatite B. L'invention concerne egalement une composition pharmaceutique contenant l'ARNdb, ou des molecules d'acides nucleiques ou des vecteurs le codant, avec un support pharmaceutiquement acceptable ; des methodes de traitement de maladies provoquees par une infection par le virus de l'hepatite B au moyen de ladite composition pharmaceutique ; et des methodes d'inhibition de l'expression d'un gene du virus de l'hepatite B dans une cellule.
Scientific reports, Apr 28, 2016
The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initi... more The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize ...
Zeitschrift für Gastroenterologie, 2015
Journal of Hepatology, 2015
POSTERS from Gpnmb − mice are able to ingest the cargo but not to process it. As in vivo model of... more POSTERS from Gpnmb − mice are able to ingest the cargo but not to process it. As in vivo model of liver fibrosis we have used the chronic administration of CCl 4. After CCl 4 withdraw mice experience a fibrotic phase dominated by inflammatory macrophages, followed by a phase of fibrosis remodelling characterised by restorative macrophages. Results: Gpnmb − mice show a higher level of plasma transaminases in the phase of fibrosis remodelling, associated with higher proliferation of hepatocytes and higher levels of inflammatory cytokines in the tissue. Concomitantly, Gpnmb − mice show a lower number of macrophages and T cells. Flow cytometry analysis unveils a skew to a pro-inflammatory phenotype in the recovering liver but not in circulating monocytes. The role of IL6 as pro-proliferative to hepatocytes is established but its regulatory role on phagocytosis is unknown. Both in vivo and in vitro IL6 shows a pro-phagocytic effect and this effect seems to be mediated by the activation of the STAT3 pathway. After a single CCl4 injection the fraction of phagocytic macrophages in the liver of Gpnmb − mice increases from 25% to 85% when IL6 is co-administered (n = 5, p < 0.01, two-way ANOVA followed by Bonferroni's post-hoc test). Conclusions: Our data suggest that phagocytosis could be the gatekeeper between the phase of initial inflammation and the phase of tissue remodelling and that IL6 could mediate this activity.
Zeitschrift für Gastroenterologie, 2014
International Journal of Infectious Diseases, 2012
Background: Hepatitis E virus (HEV)infectionis endemic in India. The disease manifestation ranges... more Background: Hepatitis E virus (HEV)infectionis endemic in India. The disease manifestation ranges fromasymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). The pathogenesis of Hepatitis E is poorly understood. High mobility group box 1 (HMGB-I) is a non-histone chromosomal protein with recently discovered proinflamatory and immunomodulatory effect in sepsis. Hypothesizing the damage in HEV is immune mediated; the present study was designed to elucidate the role of circulating HMGB1 in serum and its gene expression in peripheral blood mono-nuclear cells (PBMCs). Methods: Approximately 10 ml of venous blood was collected from 47 HEV patients (33 AVH and 14 ALF), confirmed by anti-HEV IgM and/or HEV RNA positivity. The control group (n= 20) comprised of age and sex matched apparently healthy volunteers. Serum was separated and PBMCs were isolated using Ficoll. Approximately 2x105 PBMCs/well were cultured in RPMI-1640 and pulsed with recombinant HEV ORF2 protein (452-617 a.a). Routine Biochemical investigations were performed and levels of circulating HMGB1 were estimated by quantitative micro ELISA. Gene expression levels in the patient PBMCs were checked using Real time RT-PCR. Lymphocyte proliferation was estimated using Colorimetric MTT assay. Results: Mean circulating HMGB1 levels in healthy controls (HC), AVH and ALF patient groups were found to be 18.29±16.68 ng/ml, 155.4±89.51 ng/ml and 306±70.38 ng/ml respectively. The ALF patients had significantly higher levels than AVH patients (p<0.0001). Interestingly 90% of the patients who succumbed to disease, had circulating HMGB1 levels > 250 ng/ml. However the gene expression in the PBMCs between ALF and AVH patients in comparison to HC were upregulated to 2.5 and 2.0 folds respectively. A significantly low proliferation index was observed in ALF (2.188±0.722) Vs AVH (3.040±0.667) patients (p=0.008). Strong negative correlation was found between the HMGB1 levels and prothrombin time index (PTI) in the ALF patients. Conclusion: This suggests, massive destruction of hepatocytes might lead to excessive accumulation of extracellular HMGB1 which downregulates T cell proliferation. Thus, excessive circulating HMGB1 protein might play a key role in ALF patients towards immunosupression and fulminant disease course following HEV infection.
Molecular and Cellular Biology, 2006
The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA ... more The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5 end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5 end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNAprotein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.
The Embo Journal, Jul 1, 2009
In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structu... more In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structure maps of several spliceosomal complexes. However, owing to their limited resolution, little is known at present about the location of the pre-mRNA, the spliceosomal small nuclear ribonucleoprotein or the spliceosome's active site within these structures. In this work, we used EM to localise the intron and the 5 0 and 3 0 exons of a model pre-mRNA, as well as the U2-associated protein SF3b155, in pre-catalytic spliceosomes (i.e. B complexes) by labelling them with an antibody that bears colloidal gold. Our data reveal that the intron and both exons, together with SF3b155, are located in specific regions of the head domain of the B complex. These results represent an important first step towards identifying functional sites in the spliceosome. The goldlabelling method adopted here can be applied to other spliceosomal complexes and may thus contribute significantly to our overall understanding of the pre-mRNA splicing process.
Clinical Proteomics, 2013
Background: Alzheimer's disease (AD) is the most common type of dementia affecting people over 65... more Background: Alzheimer's disease (AD) is the most common type of dementia affecting people over 65 years of age. The hallmarks of AD are the extracellular deposits known as amyloid β plaques and the intracellular neurofibrillary tangles, both of which are the principal players involved in synaptic loss and neuronal cell death. Tau protein and Aβ fragment 1-42 have been investigated so far in cerebrospinal fluid as a potential AD biomarkers. However, an urgent need to identify novel biomarkers which will capture disease in the early stages and with better specificity remains. High-throughput proteomic and pathway analysis of hippocampal tissue provides a valuable source of disease-related proteins and biomarker candidates, since it represents one of the earliest affected brain regions in AD. Results: In this study 2954 proteins were identified (with at least 2 peptides for 1203 proteins) from both control and AD brain tissues. Overall, 204 proteins were exclusively detected in AD and 600 proteins in control samples. Comparing AD and control exclusive proteins with cerebrospinal fluid (CSF) literature-based proteome, 40 out of 204 AD related proteins and 106 out of 600 control related proteins were also present in CSF. As most of these proteins were extracellular/secretory origin, we consider them as a potential source of candidate biomarkers that need to be further studied and verified in CSF samples. Conclusions: Our semiquantitative proteomic analysis provides one of the largest human hippocampal proteome databases. The lists of AD and control related proteins represent a panel of proteins potentially involved in AD pathogenesis and could also serve as prospective AD diagnostic biomarkers.
Molecular and Cellular Biology, 2006
The spliceosomal B complex is the substrate that undergoes catalytic activation leading to cataly... more The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including ϳ50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 Å and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.
The EMBO Journal, 2007
Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing... more Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.